Macrophage-mediated LDL Oxidation Research Articles

Injection of paraoxonase 1 (PON1) to mice stimulates their HDL and macrophage antiatherogenicity

We analyzed, for the first time, the effects of recombinant PON1 (rePON1) intraperitoneal injection to C₅₇BL/6 mice on their HDL and macrophage antiatherogenic properties. Thioglycolate-treated mice were injected with either saline (Control), or rePON1 (50 μg/mouse), and 20 H post injection, their blood samples and peritoneal macrophages (MPM) were collected. A significant increase in serum and HDL-PON1 arylesterase and lactonase activities was noted. Similarly, a significant increment, by 3.8 and 2.8 fold, in MPM-PON1 arylesterase and lactonase activities, respectively, as compared to the activities in control MPM was observed. The HDL from rePON1-injected mice was resistant to oxidation by copper ions as compared to control HDL. Furthermore, enrichment of the mouse HDL with rePON1 increased its ability to induce cholesterol efflux from J774A.1 macrophage cell line, and to inhibit macrophage-mediated LDL oxidation. In MPM from rePON1-injected mice vs. control MPM, there was a significant reduction in cholesterol mass, by 42%, in association with inhibition in cellular cholesterol biosynthesis rate, by 33%, and with significant stimulation, by 65%, of human HDL-mediated cholesterol efflux from the cells. We conclude that rePON1 injection to mice improved the mice HDL and MPM antiatherogenic properties, and these effects could probably lead to attenuation of atherosclerosis development.

Paraoxonase 2 (PON2) decreases high glucose-induced macrophage triglycerides (TG) accumulation, via inhibition of NADPH-oxidase and DGAT1 activity: Studies in PON2-deficient mice

ObjectiveThe present study investigates the role of paraoxonase 2 (PON2) in the attenuation of macrophage triglycerides (TG) biosynthesis, and oxidative stress, under diabetic conditions. MethodsPeritoneal macrophages (MPM) from PON2-deficient and from C57BL/6 control mice were harvested and cultured under normal (5mM) or high glucose concentration (30mM), and evaluated for cellular TG metabolism as well as for their oxidative stress. ResultsIn PON2-deficient MPM vs. control MPM, under diabetic conditions (high glucose concentration), we observed substantial increment in TG accumulation (3 fold), TG biosynthesis (2.6 fold) and microsomal diacylglycerol acyltransferase1 (DGAT1) activity (+60%). Furthermore, in these cells we have demonstrated increased oxidative stress, as expressed by significant increment in cellular oxidative stress (+25%), macrophage-mediated LDL oxidation (+41%) and expression of the receptor for advanced glycation end products – RAGE (+18%). Apocynin, an NADPH-oxidase inhibitor, abolished the increment in MPM TG accumulation, MPM TG biosynthesis, and microsomal DGAT1 activity, as a result of PON2-deficiency, under diabetic conditions. ConclusionWe conclude that PON2 has a significant protective role against macrophage triglyceride accumulation, macrophage TG biosynthesis, microsomal DGAT1 activity and macrophage oxidative stress, under high glucose concentrations. We suggest that this protective effect may be mediated by PON2 through the attenuation of NADPH-oxidase activity. The use of appropriate means to increase macrophage PON2 expression can lead to attenuation in macrophage TG accumulation and in cellular oxidative stress, under diabetic conditions, and thus may contribute to the decrement in macrophage atherogenicity and foam cell formation, attenuating the development of vascular complications in diabetes mellitus.

In Vitro antioxidant and anti-inflammatory activities of Jaceosidin from Artemisia princeps Pampanini cv. Sajabal

Oxidized low-density lipoprotein (oxLDL) plays a key role in the inflammatory processes of atherosclerosis. Jaceosidin isolated from the methanolic extracts of the aerial parts of Artemisia princeps Pampanini cv. Sajabal was tested for antioxidant and anti-inflammatory activities. Jaceosidin inhibited the Cu(2+)-mediated LDL oxidation with IC(50) values of 10.2 microM in the thiobarbituric acid-reactive substances (TBARS) assay as well as the macrophage-mediated LDL oxidation. The antioxidant activities of jaceosidin were exhibited in the conjugated diene production, relative electrophoretic mobility, and apoB-100 fragmentation on copper-mediated LDL oxidation. Jaceosidin also inhibited the generation of reactive oxygen species (ROS) concerning in regulation of NF-kappaB signaling. And jaceosidin inhibited nuclear factor-kappa B (NF-kappaB) activity, nitric oxide (NO) production, and suppressed expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages.

Low-density lipoprotein (LDL)-antioxidant lignans from Myristica fragrans seeds

Six diarylbutane lignans 1-5 and one aryltetralin lignan 6 were isolated from the methanol (95%) extracts of Myristica fragrans seeds and then 7-methyl ether diarylbutane lignan 4 has proven to be new a compound. Their compounds 1-7 were evaluated for LDL-antioxidant activity to identify the most potent LDL-antioxidant 3 with an IC50 value of 2.6 microM in TBARS assay. Due to its potency, compound 3 was tested for complementary in vitro investigations, such as lag time (140 min at 1.0 microM), relative electrophoretic mobility (REM) of ox-LDL (inhibition of 80% at 20 microM and 72% at 10 microM), and fragmentation of apoB-100 (inhibition of 93% at 20 microM) on copper-mediated LDL oxidation. In macrophage-mediated LDL oxidation, the TBARS formation was also inhibited by compound 3.

Lysophosphatidylcholine (LPC) attenuates macrophage-mediated oxidation of LDL

We have previously shown that paraoxonase 1 action on macrophages produced lysophosphatidylcholine (LPC) and significantly decreased cell-mediated LDL oxidation. Thus, in the present study, we questioned whether LPC can directly inhibit macrophage-mediated oxidation of LDL. Addition of increasing LPC concentrations (0–5 μM) to J774A.1 macrophages, mouse peritoneal macrophages (MPM), or to human monocytes-derived macrophages (HMDM) resulted in up to 83%, 67%, and 75% inhibition in cell-mediated oxidation of LDL, respectively. The mechanism for this LPC effect involves up to 60% inhibition of superoxide anion release from MPM in response to phorbol ester (PMA), 26% inhibition of PMA-induced NADPH oxidase activation (p47phox translocation from the cytosol to the plasma membrane), and a 2-fold stimulation of the macrophage paraoxonase 2 (PON2) lactonase activity. We thus conclude that inhibition of macrophage-mediated oxidation of LDL by LPC can contribute to attenuation of macrophage foam cell formation and atherosclerotic lesion development.

Macrophage-specific expression of group IIA sPLA2 results in accelerated atherogenesis by increasing oxidative stress

Group IIA secretory phospholipase A2 (sPLA2) is an acute-phase protein mediating decreased plasma HDL cholesterol and increased atherosclerosis. This study investigated the impact of macrophage-specific sPLA2 overexpression on lipoprotein metabolism and atherogenesis. Macrophages from sPLA2 transgenic mice have 2.5 times increased rates of LDL oxidation (thiobarbituric acid-reactive substances formation) in vitro (59 +/- 5 vs. 24 +/- 4 nmol malondialdehyde/mg protein; P < 0.001) dependent on functional 12/15-lipoxygenase (12/15-LO). Low density lipoprotein receptor-deficient (LDLR-/-) mice were transplanted with bone marrow from either sPLA2 transgenic mice (sPLA2--> LDLR-/-; n = 19) or wild-type C57BL/6 littermates (C57 BL/6-->LDLR-/-; n = 19) and maintained for 8 weeks on chow and then for 9 weeks on a Western-type diet. Plasma sPLA2 activity and plasma lipoprotein profiles were not significantly different between sPLA2-->LDLR-/- and C57BL/6-->LDLR-/- mice. Aortic root atherosclerosis was increased by 57% in sPLA2-->LDLR-/- mice compared with C57BL/6-->LDLR-/- controls (P < 0.05). Foam cell formation in vitro and in vivo was increased significantly. Urinary, plasma, and aortic levels of the isoprostane 8,12-iso-iPF2alpha-VI and aortic levels of 12/15-LO reaction products were each significantly higher (P < 0.001) in sPLA2-->LDLR-/- compared with C57BL/6-->LDLR-/- mice, indicating significantly increased in vivo oxidative stress in sPLA2--> LDLR-/-. These data demonstrate that macrophage-specific overexpression of human sPLA2 increases atherogenesis by directly modulating foam cell formation and in vivo oxidative stress without any effect on systemic sPLA2 activity and lipoprotein metabolism.

Grape Powder Polyphenols Attenuate Atherosclerosis Development in Apolipoprotein E Deficient (E0) Mice and Reduce Macrophage Atherogenicity

The beneficial health effects of red wine have been attributed to the antioxidant activity of its polyphenols. The present study investigated the effects of a standardized freeze-dried powder made from fresh grapes, rich in grape-specific polyphenols and free of alcohol, on oxidative stress, atherogenicity of macrophages, and the development of atherosclerotic lesions in apolipoprotein E deficient (E(0)) mice. Thirty E(0) mice were assigned to 3 groups. Mice consumed water alone (control), 150 mug total polyphenols/d in the form of grape powder (grape powder), or the equivalent amount of glucose and fructose (placebo) in drinking water for 10 wk. Consumption of grape powder reduced the atherosclerotic lesion area by 41% (P < 0.0002) compared to the control or placebo mice. The antiatherosclerotic effect was at least partly due to a significant 8% reduction in serum oxidative stress, an up to 22% increase in serum antioxidant capacity, a significant 33% reduction in macrophage uptake of oxidized LDL, and a 25% decrease in macrophage-mediated oxidation of LDL relative to controls. Grape powder directly protected both plasma LDL and macrophages from oxidative stress in vitro. We conclude that polyphenols from fresh grape powder directly affect macrophage atherogenicity by reducing macrophage-mediated oxidation of LDL and cellular uptake of oxidized LDL. Both of these processes can eventually reduce macrophage cholesterol accumulation and foam cell formation and hence attenuate atherosclerosis development.

Secreted apolipoprotein E reduces macrophage-mediated LDL oxidation in an isoform-dependent way.

As an inflammatory cell, the macrophage produces various oxidizing agents, such as free radical species. These can modify LDL as a secondary effect and doing so may favor atherogenic processes. Any molecule able to counteract these reactions would be of much benefit, especially if secreted by the macrophage itself at the lesion site. Such is the case for apolipoprotein E (apoE), which has been shown to exert antioxidant properties in some studies, mostly in relation to Alzheimer's disease. In this study, we assessed the antioxidant potential of the various isoforms of apoE (E2, E3, and E4) using a metal-induced LDL oxidation system with exogenous recombinant apoE and an in vitro model of macrophage-mediated LDL oxidation. We found that all three isoforms had an antioxidant capacity. However, whereas apoE2 was the most protective isoform in the cell-free system, the opposite was observed in apoE-transfected J774 macrophages. In the latter model, cellular cholesterol efflux was found to be more important with apoE2, possibly explaining the larger quantity of oxidative indices observed in the medium. It is proposed that the antioxidant property of apoE results from a balance between direct apoE antioxidant capacities, such as the ability to trap free radicals, and potentially pro-oxidative indirect events associated with cholesterol efflux from cells. Our observations add to the therapeutic potential of apoE. However, they also suggest the need for more experiments in order to achieve careful selection of the apoE isoform to be targeted, especially in the perspective of apoE transgene use.

Inducible nitric oxide synthase knockout mouse and low-density lipoprotein oxidation

We have presented experimental procedures that examine macrophage-mediated LDL oxidation using Ham's F-10 medium. By comparing iNOS-/- and iNOS+/+ macrophages, an antioxidant effect for NO and a prooxidant effect for IFN-gamma were demonstrated. The methods outlined here should allow for the investigation on the mechanism of in vitro LDL oxidation and how the macrophage-mediated LDL oxidation process is affected by various factors, one of which was the effect of iNOS induction by IFN-gamma.

Contribution of HDL-apolipoproteins to the inhibition of low density lipoprotein oxidation and lipid accumulation in macrophages.

High-density lipoprotein (HDL) is known as a protective factor against atherosclerosis. However, whether HDL-apolipoproteins (apo-HDL) contribute to the protection in arterial cells remains unclear. The localization patterns of human apolipoproteins in atherosclerotic arteries were determined using immunohistochemical examination. The results indicate that several apolipoproteins are retained in component cells of the coronary artery walls. To elucidate the possible roles of apo-HDL in the protection of atherosclerotic lesion formation, we investigated the effects of apo-HDL on the formation of conjugated diene (CD) in a cell-free system and thiobarbituric acid-reactive substances (TBARS) in the medium of a macrophage-mediated LDL oxidation system. The results showed that apo-HDL significantly exerted an inhibitory effect on LDL lipid oxidation in vitro. In addition, apo-HDL decreased cholesterol influx but enhanced cholesterol efflux from J774 macrophages in a dose-dependent manner. These results are consistent with the notion that there is reduced intracellular lipid accumulation in apo-HDL treated macrophages. These data provide a direct evidence for apo-HDL in protecting LDL from oxidative modification and in reducing the accumulation of cholesterol and lipid droplets by J774 macrophages.

Phenolic compounds contained in extra-virgin olive oil inhibit macrophage-mediated oxidation of LDL: effects on intracellular redox balance

Phenolic compounds contained in extra-virgin olive oil inhibit macrophage-mediated oxidation of LDL: effects on intracellular redox balance

Inducible Nitric Oxide Synthase Knockout Mouse Macrophages Disclose Prooxidant Effect of Interferon-γ on Low-Density Lipoprotein Oxidation

To test our hypothesis that interferon-γ (IFN-γ) has a direct prooxidant effect on macrophage-mediated LDL oxidation behind its antioxidant effect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS+/+) or iNOS knockout mouse (iNOS−/−) macrophages preincubated with IFN-γ or IFN-γ plus lipopolysaccharide (IFN-γ/LPS) for 24 h. LDL oxidation was measured in terms of formation of thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrophages were measured by using Ellman's assay, the Griess reagent, and the SOD-inhibitable cytochrome c reduction method, respectively. IFN-γ alone or combined with LPS induced iNOS expression and increased nitrite production in iNOS+/+ macrophages, but not in iNOS−/− macrophages. TBARS formation from LDL was suppressed in IFN-γ- and IFN-γ/LPS-treated iNOS+/+ macrophages but was increased in IFN-γ-treated iNOS−/− macrophages. In the presence of NG-monomethyl-l-arginine (l-NMMA), a NOS inhibitor, the suppressive effect of IFN-γ and IFN-γ/LPS was abolished and TBARS formation was even increased to a level above that of untreated iNOS+/+ macrophage. NOC 18, an NO donor, dose dependently inhibited macrophage-mediated LDL oxidation. IFN-γ increased superoxide and thiol productions in both types of macrophages. We conclude that IFN-γ promotes macrophage-mediated LDL oxidation by stimulating superoxide and thiol production under conditions where iNOS-catalyzed NO release is restricted.

Macrophage-mediated oxidation of LDL may be counteracted by phenolic compounds contained in extra-virgin olive oil

Macrophage-mediated oxidation of LDL may be counteracted by phenolic compounds contained in extra-virgin olive oil

Involvement of transplasma-membrane electron transport in macrophage-mediated LDL oxidation

Involvement of transplasma-membrane electron transport in macrophage-mediated LDL oxidation

Antioxidant BO-653 and human macrophage-mediated LDL oxidation

Oxidation of LDL is now widely accepted to be involved in atherogenesis. The aim of this study was to examine the effect of BO-653, a strong radical scavenger and antioxidant, on oxidation of LDL by human macrophages in vitro. Fifty μg/ml LDL protein was incubated with macrophages in Ham's F10 medium, supplemented with additional Fe2+, for up to 48h. Then the medium was analysed by LDL agarose gel electrophoresis, the thiobarbituric acid assay and gas chromatography. In the absence of added exogenous antioxidants, after 24 h LDL oxidation produced 30.48 nmoles MDA equivalents/mg LDL protein and a relative electrophoretic mobility of 4.74. Linoleic acid (18:2), arachidonic acid (20:4) and cholesterol were depleted and 7β-hydroxycholesterol was generated. BO-653 completely inhibited this cell-mediated oxidation of LDL in concentrations as low as 5 μM, being more effective than either α-tocopherol or probucol, which completely inhibited oxidation at 200 and 80 μM and only partially at 80 and 8 μM, respectively. This inhibition of cell-mediated LDL oxidation was not due to toxicity, as α-tocopherol, probucol and BO-653 were not toxic for the macrophages at the concentrations tested. Eighty μM α-tocopherol, 8 μM probucol and 5 μM BO-653 significantly reduced the toxicity to the oxidising culture caused by LDL oxidation. The results show that in this system BO-653 is a more effective antioxidant than α-tocopherol or probucol.

Inhibition of LDL oxidation by melatonin requires supraphysiologic concentrations

Melatonin has been suggested as a potent antioxidant that may protect against development of atherosclerosis and cancer; however, these effects are unproven and controversial. The antioxidant capacity of melatonin was tested in comparison with alpha-tocopherol, ascorbic acid, and the melatonin precursors tryptophan and serotonin, by measuring inhibition of metal ion-mediated and human macrophage-mediated oxidation of LDL. Melatonin had weak antioxidant activity that was detectable only at concentrations 10000- to 100000-fold higher than physiologic concentrations. These results were comparable with published data showing that the radical scavenging activity of melatonin requires markedly supraphysiologic concentrations. In contrast, alpha-tocopherol was 50- to 100-fold more potent and was efficacious at physiologic concentrations. Ascorbic acid and tryptophan also were active at physiologic concentrations and were significantly more potent than melatonin. In summary, extremely supraphysiologic concentrations of melatonin had only weak antioxidant activity, which was surpassed by alpha-tocopherol, ascorbic acid, and tryptophan.

Iron and LDL-oxidation in atherogenesis.

It has been proposed that the development of atherosclerosis may be linked to the size of the body iron stores. The exact role of iron in the initiation and progression of atherogenesis is, however, still unknown. As a result of increasing support for the LDL-oxidation hypothesis, much additional knowledge about the relation between iron and atherosclerosis has recently been gained. This review presents the current evidence on the role of iron--being a potent catalyst of oxidative reactions--and macrophage-mediated LDL-oxidation in atherogenesis. The authors hypothesize that iron, as a possible central intermediary, may play an important role in cell-mediated LDL-oxidation.

Structural Aspects of The Inhibitory Effect of Glabridin on LDL Oxidation

The inhibitory effects of glabridin, an isoflavan isolated from licorice ( Glycyrrhiza glabra) root, and its derivatives on the oxidation of LDL induced by copper ions or mediated by macrophages were studied, in order to evaluate the contribution of the different parts of the isoflavan molecule to its antioxidant activity. The peak potential (E 1/2 ) of the isoflavan derivatives, their radical scavenging capacity toward 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and their ability to chelate heavy metals were also analyzed and compared to their inhibitory activity on LDL oxidation. In copper ion-induced LDL oxidation, glabridin (1), 4′- O-methylglabridin (2), hispaglabridin A (3), and hispaglabridin B (4), which have two hydroxyl groups at positions 2′ and 4′ or one hydroxyl at position 2′ on ring B, successfully inhibited the formation of conjugated dienes, thiobarbituric acid reactive substances (TBARS) and lipid peroxides, and inhibited the electrophoretic mobility of LDL under oxidation. Compounds 1–3 exhibited similar activities, whereas compound 4 was less active. In macrophage-mediated LDL oxidation, the TBARS formation was also inhibited by these isoflavans (1–4) at a similar order of activity to that obtained in copper ion-induced LDL oxidation. On the other hand, 2′- O-methylglabridin (5), a synthesized compound, whose hydroxyl at 2′-position is protected and the hydroxyl at 4′-position is free, showed only minor inhibitory activity in both LDL oxidation systems. 2′,4′- O-Dimethylglabridin (6), whose hydroxyls at 2′- and 4′-positions are both protected, was inactive. Resorcinol (7), which is identical to the phenolic B ring in glabridin, presented low activity in these oxidation systems. The isoflavene glabrene (8), which contains an additional double bond in the heterocyclic C ring, was the most active compound of the flavonoid derivatives tested in both oxidation systems. The peak potential of compounds 1–5 (300 μM), tested at pH 7.4, was similar (425–530 mV), and that for compound 6 and 8 was 1078 and 80 mV, respectively. Within 30 min of incubation, compounds 1, 2, 3, 4, 8 scavenged 31%, 16%, 74%, 51%, 86%, respectively, of DPPH radical, whereas compounds 5 and 6, which almost did not inhibit LDL oxidation, also failed to scavenge DPPH. None of the isoflavan derivatives nor the isoflavene compound were able to chelate iron, or copper ions. These results suggest that the antioxidant effect of glabridin on LDL oxidation appears to reside mainly in the 2′ hydroxyl, and that the hydrophobic moiety of the isoflavan is essential to obtain this effect. It was also shown that the position of the hydroxyl group at B ring significantly affected the inhibitory efficiency of the isoflavan derivatives on LDL oxidation, but did not influence their ability to donate an electron to DPPH or their peak potential values.

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.

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, beta-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 lipoprotein's lipid peroxides. The present review article summarizes the above issues with an emphasis on our own data.