Nitro-Oleic Acid Reduces J774A.1 Macrophage Oxidative Status and Triglyceride Mass: Involvement of Paraoxonase2 and Triglyceride Metabolizing Enzymes.

Nitro-fatty acids possess anti-atherogenic properties, but their effects on macrophage oxidative status and lipid metabolism that play important roles in atherosclerosis development are unclear. This study compared the effects of nitro-oleic acid (OLA-NO2) with those of native oleic acid (OLA) on intracellular reactive oxygen species (ROS) generation, anti-oxidants and metabolism of triglycerides and cholesterol in J774A.1 macrophages. Upon incubating the cells with physiological concentrations of OLA-NO2 (0-1 µM) or with equivalent levels of OLA, ROS levels measured by 2, 7-dichlorofluorescein diacetate, decreased dose-dependently, but the anti-oxidative effects of OLA-NO2 were significantly augmented. Copper ion addition increased ROS generation in OLA treated macrophages without affecting OLA-NO2 treated cells. These effects could be attributed to elevated glutathione levels and to increased activity and expression of paraoxonase2 that were observed in OLA-NO2 vs OLA treated cells. Beneficial effects on triglyceride metabolism were noted in OLA-NO2 vs OLA treated macrophages in which cellular triglycerides were reduced due to attenuated biosynthesis and accelerated hydrolysis of triglycerides. Accordingly, OLA-NO2 treated cells demonstrated down-regulation of diacylglycerol acyltransferase1, the key enzyme in triglyceride biosynthesis, and increased expression of hormone-sensitive lipase and adipose triglyceride lipase that regulate triglyceride hydrolysis. Finally, OLA-NO2 vs OLA treatment resulted in modest but significant beneficial effects on macrophage cholesterol metabolism, reducing cholesterol biosynthesis rate and low density lipoprotein influx into the cells, while increasing high density lipoprotein-mediated cholesterol efflux from the macrophages. Collectively, compared with OLA, OLA-NO2 modestly but significantly reduces macrophage oxidative status and cellular triglyceride content via modulation of cellular anti-oxidants and triglyceride metabolizing enzymes.

Silicon dioxide nanoparticles increase macrophage atherogenicity: Stimulation of cellular cytotoxicity, oxidative stress, and triglycerides accumulation.

Nanoparticle research has focused on their toxicity in general, while increasing evidence points to additional specific adverse effects on atherosclerosis development. Arterial macrophage cholesterol and triglyceride (TG) accumulation and foam cell formation are the hallmark of early atherogenesis, leading to cardiovascular events. To investigate the in vitro atherogenic effects of silicon dioxide (SiO2 ), J774.1 cultured macrophages (murine cell line) were incubated with SiO2 nanoparticle (SP, d = 12 nm, 0-20 µg/mL), followed by cellular cytotoxicity, oxidative stress, TG and cholesterol metabolism analyses. A significant dose-dependent increase in oxidative stress (up to 164%), in cytotoxicity (up to 390% measured by lactate dehydrogenase (LDH) release), and in TG content (up to 63%) was observed in SiO2 exposed macrophages compared with control cells. A smaller increase in macrophage cholesterol mass (up to 22%) was noted. TG accumulation in macrophages was not due to a decrease in TG cell secretion or to an increased TG biosynthesis rate, but was the result of attenuated TG hydrolysis secondary to decreased lipase activity and both adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) protein expression (by 42 and 25%, respectively). Overall, SPs showed pro-atherogenic effects on macrophages as observed by cytotoxicity, increased oxidative stress and TG accumulation. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 713-723, 2016.

In vitro effects of exogenous carbon monoxide on oxidative stress and lipid metabolism in macrophages.

Carbon monoxide (CO) is a major constituent of traffic-related air pollution and is also produced endogenously under conditions of oxygen-mediated stress. It has been shown to affect both oxidative stress and inflammation. However, its role in lipid metabolism has been neglected. Using short exposure times, the effect of CO on J774A.1 macrophage atherogenic functions was investigated up to 16 h after exposure. Exposure of macrophages was found to be pro-atherogenic as it significantly increased triglyceride mass, up to 60%, and decreased high-density lipoprotein-mediated cholesterol efflux, up to 27%. In contrast, paraoxonase 2 lactonase activity was increased, up to 65%, and cellular oxidative stress was attenuated by 29%, compared with the control cells. The above results on lipid metabolism may lead to arterial macrophage foam cell formation, the hallmark of early atherogenesis.

Selective oxidative stress and cholesterol metabolism in lipid-metabolizing cell classes: Distinct regulatory roles for pro-oxidants and antioxidants.

Atherogenesis is associated with macrophage cholesterol and oxidized lipids accumulation and foam cell formation. However, two other major lipid-metabolizing cell classes, namely intestinal and liver cells, are also associated with atherogenesis. This study demonstrates that manipulations of cellular oxidative stress (by fatty acids, glucose, low-density lipoprotein, angiotensin II, polyphenolic antioxidants, or the glutathione/paraoxonase 1 systems) have some similar, but also some different effects on cholesterol metabolism in macrophages (J774A.1) versus intestinal cells (HT-29) versus liver cells (HuH7). Cellular oxidative stress was ≈3.5-folds higher in both intestinal and liver cells versus macrophages. In intestinal cells or liver cells versus macrophages, the cholesterol biosynthesis rate was increased by 9- or 15-fold, respectively. In both macrophages and intestinal cells C-18:1 and C-18:2 but not C-18:0, fatty acids significantly increased oxidative stress, whereas in liver cells oxidative stress was significantly decreased by all three fatty acids. In liver cells, trans C-18:1 versus cis C-18:1, unlike intestinal cells or macrophages, significantly increased cellular oxidative stress and cellular cholesterol biosynthesis rate. Pomegranate juice (PJ), red wine, or their phenolics gallic acids or quercetin significantly reduced cellular oxidation mostly in macrophages. Recombinant PON1 significantly decreased macrophage (but not the other cells) oxidative stress by ≈30%. We conclude that cellular atherogenesis research should look at atherogenicity, not only in macrophages but also in intestinal and liver cells, to advance our understanding of the complicated mechanisms behind atherogenesis. © 2015 BioFactors, 41(4):273-288, 2015.

High intrinsic aerobic capacity and pomegranate juice are protective against macrophage atherogenecity: studies in high- vs. low-capacity runner (HCR vs. LCR) rats.

We studied the rat model system of high- vs. low-capacity runner (HCR vs. LCR) rats to question the atherogenic properties (oxidative stress, triglycerides and cholesterol metabolism) in the rat macrophages, serum, liver and heart. Half of the LCR or HCR rats consumed pomegranate juice (PJ; 15 µmol of gallic acid equivalents/rat/day) for 3 weeks and were compared to placebo-treated rats. At the end of the study blood samples, peritoneal macrophages (RPM), livers, and hearts were harvested from the rats. RPM harvested from HCR vs. LCR demonstrated reduced cellular oxidation (21%), increased paraoxonase 2 activity (28%) and decreased triglycerides mass (44%). Macrophage uptake rates of fluorescein-isothiocyanate-labeled low-density lipoprotein (LDL) or oxidized LDL were significantly lower, by 37% or by 18%, respectively, in HCR vs. LCR RPM. PJ consumption significantly decreased all the above atherogenic parameters with more substantial beneficial effects observed in the LCR vs. the HCR rats (~80% vs. ~40% improvement, respectively). Similar hypo-triglyceridemic pattern was noted in serum from HCR vs. LCR. In contrast to the above results, liver oxidation and triglycerides mass were both minimally increased in HCR vs. LCR rats by 31% and 28%, respectively. In the heart, lipid content was very low, and interestingly, an absence of any significant oxidative stress, along with modest triglyceride accumulation, was observed. We conclude that HCR vs. LCR rats demonstrate reduced atherogenicity, mostly in their macrophages. PJ exerts a further improvement, mostly in macrophages from LCR rats.

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.

Human carotid atherosclerotic lesion protein components decrease cholesterol biosynthesis rate in macrophages through 3-hydroxy-3-methylglutaryl-CoA reductase regulation.

Atherosclerosis is characterized by the formation of cholesterol-loaded macrophages, which are turned into foam cells, the hallmark of early atherogenesis. As part of ongoing research on the interactions among human carotid lesion components and blood elements, the effect of plaque homogenate on macrophage cholesterol biosynthesis rate was examined. Human carotid plaques were ground, extracted with phosphate-buffered saline (homogenate), and then added to the macrophage medium. This extract decreased macrophage cholesterol biosynthesis rate up to 50% in a dose-dependent manner. Cholesterol or lipoproteins were separated from the homogenate and added to the MQ medium. Unlike the homogenate, neither free cholesterol nor the lipoproteins were able to inhibit cholesterol biosynthesis rate under the above experimental concentration, suggesting that the homogenate-induced cholesterol biosynthesis inhibition in our experimental system was not owing to the feedback inhibition of cholesterol. Furthermore, the homogenate remaining after lipoprotein removal (lipoprotein-deficient homogenate) also decreased cholesterol biosynthesis rate, whereas boiled homogenate or phospholipids extracted from the homogenate decreased macrophage cholesterol biosynthesis rate only partially. Finally, cholesterol biosynthesis inhibition was achieved only upon using the precursor [(3)H]acetate, but not [(14)C]mevalonate, suggesting that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA Reductase), the rate-limiting enzyme in the cholesterol biosynthesis pathway, is involved in the above antiatherogenic effect of the homogenate, whereas the treatment with homogenate decreased HMGCoA Reductase mRNA. Proteins and phospholipids from human carotid lesion homogenate decrease cholesterol biosynthesis rate in macrophages secondary to HMGCoA Reductase feedback regulation. Such an effect may delay foam cell formation and atherosclerosis progression.

HDL3 stimulates paraoxonase 1 antiatherogenic catalytic and biological activities in a macrophage model system: in vivo and in vitro studies.

We analyzed in-vivo and in-vitro high density lipoprotein (HDL) effects on paraoxonase 1 (PON1) antiatherogenic properties in serum and in macrophages. Intraperitoneal injection to C57BL/6 mice of recombinant PON1 (rePON1) + HDL, in comparison to HDL or to rePON1 alone, significantly increased serum PON1 arylesterase activity (by 20%), and serum-mediated cholesterol efflux from J774A.1 macrophages (by 18%). Similarly, in peritoneal macrophages (MPM) harvested from mice injected with HDL + rePON1 versus rePON1 alone, we observed reduction in oxidative stress (by 11%), increase in cellular PON1 activity (by 14%) and in HDL-mediated cholesterol efflux (by 38%). Incubation of serum or HDL with rePON1, substantially increased PON1 arylesterase activity, two-fold more than the expected additive values. HDL2 and HDL3 increased PON1 activity by 199% or 274%, respectively. Macrophage (J774A.1) cholesterol efflux rate significantly increased by HDL3 + rePON1 versus HDL3 alone (by 19%), but not by HDL2 + rePON1 versus HDL2 alone. Oxidation of HDL3 reduced its ability to induce macrophage cholesterol efflux, and abolished HDL3 stimulatory effects on rePON1. Addition of exogenous polyphenol quercetin (60 µM), but not phosphatidylcholine or apolipoprotein A1, to HDL + rePON1 increased PON1 activity (by 404%), increased the ability to reduce oxidative stress in J774A.1 macrophages (by 53%) and to stimulate macrophage cholesterol efflux (by 14%). Upon adding the hypocholesterolemic drug simvastatin (15 µg/mL) to HDL + rePON1, PON1 activity and the ability to induce macrophage cholesterol efflux increased, in comparison to HDL + rePON1. We thus concluded that HDL (mostly HDL3), stimulates PON1 antiatherogenic activities in macrophages, and these PON1 activities were further stimulated by quercetin, or by simvastatin.

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.

[Addition of pomegranate juice to statin inhibits cholesterol accumulation in macrophages: protective role for the phytosterol beta-sitosterol and for the polyphenolic antioxidant punicalagin].

Macrophage cholesterol and oxidized lipids accumulation and foam cell formation occur in the early stages of atherosclerosis development. In the current study we used the J774A.1 murine macrophage cell line in order to analyze two atherogenic functions: a. the ability of the cells to produce reactive oxygen species (ROS), and to increase cellular oxidative stress, and b. the ability of the cells to synthesize cholesterol, leading to cholesterol accumulation in the cells. The addition of punicalagin, or beta-sitosterol, or pomegranate juice (which contains both of the above) to simvastatin, significantly improved the statin's ability to inhibit macrophage cholesterol biosynthesis. Furthermore, the addition of pomegranate juice (or punicalagin, but not beta sitosterol) to simvastatin significantly increased the statin ability to protect the cells from oxidative stress. Taken together, the current research provides evidence for the additional cardio protection of statins, that is provided by pomegranate juice antioxidant and hypocholesterolemic effects. The use of statins in combination with pomegranate juice in hypercholesterolemic patients, may allow for the use of lower dosages of statin in order to prevent statin deleterious side effects.

Pomegranate for your cardiovascular health.

Pomegranate is a source of some very potent antioxidants (tannins, anthocyanins) which are considered to be also potent anti-atherogenic agents. The combination of the above unique various types of pomegranate polyphenols provides a much wider spectrum of action against several types of free radicals. Indeed, pomegranate is superior in comparison to other antioxidants in protecting low-density lipoprotein (LDL, "the bad cholesterol") and high-density lipoprotein (HDL, "the good cholesterol") from oxidation, and as a result it attenuates atherosclerosis development and its consequent cardiovascular events. Pomegranate antioxidants are not free, but are attached to the pomegranate sugars, and hence were shown to be beneficial even in diabetic patients. Furthermore, pomegranate antioxidants are unique in their ability to increase the activity of the HDL-associated paraoxonase 1 (PON1), which breaks down harmful oxidized lipids in lipoproteins, in macrophages, and in atherosclerotic plaques. Finally, unique pomegranate antioxidants beneficially decrease blood pressure. All the above beneficial characteristics make the pomegranate a uniquely healthy fruit.

Date (Phoenix dactylifera L.) fruit soluble phenolics composition and anti-atherogenic properties in nine Israeli varieties.

Date (Phoenix dactylifera L.) fruit soluble phenolics composition and anti-atherogenic properties were examined in nine diverse Israeli grown varieties. Ethanol and acetone extracts of 'Amari', 'Barhi', 'Deglet Noor', 'Deri', 'Hadrawi', 'Hallawi', 'Hayani', 'Medjool', and 'Zahidi' fruit were analyzed for phenolics composition by RP-HPLC and tested for anti-atherogenicity by measuring their effects on LDL susceptibility to copper ion- and free radical-induced oxidation, and on serum-mediated cholesterol efflux from macrophages. The most frequently detected phenolics were hydroxybenzoates, hydroxycinnamates, and flavonols. Significant differences in phenolics composition were established between varieties as well as extraction solvents. All extracts inhibited LDL oxidation, and most extracts also stimulated cholesterol removal from macrophages. Considerable varietal differences were measured in the levels of the bioactivities. Also, acetone extracts exhibited a significantly higher anti-atherogenic potency for most varieties. The presence of soluble ingredients with anti-atherogenic capacities in dates and the possible involvement of phenolics are discussed.

Pomegranate phytosterol (ß-sitosterol) and polyphenolic antioxidant (punicalagin) addition to statin, significantly protected against macrophage foam cells formation.

OBJECTIVE: To assess the anti-atherogenic effects on macrophage cholesterol biosynthesis rate, and on cellular oxidative stress by the combination of simvastatin with a potent polyphenolic antioxidant (punicalagin), or with a phytosterol (ß-sitosterol), or with pomegranate juice (POM, that contains both of them). METHODS AND RESULTS: Simvastatin (15 µg/ml) decreased J774A.1 macrophage cholesterol biosynthesis rate by 42% as compared to control cells. The addition to the statin of either punicalagin (15 or 30 µM), or ß-sitosterol (50 or 100 µM), increased the inhibitory effect of the statin up to 62% or 57%, respectively. Similarly, the combination of POM and simvastatin, resulted in an inhibitory effect up to 59%. While simvastatin inhibited the rate limiting enzyme HMGCoA-reductase, punicalagin, ß-sitosterol or POM inhibited macrophage cholesterol biosynthesis downstream to mevalonate. Simvastatin (15 µg/ml) also modestly decreased macrophage reactive oxygen species (ROS) formation by 11%. In the presence of punicalagin (15 or 30 µM) however, a remarkable further inhibition was noted (by 61% or 79%, respectively). Although ß-sitosterol alone showed some pro-oxidant activity, the combination of simvastatin, ß-sitosterol and punicalagin, clearly demonstrated a remarkable 73% reduction in ROS production. Similarly, simvastatin + POM decreased the extent of ROS formation by up to 63%. These improved antioxidant effects of the combinations could be related to various anti-oxidative properties of the different compounds, including free radicals scavenging capacity, upregulation of paraoxonase 2, and stimulation of reduced glutathione. CONCLUSION: The combination of simvastatin with potent antioxidant and phytosterol (such as present in pomegranate) could lead to attenuation of macrophage foam cell formation and atherogenesis.

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.

Triglyceride accumulation in macrophages upregulates paraoxonase 2 (PON2) expression via ROS-mediated JNK/c-Jun signaling pathway activation.

The aim of this study was to analyze the effect and mechanism of action of macrophage triglyceride accumulation on cellular PON2 expression. Incubation of J774A.1 (murine macrophages) with VLDL (0-75 µg protein/mL) significantly and dose-dependently increased cellular triglyceride mass, and reactive oxygen species (ROS) formation, by up to 3.3- or 1.8-fold, respectively. PON2 expression (mRNA, protein, activity) in cells treated with VLDL (50 µg protein/mL) was higher by 2- to 3-fold, as compared with control cells. Similar effects were noted upon using THP-1 (human macrophages). Incubation of macrophages with synthetic triglyceride or triglyceride fraction from carotid lesion resulted in similar effects, as shown for VLDL. Upon using specific inhibitors of MEK1/2 (UO126, 10 µM), p38 (SB203580, 10 µM), or JNK (SP600125, 20 µM), we demonstrated that MEK, as well as JNK, but not p38, are involved in VLDL-induced macrophage PON2 upregulation. VLDL activated JNK (but not ERK), which resulted in c-Jun phosphorylation. This signaling pathway is probably activated by ROS, since the antioxidant reduced glutathione (GSH), significantly decreased VLDL-induced macrophage ROS formation, c-Jun phosphorylation and PON2 overexpression. We conclude that macrophage triglyceride accumulation upregulates PON2 expression via MEK/ JNK/c-Jun pathway, and these effects could be related, at least in part, to cellular triglycerides-induced ROS formation. ©

VLDL triglycerides inhibit HDL-associated paraoxonase 1 (PON1) activity: in vitro and in vivo studies.

We analyzed, for the first time, both in vitro and in vivo, the effect of very low density lipoprotein (VLDL), or of pure triglycerides, on high-density lipoprotein (HDL)-associated paraoxonase1 (PON1) catalytic activities. Incubation of serum or HDL from healthy subjects with VLDL (0-330 µg protein/mL) significantly decreased serum PON1 lactonase or arylesterase activities by up to 11% or 24%, and HDL-associated PON1 lactonase or arylesterase activities by up to 32% or 46%, respectively, in a VLDL dose-dependent manner. VLDL (0-660 µg protein/mL) also inhibited recombinant PON1 (rePON1) lactonase or arylesterase activities by up to 20% or 42%, respectively. Similar inhibitory effect was noted upon rePON1 incubation with pure triglyceride emulsion. Bezafibrate therapy to three hypertriglyceridemic patients (400 mg/day, for one month) significantly decreased serum triglyceride concentration by 67%, and increased serum HDL cholesterol levels by 48%. PON1 arylesterase or paraoxonase activities in the patients' HDL fractions after drug therapy were significantly increased by 86-88%, as compared to PON1 activities before treatment. Similarly, HDL-PON1 protein levels significantly increased after bezafibrate therapy. Finally, bezafibrate therapy improved HDL biological activity, as HDL obtained after drug therapy showed increased ability to induce cholesterol efflux from J774A.1 macrophages, by 19%, as compared to HDL derived before therapy. We thus conclude that VLDL triglycerides inhibit PON1 catalytic activities, and bezafibrate therapy significantly improved HDL-PON1 catalytic and biological activities.

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.

Pomegranate juice protects macrophages from triglyceride accumulation: inhibitory effect on DGAT1 activity and on triglyceride biosynthesis.

BACKGROUND/AIMS: To analyze the effects of pomegranate juice (PJ) and punicalagin on macrophage triglyceride metabolism. METHODS: Triglyceride metabolism was analyzed in PJ- or punicalagin-treated J774A.1 macrophages or in mouse peritoneal macrophages (MPM) harvested from C57BL/6 mice or from paraoxonase 2 (PON2)-deficient mice. RESULTS: PJ (0-50 µM) significantly and dose-dependently decreased the triglyceride content and triglyceride biosynthesis rate in J774A.1 macrophages or in C57BL/6 MPM by about 30%. Similarly, punicalagin, the major PJ polyphenol, inhibited the MPM triglyceride biosynthesis rate by 40%. The triglyceride hydrolytic rate, however, was not significantly affected by PJ or punicalagin. The activity of diacylglycerol acyltransferase 1 (DGAT1; the rate-limiting enzyme in triglyceride biosynthesis) was significantly inhibited, by 54%, in C57BL/6 MPM that were treated with 50 µM PJ or punicalagin, with no significant effect on DGAT1 mRNA or protein expression. Both PJ and punicalagin increased (1.7-fold) MPM PON2 mRNA expression, and PON2 was previously shown to inhibit DGAT1 activity. However, the addition of PJ or punicalagin (50 µM) to microsomes from PON2-deficient MPM still resulted in a significant reduction (50-58%) in DGAT1 activity. CONCLUSIONS: We conclude that the inhibitory effect of PJ on triglyceride biosynthesis could be attributed to a direct effect of PJ on DGAT1 activity.

Macrophage endoplasmic reticulum (ER) proteins and reducing elements stabilize paraoxonase 2 (PON2).

OBJECTIVE: To analyze the ability of macrophage sub-cellular fractions to stabilize paraoxonase 2 (PON2). METHODS: Nuclei, mitochondria, lysosomes, endoplasmic reticulum (ER) and cytosol were isolated from J774A.1 macrophage cell line and incubated with recombinant PON2. RESULTS: Among the fractions analyzed the ER contains the highest PON2 lactonase activity, and was the most potent one in stabilizing recombinant PON2 (rePON2). Whereas control rePON2 activity was decreased by 40% after 20 h of incubation at 37°C, in the presence of ER it decreased by only 15%. This effect could be attributed to the ER aqueous phase, and not to the ER lipids. The ER proteins fraction was responsible for PON2 stabilization, since heated ER or proteinase K-treated ER was not able to protect rePON2 from inactivation, while the protein fraction (after ammonium sulfate precipitation) completely prevented rePON2 inactivation. Since in the macrophage ER, there are increased levels of NADPH, secondary to glutathione reductase deficiency, we next studied the effect of the redox environment on PON2 inactivation. Incubation of rePON2 with DTT protected PON2 from inactivation. Similarly, NADPH, but not NADP, significantly increased rePON2 lactonase activity by up to 19%, after 20h of incubation as compared to control rePON2. Unlike ER from non-treated macrophages, ER harvested from oxidized-, or from cholesterol loaded-macrophages showed a significant lower basal PON2 lactonase activity, and did not protect PON2 from inactivation but rather increased it. CONCLUSION: Under normal conditions macrophage ER stabilizes PON2 activity, and this effect could be attributed to ER proteins and redox status.