Induction of glutathione biosynthesis by glycine-based treatment mitigates atherosclerosis.

Lower circulating levels of glycine are consistently reported in association with cardiovascular disease (CVD), but the causative role and therapeutic potential of glycine in atherosclerosis, the underlying cause of most CVDs, remain to be established. Here, following the identification of reduced circulating glycine in patients with significant coronary artery disease (sCAD), we investigated a causative role of glycine in atherosclerosis by modulating glycine availability in atheroprone mice. We further evaluated the atheroprotective potential of DT-109, a recently identified glycine-based compound with dual lipid/glucose-lowering properties. Glycine deficiency enhanced, while glycine supplementation attenuated, atherosclerosis development in apolipoprotein E-deficient (Apoe-/-) mice. DT-109 treatment showed the most significant atheroprotective effects and lowered atherosclerosis in the whole aortic tree and aortic sinus concomitant with reduced superoxide. In Apoe-/- mice with established atherosclerosis, DT-109 treatment significantly reduced atherosclerosis and aortic superoxide independent of lipid-lowering effects. Targeted metabolomics and kinetics studies revealed that DT-109 induces glutathione formation in mononuclear cells. In bone marrow-derived macrophages (BMDMs), glycine and DT-109 attenuated superoxide formation induced by glycine deficiency. This was abolished in BMDMs from glutamate-cysteine ligase modifier subunit-deficient (Gclm-/-) mice in which glutathione biosynthesis is impaired. Metabolic flux and carbon tracing experiments revealed that glycine deficiency inhibits glutathione formation in BMDMs while glycine-based treatment induces de novo glutathione biosynthesis. Through a combination of studies in patients with CAD, in vivo studies using atherosclerotic mice and in vitro studies using macrophages, we demonstrated a causative role of glycine in atherosclerosis and identified glycine-based treatment as an approach to mitigate atherosclerosis through antioxidant effects mediated by induction of glutathione biosynthesis.

Exogenous (Pomegranate Juice) or Endogenous (Paraoxonase1) Antioxidants Decrease Triacylglycerol Accumulation in Mouse Cardiovascular Disease-Related Tissues.

The polyphenol-rich pomegranate juice (PJ) and the high-density lipoprotein (HDL)-associated paraoxonase1 (PON1) are known as potent atheroprotective antioxidants, but their effects on other tissues related to cardiovascular disease (CVD) remain unknown. The current study aimed to investigate the effects of treating mice with PJ or recombinant PON1 (rePON1) on the oxidation and lipid status of CVD-related tissues: serum, aorta, heart, liver, kidney, visceral, and subcutaneous adipose tissues (VAT and SAT). Both PJ consumption and rePON1 injection decreased the serum levels of thiobarbituric acid-reactive substances (16% and 19%) and triacylglycerols (TAG, 24% and 27%), while only rePON1 increased the levels of thiol groups (35%) and decreased serum cholesterol (15%). Both PJ and rePON1 significantly decreased aortic cholesterol (38% and 32%) and TAG (62% and 58%) contents in association with downregulation of the key TAG biosynthetic enzyme diacylglycerol O-acyltransferase 1 (DGAT1, 71% and 65%), while only PJ decreased aortic lipid peroxides (47%). Substantial TAG-lowering effects of both PJ and rePON1 were observed also in the heart (31% and 42%), liver (34% and 42%), and kidney (42% and 57%). In both VAT and SAT, rePON1 decreased the levels of lipid peroxides (28% and 25%), while PJ decreased the TAG content (22% and 18%). Ex vivo incubation of SAT with serum derived from mice that consumed PJ or injected with rePON1 decreased SAT lipid peroxides (35% or 28%) and TAG mass (12% or 10%). These novel findings highlight potent TAG-lowering properties of exogenous (PJ) and endogenous (PON1) antioxidants in tissues associated with CVD.

Leucine supplementation attenuates macrophage foam-cell formation: Studies in humans, mice, and cultured macrophages.

Whereas atherogenicity of dietary lipids has been largely studied, relatively little is known about the possible contribution of dietary amino acids to macrophage foam-cell formation, a hallmark of early atherogenesis. Recently, we showed that leucine has antiatherogenic properties in the macrophage model system. In this study, an in-depth investigation of the role of leucine in macrophage lipid metabolism was conducted by supplementing humans, mice, or cultured macrophages with leucine. Macrophage incubation with serum obtained from healthy adults supplemented with leucine (5 g/d, 3 weeks) significantly decreased cellular cholesterol mass by inhibiting the rate of cholesterol biosynthesis and increasing cholesterol efflux from macrophages. Similarly, leucine supplementation to C57BL/6 mice (8 weeks) resulted in decreased cholesterol content in their harvested peritoneal macrophages (MPM) in relation with reduced cholesterol biosynthesis rate. Studies in J774A.1 murine macrophages revealed that leucine dose-dependently decreased cellular cholesterol and triglyceride mass. Macrophages treated with leucine (0.2 mM) showed attenuated uptake of very low-density lipoproteins and triglyceride biosynthesis rate, with a concurrent down-regulation of diacylglycerol acyltransferase-1, a key enzyme catalyzing triglyceride biosynthesis in macrophages. Similar effects were observed when macrophages were treated with α-ketoisocaproate, a key leucine metabolite. Finally, both in vivo and in vitro leucine supplementation significantly improved macrophage mitochondrial respiration and ATP production. The above studies, conducted in human, mice, and cultured macrophages, highlight a protective role for leucine attenuating macrophage foam-cell formation by mechanisms related to the metabolism of cholesterol, triglycerides, and energy production. © 2018 BioFactors, 44(3):245-262, 2018.

Atherogenicity of amino acids in the lipid-laden macrophage model system in vitro and in atherosclerotic mice: a key role for triglyceride metabolism.

Atherosclerosis-related research has focused mainly on the effects of lipids on macrophage foam cell formation and atherogenesis, whereas the role of amino acids (AAs) was understudied. The current study aimed to identify anti- or pro-atherogenic AA in the macrophage model system and to elucidate the underlying metabolic and molecular mechanisms. J774A.1 cultured macrophages were treated with increasing concentrations of each 1 of the 20 AAs. Macrophage atherogenicity was assessed in terms of cellular toxicity, generation of reactive oxygen species (ROS) and cellular cholesterol or triglyceride content. At nontoxic concentrations (up to 1 mM), modest effects on ROS generation or cholesterol content were noted, but six specific AAs significantly affected macrophage triglyceride content. Glycine, cysteine, alanine and leucine significantly decreased macrophage triglyceride content (by 24%-38%), through attenuated uptake of triglyceride-rich very low-density lipoprotein (VLDL) by macrophages. In contrast, glutamate and glutamine caused a marked triglyceride accumulation in macrophages (by 107% and 129%, respectively), via a diacylglycerol acyltransferase-1 (DGAT1)-dependent increase in triglyceride biosynthesis rate with a concurrent maturation of the sterol regulatory element-binding protein-1 (SREBP1). Supplementation of apolipoprotein E-deficient (apoE-/-) mice with glycine for 40 days significantly decreased the triglyceride levels in serum and in peritoneal macrophages (MPMs) isolated from the mice (by 19%). In contrast, glutamine supplementation significantly increased MPM ROS generation and the accumulation of cholesterol and that of triglycerides (by 48%), via enhanced uptake of LDL and VLDL. Altogether, the present findings reveal some novel roles for specific AA in macrophage atherogenicity, mainly through modulation of cellular triglyceride metabolism.

Supplementation with linoleic acid-rich soybean oil stimulates macrophage foam cell formation via increased oxidative stress and diacylglycerol acyltransferase1-mediated triglyceride biosynthesis.

During the last decades there has been a staggering rise in human consumption of soybean oil (SO) and its major polyunsaturated fatty acid linoleic acid (LA). The role of SO or LA in cardiovascular diseases is highly controversial, and their impact on macrophage foam cell formation, the hallmark of early atherogenesis, is unclear. To investigate the effects of high SO or LA intake on macrophage lipid metabolism and the related mechanisms of action, C57BL/6 mice were orally supplemented with increasing levels of SO-based emulsion or equivalent levels of purified LA for 1 month, followed by analyses of lipid accumulation and peroxidation in aortas, serum and in peritoneal macrophages (MPM) of the mice. Lipid peroxidation and triglyceride mass in aortas from SO or LA supplemented mice were dose-dependently and significantly increased. In MPM from SO or LA supplemented mice, lipid peroxides were significantly increased and a marked accumulation of cellular triglycerides was found in accordance with enhanced triglyceride biosynthesis rate and overexpression of diacylglycerol acyltransferase1 (DGAT1), the key enzyme in triglyceride biosynthesis. In cultured J774A.1 macrophages treated with SO or LA, triglyceride accumulated via increased oxidative stress and a p38 mitogen-activated protein kinase (MAPK)-mediated overexpression of DGAT1. Accordingly, anti-oxidants (pomegranate polyphenols), inhibition of p38 MAPK (by SB202190) or DGAT1 (by oleanolic acid), all significantly attenuated SO or LA-induced macrophage triglyceride accumulation. These findings reveal novel mechanisms by which supplementation with SO or LA stimulate macrophage foam cell formation, suggesting a pro-atherogenic role for overconsumption of SO or LA. © 2016 BioFactors, 43(1):100-116, 2017.

Pomegranate Juice Polyphenols Induce Macrophage Death via Apoptosis as Opposed to Necrosis Induced by Free Radical Generation: A Central Role for Oxidative Stress.

At high concentrations, polyphenols induce cell death, and the polyphenols-rich pomegranate juice (PJ), known for its antioxidative/antiatherogenic properties, can possibly affect cell death, including macrophage death involved in atherogenesis. In the present study, apoptotic/necrotic macrophage death was analyzed in J774A.1 macrophages and in peritoneal macrophages isolated from atherosclerotic apoE-/- mice treated with PJ. The effects of PJ were compared with those of the free radical generator 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH). Both PJ and AAPH significantly increased J774A.1 macrophage death; however, flow cytometric and microscopic analyses using annexin V/propidium iodide revealed that PJ increased the early apoptosis of the macrophage dose dependently (up to 2.5-fold, P < 0.01), whereas AAPH caused dose-dependent increases in late apoptosis/necrosis (up to 12-fold, P < 0.001). Unlike PJ, AAPH-induced macrophage death was associated with increased intracellular oxidative stress (up to 7-fold, P < 0.001) and with lipid stress demonstrated by triglyceride accumulation (up to 3-fold, P < 0.01) and greater chromatic vesicle response to culture medium (up to 5-fold, P < 0.001). Accordingly, recombinant paraoxonase 1, which hydrolyzes oxidized lipids, attenuated macrophage death induced by AAPH, but not by PJ. Similar apoptotic and oxidative effects were found in macrophages from apoE-/- mice treated with PJ or AAPH. As macrophage apoptotic/necrotic death has considerable impact on atherosclerosis progression, these findings may provide novel mechanisms for the antiatherogenicity of PJ.

Effects of Pomegranate Extract Supplementation on Cardiovascular Risk Factors and Physical Function in Hemodialysis Patients.

The purpose of this study was to evaluate the effects of oral supplementation with pomegranate extract on cardiovascular risk, physical function, oxidative stress, and inflammation in hemodialysis (HD) patients. Thirty-three HD subjects were randomized to the pomegranate (POM) or placebo (CON) group. Patients in POM ingested a 1000 mg capsule of a purified pomegranate polyphenol extract 7 days/week for 6 months. Individuals in CON ingested a noncaloric placebo capsule using the same protocol. Measurements were conducted at baseline and repeated 6 months following the start of the intervention. Brachial blood pressure (BP) was obtained using an automatic digital BP monitor. Cardiovascular risk was assessed using ultrasound and arterial tonometry. Blood samples were collected for the measurements of circulating markers of inflammation, oxidative stress, and antioxidant capacity. Muscle strength and physical function were assessed by isokinetic dynamometry, a validated shuttle walk test, and a battery of tests to assess functional fitness. Systolic blood pressure and diastolic blood pressure were reduced by 24 ± 13.7 and 10 ± 5.3 mmHg, respectively, in POM (P < .05). However, the BP differences in POM were no longer significant after controlling for baseline BP. The paraoxonase-1 activity increased by 26.6% (P < .05) in POM, compared to no significant change in CON. However, pomegranate supplementation had no effect on other markers of cardiovascular disease risk, inflammation and oxidative stress, or measures of physical function and muscle strength. While pomegranate extract supplementation may reduce BP and increase the antioxidant activity in HD patients, it does not improve other markers of cardiovascular risk, physical function, or muscle strength.

Anti-atherogenic properties of date vs. pomegranate polyphenols: the benefits of the combination.

Hydrolysable tannin polyphenols in pomegranate and phenolic acids in date fruit and seeds are potent antioxidants and anti-atherogenic agents, and thus, in the present study we investigated the possible benefits of combining them in vivo in atherosclerotic apolipoprotein E KO (E(0)) mice, compared with the individual fruit. In vitro studies revealed that the date seed extract contains more polyphenols than Amari or Hallawi date extracts, and possesses a most impressive free radical scavenging capacity. Similarly, pomegranate juice (PJ), punicalagin, punicalain, gallic acid, and urolithins A and B are very potent antioxidants. E(0) mice consumed 0.5 µmol gallic acid equivalents (GAE) per mouse per day of PJ, Hallawi extract, date seed extract, or a combination for 3 weeks. Consumption of the combination was the most potent treatment, as it decreased serum cholesterol and triglyceride levels, and increased serum paraoxonase 1 (PON1) activity. Consumption of the combination also significantly reduced mouse peritoneal macrophage (MPM) oxidative stress, MPM cholesterol content, and MPM LDL uptake. Finally, the lipid peroxide content in the aortas of the mice significantly decreased, and the PON lactonase activity of the aortas increased after treatment with the combination. We thus conclude that consumption of pomegranate, together with date fruit and date seeds, has the most beneficial anti-atherogenic effects on E(0) mice serum, macrophages, and aortas, probably due to their unique and varied structures.

Antioxidant and antiatherogenic properties of phenolic acid and flavonol fractions of fruits of 'Amari' and 'Hallawi' date (Phoenix dactylifera L.) varieties.

Date (Phoenix dactylifera L.) fruit phenolic-acid or flavonol fractions were examined in vitro for antioxidant and antiatherogenic properties. Two fractions of each subgroup were prepared from two date varieties, 'Amari' and 'Hallawi', by solid phase extraction on C18. The fractions were analyzed for phenolics composition by RP-HPLC and tested for ferric-reducing antioxidant power, free radical scavenging capacity, inhibition of Cu(2+)-induced LDL oxidation, and enhancement of HDL-mediated cholesterol efflux from macrophages. All four fractions exhibited variable capacities to reduce ferric ions, scavenge radicals, and inhibit LDL oxidation. Flavonol fractions were considerably better inhibitors of LDL oxidation compared to phenolic acid fractions, with IC50's of 9-31 nmol GAE mL(-1) compared to 85-116 nmol GAE mL(-1), respectively. Only the flavonol fractions stimulated cholesterol removal from macrophages. Within each subgroup, the levels of all the activities varied with fraction composition. The results demonstrated strong structure-activity relationships for date phenolics and identified date flavonols as potential antiatherogenic bioactives.

Colorimetric polymer assay for the diagnosis of plasma lipids atherogenic quality in hypercholesterolemic patients.

OBJECTIVE: Hypercholesterolemia (increased blood cholesterol level) is considered a major risk factor for developing atherosclerotic diseases. As such, alerting individuals on hypercholesterolemic conditions is a crucial component in averting onset of atherosclerosis and its outcome-cardiovascular diseases. While common diagnostic tools such as cholesterol and lipoproteins determination are widely employed for hypercholesterolemia screening, their effectiveness has been questioned since they do not shed light on critical physiological factors like lipid oxidation and inflammation levels, which constitute prominent determinants for development of atherosclerotic diseases. The objective of this study is to develop a simple assay for identifying hypercholesterolemia, and assessing the impact of therapeutic treatments. METHODS: We developed a diagnostic assay based upon color transformations of polydiacetylene, a unique conjugated polymer, upon interactions with blood plasma obtained from healthy individuals, hypercholesterolemic patients, hypercholesterolemic patients treated with statin, and hypercholesterolemic patients treated with statin together with pomegranate extracts. The color transformations of the polymer were monitored through desktop color scanning combined with colorimetric image analysis. RESULTS: We show that the colorimetric assay was able to distinguish among plasma. Bio-analytical characterization reveals that the distinct colorimetric responses likely arise from interactions with plasma lipoproteins. Importantly, the colorimetric changes are not simply correlated with the relative abundance of cholesterol (or other lipids) in the plasma of hypercholesterolemic or healthy patients, but also reflect the presence of oxidized and inflamed species. CONCLUSIONS: This paper introduces a simple color assay for detection of hypercholesterolemia and monitoring the effect of therapies directed at mitigating this physiological condition. The colorimetric system might constitute a novel platform for assessing patient vulnerability towards the development of atherosclerosis.

Pomegranate juice polyphenols induce a phenotypic switch in macrophage polarization favoring a M2 anti-inflammatory state.

It was documented that pomegranate has anti-inflammatory effects. In this study, we investigated a direct effect of pomegranate juice (PJ) and its polyphenols on macrophage inflammatory phenotype. In vitro, PJ and its major polyphenols dose-dependently attenuated macrophage response to M1 proinflammatory activation in J774.A1 macrophage-like cell line. This was evidenced by a significant decrease in TNFα and IL-6 secretion in response to stimulation by IFNγ and Lipopolysaccharide. In addition, PJ and punicalagin dose-dependently promoted the macrophages toward a M2 anti-inflammatory phenotype, as determined by a significant increase in the spontaneous secretion of IL-10. In mice, supplementation with dietary PJ substantially inhibited the M2 to M1 macrophage phenotypic shift associated with age, toward a favorable anti-inflammatory M2 phenotype. This effect was also reflected in the mice atherosclerotic plaques, as evaluated by the distinct expression of arginase isoforms. PJ consumption inhibited the increment of arginase II (Arg II, M1) mRNA expression during aging, and maintained the levels of Arg I (M2) expression similar to those in young mice aorta. This study demonstrates, for the first time, that pomegranate polyphenols directly suppress macrophage inflammatory responses and promote M1 to M2 switch in macrophage phenotype. Furthermore, this study indicates that PJ consumption may inhibit the progressive proinflammatory state in the aorta along atherosclerosis development with aging, due to a switch in macrophage phenotype from proinflammatory M1 to anti-inflammatory M2.

Oxidative stress and nephrolithiasis: a comparative pilot study evaluating the effect of pomegranate extract on stone risk factors and elevated oxidative stress levels of recurrent stone formers and controls.

Previous studies have linked oxidative stress and nephrolithiasis. Animal studies have demonstrated that pomegranate juice may play a role in preventing stone formation. We examined differences between recurrent stone formers (RSFs) and non-stone formers (NSFs) regarding oxidative stress and the effect of pomegranate administration on risk factors for nephrolithiasis. RSFs were recruited prospectively and matched to a group of NSFs. Subjects submitted urine and blood samples prior to and after receiving pomegranate polyphenol extract (1,000 mg) for 90 days. Serum and urine samples were analyzed for stone risk and oxidative stress. Thirty subjects completed the study. RSFs had significantly higher levels of oxidative stress at baseline as measured by urinary 8-hydroxy-deoxyguanosine (p < 0.0001), 2.2'-azobis (2-amidinopropane) hydrochloride-induced serum lipid peroxidation [increased levels of lipid peroxides (p = 0.0002), and thiobarbituric acid reactive substances (p = 0.002)], but not by serum paraoxonase1 (PON1) arylesterase activity (p > 0.99), or by highly sensitive C-reactive protein (p > 0.99). Following pomegranate supplementation, there was a 10 % increase in PON1 activity in RSFs (p = 0.007), which correlated with a trend toward decreasing values of supersaturation of calcium oxalate (SSCaOx; p = 0.05). RSFs have markedly higher levels of oxidative stress than NSFs. While the ability to prevent stone formation through supplementation cannot be determined in this pilot study, supplementation with pomegranate extract does not increase the risk of stones and may confer some benefit in lowering SSCaOX in those patients with increased PON-1 levels following supplementation, confirming findings of previous animal models.

Pomegranate extract (POMx) decreases the atherogenicity of serum and of human monocyte-derived macrophages (HMDM) in simvastatin-treated hypercholesterolemic patients: a double-blinded, placebo-controlled, randomized, prospective pilot study.

OBJECTIVE: To analyze pomegranate extract (POMx) effects on serum and on human HMDM atherogenicity in simvastatin - treated hypercholesterolemic patients. METHODS AND RESULTS: Patients were randomly assigned to receive either simvastatin (20 mg/day) + vegan placebo pill (n = 11), or simvastatin (20 mg/day) + POMx pill (1g/day, n = 12). Fasting blood samples were collected at baseline and after 1 and 2 months of therapy. HMDM were collected from 3 patients in each group at baseline and after 2 months of therapy, as well as from 3 healthy subjects. After 2 months of therapy, serum LDL-cholesterol levels significantly decreased, by 23%, in the simvastatin + placebo group, and by 26% in the simvastatin + POMx group. Simvastatin + POMx therapy increased serum thiols concentration by 6%. Patients' HMDM reactive oxygen species (ROS) levels were significantly increased, by 69%, vs. healthy subjects HMDM. After 2 months of therapy, HMDM ROS levels decreased by 18% in the simvastatin + placebo group, whereas in the simvastatin + POMx group it decreased by up to 30%. A novel finding was the triglycerides levels in the patients' HMDM at baseline which were significantly higher, by 71%, vs. healthy subjects HMDM. The simvastatin + POMx, but not the simvastatin + placebo therapy, significantly reduced macrophage triglycerides content by 48%, vs. baseline levels. In addition, whereas the simvastatin + placebo therapy significantly decreased the patients' HMDM cholesterol biosynthesis rate by 33%, the simvastatin + POMx therapy further decreased it, by 44%. CONCLUSION: The addition of POMx to simvastatin therapy in hypercholesterolemic patients improved oxidative stress and lipid status in the patient's serum and in their HMDM. These anti-atherogenic effects could reduce the risk for atherosclerosis development.

Pomegranate Protection against Cardiovascular Diseases.

The current paper summarizes the antioxidative and antiatherogenic effects of pomegranate polyphenols on serum lipoproteins and on arterial macrophages (two major components of the atherosclerotic lesion), using both in vitro and in vivo humans and mice models. Pomegranate juice and its by-products substantially reduced macrophage cholesterol and oxidized lipids accumulation, and foam cell formation (the hallmark of early atherogenesis), leading to attenuation of atherosclerosis development, and its consequent cardiovascular events.

The Middle Eastern and biblical origins of the Mediterranean diet.

OBJECTIVE: To place the Mediterranean diet (MedDi) in the context of the cultural history of the Middle East and emphasise the health effects of some of the biblical seven species - wheat, barley, grapes, figs, pomegranates, olives and date honey. DESIGN: Review of the literature concerning the benefits of these foods. SETTING: Middle East and Mediterranean Basin. SUBJECTS: Mediterranean populations and clinical studies utilising the MedDi. RESULTS AND CONCLUSIONS: The MedDi has been associated with lower rates of CVD, and epidemiological evidence promotes the benefits of consuming fruit and vegetables. Recommended foods for optimal health include whole grain, fish, wine, pomegranates, figs, walnuts and extra virgin olive oil. The biblical traditional diet, including the seven species and additional Mediterranean fruits, has great health advantages, especially for CVD. In addition to the diet, lifestyle adaptation that involves increasing physical activity and organised meals, together with healthy food choices, is consistent with the traditional MedDi. The MedDi is a manageable, lifestyle-friendly diet that, when fortified with its biblical antecedent attributes, may prove to be even more enjoyable and considerably healthier in combating the obesogenic environment and in decreasing the risks of the non-communicable diseases of modern life than conventional, modern dietary recommendations. The biblical seven species, together with other indigenous foods from the Middle East, are now scientifically recognised as healthy foods, and further improve the many beneficial effects of the MedDi.

Consumption of pomegranate decreases serum oxidative stress and reduces disease activity in patients with active rheumatoid arthritis: a pilot study.

BACKGROUND: Pomegranate extract (POMx) consumption has been shown to reduce the incidence and severity of collagen-induced arthritis in mice. OBJECTIVES: To investigate whether pomegranate consumption affects disease activity in patients with rheumatoid arthritis (RA), in relation to their serum oxidative status. METHODS: In this pilot 12 week open-labeled study eight patients with active RA consumed POMx (10 ml/day) for 12 weeks. Patients' joint status and serum oxidative status (lipid peroxidation, total thiols group, paraoxonase 1 activity) were evaluated at baseline and at week 12. RESULTS: Six patients completed the study. POMx consumption significantly (P < 0.02) reduced the composite Disease Activity Index (DAS28) by 17%, which could be related mostly to a significant (P < 0.005) reduction in the tender joint count (by 62%). These results were associated with a significant (P < 0.02) reduction in serum oxidative status and a moderate but significant (P < 0.02) increase in serum high density lipoprotein-associated paraoxonase 1 (PON1) activity. The addition of POMx to serum from RA patients reduced free radical-induced lipid peroxidation by up to 25%. CONCLUSIONS: The pomegranate consumption reduced DAS28 in RA patients, and this effect could be related to the antioxidative property of pomegranates. Dietary supplementation with pomegranates may be a useful complementary strategy to attenuate clinical symptoms in RA patients.

Paraoxonase 1 interactions with HDL, antioxidants and macrophages regulate atherogenesis - a protective role for HDL phospholipids.

Macrophage cholesterol accumulation and foam cell formation is the hallmark of early atherogenesis. In addition to macrophages, at least three more major players regulate atherosclerosis development; paraoxonase 1 (PON1), antioxidants, and HDL. PON1 is an HDL-associated lactonase which posses antioxidant and anti-atherogenic properties. PON1 protects against macrophage-mediated LDL oxidation, and increases HDL binding to macrophages which, in turn, stimulates HDL's ability to promote cholesterol efflux. These two major anti-atherogenic properties of HDL (and of PON1) require, at least in part, macrophage binding sites for HDL-associated PON1. Indeed, PON1, as well as HDL-associated PON1, specifically binds to macrophages, leading to anti-atherogenic effects. Macrophage PON1 binding sites may thus be a target for future cardioprotection therapy. Studying the interactions among PON1, antioxidants, and macrophages can thus assist in achieving appropriate treatment and prevention of atherosclerosis.

Increased macrophage cholesterol biosynthesis and decreased cellular paraoxonase 2 (PON2) expression in Delta6-desaturase knockout (6-DS KO) mice: beneficial effects of arachidonic acid.

OBJECTIVE: To analyze the possible role of arachidonic acid (AA) in macrophage cholesterol biosynthesis and in PON2 expression. METHODS AND RESULTS: We used peritoneal macrophages (MPM) from the 6-DS KO mice that were fed a diet without or with AA. Macrophage cholesterol biosynthesis rate and HMGCoA-reductase mRNA levels were substantially increased, by 98% and 67%, respectively, in MPM from 6-DS KO vs. control (C57BL/6) mice. Furthermore, in the 6-DS KO vs. control mice MPM PON2 expression (mRNA and lactonase activity) was substantially decreased. In line with the above results, AA supplementation to 6-DS KO mice significantly decreased MPM cholesterol biosynthesis rate and HMGCoA-reductase mRNA levels, by 45% and by 4-fold respectively, and increased MPM PON2 lactonase activity and PON2 mRNA, by 119% and 2.3-fold, respectively. Similarly, incubation of control mice MPM or J774A.1 with AA, significantly and dose-dependently decreased cellular cholesterol biosynthesis rate, and increased PON2 expression. These effects were specific for AA since incubation of the cells with docosahexaenoic acid (DHA, another product of 6-DS) had no significant effects on cholesterol biosynthesis rate, and on PON2 activity. CONCLUSIONS: AA decreased macrophage cholesterol biosynthesis rate, and increased PON2 expression. These effects could protect the cells from cholesterol accumulation and oxidation, and from foam cell formation, the hallmark of early atherogenesis.

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.

Effects of consumption of pomegranate juice on carotid intima-media thickness in men and women at moderate risk for coronary heart disease.

This randomized, double-blind, parallel trial assessed the influence of pomegranate juice consumption on anterior and posterior carotid intima-media thickness (CIMT) progression rates in subjects at moderate risk for coronary heart disease. Subjects were men (45 to 74 years old) and women (55 to 74 years old) with > or =1 major coronary heart disease risk factor and baseline posterior wall CIMT 0.7 to 2.0 mm, without significant stenosis. Participants consumed 240 ml/day of pomegranate juice (n = 146) or a control beverage (n = 143) for up to 18 months. No significant difference in overall CIMT progression rate was observed between pomegranate juice and control treatments. In exploratory analyses, in subjects in the most adverse tertiles for baseline serum lipid peroxides, triglycerides (TGs), high-density lipoprotein (HDL) cholesterol, TGs/HDL cholesterol, total cholesterol/HDL cholesterol, and apolipoprotein-B100, those in the pomegranate juice group had significantly less anterior wall and/or composite CIMT progression versus control subjects. In conclusion, these results suggest that in subjects at moderate coronary heart disease risk, pomegranate juice consumption had no significant effect on overall CIMT progression rate but may have slowed CIMT progression in subjects with increased oxidative stress and disturbances in the TG-rich lipoprotein/HDL axis.