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M Aviram

Publications and source records attributed to M Aviram.

At least 37 records · Page 2Linked to original sources
PubMed2000

Losartan inhibits cellular uptake of oxidized LDL by monocyte-macrophages from hypercholesterolemic patients.

Angiotensin II (Ang II) and oxidized LDL (Ox-LDL) are risk factors for atherosclerosis, and both of them contribute to macrophage cholesterol accumulation, the hallmark of early atherosclerosis. As Ang II was shown to increase macrophage uptake of Ox-LDL, we investigated the effect of losartan, an Ang II receptor antagonist with antiatherogenic properties, on the cellular uptake of Ox-LDL by human monocyte-derived macrophages (HMDM) from hypercholesterolemic patients. Eight normotensive hypercholesterolemic patients were treated with losartan (50 mg/day) for a period of 4 weeks. Losartan therapy did not significantly affect the degradation of native LDL by the patients' HMDM. However, losartan therapy significantly reduced HMDM uptake of Ox-LDL as shown by a 78% reduction in Ox-LDL cell-association and a 21% reduction in Ox-LDL degradation. CD36 (an Ox-LDL receptor) mRNA expression in HMDM obtained after losartan treatment was decreased by 54% compared to HMDM obtained before treatment. The ability of losartan to inhibit HMDM CD36 mRNA expression and, hence, Ox-LDL uptake and macrophage foam cell formation is probably related to the blockage of Ang II binding to the cell surface and thus to the prevention of Ang II atherogenic effects.

Angiotensin II↗
PubMed2000

Human serum paraoxonases (PON1) Q and R selectively decrease lipid peroxides in human coronary and carotid atherosclerotic lesions: PON1 esterase and peroxidase-like activities.

BACKGROUND: Human serum paraoxonase (PON1) exists in two polymorphic forms: one that differs in the amino acid at position 192 (glutamine and arginine, Q and R, respectively) and the second one that differs in the amino acid at position 55 (methionine and leucine, M and L, respectively). PON1 protects LDL from oxidation, and during LDL oxidation, PON1 is inactivated. METHODS AND RESULTS: The present study compared PON1 isoforms Q and R for their effect on lipid peroxide content in human coronary and carotid lesions. After 24 hours of incubation with PON1Q or PON1R (10 arylesterase units/mL), lipid peroxides content in both coronary and carotid lesion homogenates (0.1 g/mL) was reduced up to 27% and 16%, respectively. The above incubation was associated with inactivation of PON1Q and PON1R by 15% and 45%, respectively. Lesion cholesteryl linoleate hydroperoxides and cholesteryl linoleate hydroxides were hydrolyzed by PON1 to yield linoleic acid hydroperoxides and linoleic acid hydroxides. Furthermore, lesion and pure linoleic acid hydroperoxides were reduced to yield linoleic acid hydroxides. These results thus indicate that PON1 demonstrates esterase-like and peroxidase-like activities. Recombinant PON1 mutants in which the PON1-free sulfhydryl group at cysteine-284 was replaced with either alanine or serine were no longer able to reduce lipid peroxide content in carotid lesions. CONCLUSIONS: We conclude that PON1 may be antiatherogenic because it hydrolyzes lipid peroxides in human atherosclerotic lesions.

Arteriosclerosis↗
PubMed2000

Enhanced lipid oxidation by oxidatively modified myoglobin: role of protein-bound heme.

The formation of oxidized low density lipoprotein (LDL) is believed to play a significant role in the pathogenesis of atherosclerosis. Myoglobin in the presence of H(2)O(2) has been shown to catalyze LDL oxidation in vitro. It is established that an oxidatively altered form of myoglobin (Mb-H), which contains a prosthetic heme covalently crosslinked to the apoprotein, is a major product in the reaction of native myoglobin with peroxides. In the current study, we have shown for the first time that Mb-H, in the absence of exogenously added peroxides, oxidizes LDL and purified lipids, as determined by the formation of conjugated dienes, lipid peroxides, and thiobarbituric acid reactive substances. Moreover, the rate of oxidation of pure phosphatidylcholine by Mb-H was found to be at least sevenfold greater than that observed for native myoglobin. The current study strongly suggests a role for Mb-H in the lipid peroxidation observed with myoglobin.

Animals↗
PubMed2000

Oxidized monocyte-derived macrophages in aortic atherosclerotic lesion from apolipoprotein E-deficient mice and from human carotid artery contain lipid peroxides and oxysterols.

Oxidative stress is thought to play an important role in atherogenesis. The present study demonstrated, for the first time, that macrophages (originally derived from blood monocytes) isolated from aortas of the atherosclerotic apolipoprotein E deficient (E degrees ) mice or from human carotid artery, are oxidized as they contain lipid peroxides and oxysterols. The major oxysterol in arterial macrophages was found to be 7-ketocholesterol (51% of total oxysterols). To find out whether lipid peroxidation of monocytes occurs in vivo already in the blood, we analyzed the oxidative state of monocytes derived from E degrees mice in comparison to monocytes from control mice. Cellular lipid peroxides and total oxysterols were four and sevenfold higher respectively, in monocytes derived from E degrees mice in comparison to monocytes from control mice. The results of the present study thus demonstrated the presence of lipid-peroxidized monocytes already in the blood, which are further oxidized in the arterial wall after their conversion into macrophages. The arterial oxidized macrophages could be considered key contributors to foam cell formation, the hallmark of early atherosclerosis.

Animals↗
PubMed2000

Serum lipase levels as a diagnostic marker in cystic fibrosis patients with normal or borderline sweat tests.

Patients with normal or borderline sweat test present a diagnostic challenge. In spite of the availability of different methods such as genetic analysis and measurements of nasal potential difference, uncertainty in diagnosing cystic fibrosis (CF) in some patients still exists. Neonates with CF have high serum lipase levels, which decline over time in pancreatic-insufficient patients, whereas pancreatic-sufficient patients demonstrate high serum lipase levels beyond infancy. Because patients with borderline or normal sweat test are almost always pancreatic sufficient, this study was aimed to assess whether serum lipase levels may be of help in establishing the diagnosis of CF in these patients. Serum lipase levels were measured in 100 CF patients and in 17 healthy individuals. Patients were grouped according to their genotype. Group A patients (n = 70) carried two mutations previously found to be associated with a pathologic sweat test and pancreatic insufficiency (delta F508, W1282X, G542X, N1303K, S549R). Group B (n = 30) were compound heterozygote patients who carried one mutation known to cause mild disease with borderline or normal sweat tests and pancreatic sufficiency (3849+10kb C-->T, 5T). Group C included 17 healthy controls. Serum lipase levels ranged between 2 and 104.4 U/L (mean +/- SD 16.9 +/- 14.7), 6.1-200 U/L (mean +/- SD 53.9 +/- 47.9), and 8.5-27.8 U/L (mean +/- SD 16.9 +/- 5.1) in Groups A, B, and C, respectively, with some overlapping between groups. The distribution of lipase levels was significantly different in Group B vs Groups A and C (P < 0.01). High lipase levels were found in 63.3% (19/30) of Group B patients, but in only 4.3% (3/70) and 0% (0/17) of Group A and C, respectively. Lipase levels were found to be inversely related to sweat chloride concentrations (r = -0.19, P < 0.05). Patients with borderline or normal sweat tests had high lipase levels, whereas low lipase levels were associated with pathologic sweat tests. Our findings indicate that the serum lipase level is genetically determined and that it has a useful role in the diagnosis of CF. Thus, in patients with borderline sweat tests and high lipase levels, the diagnosis of CF should be considered.

Adult↗
PubMed2000

Macrophage plasma membrane chondroitin sulfate proteoglycan binds oxidized low-density lipoprotein.

Lipoprotein interactions with macrophage proteoglycans (PGs) is believed to play an important role in the cellular uptake of lipoproteins and in macrophage cholesterol accumulation. Recently, we have shown the participation of macrophage plasma membrane glycosaminoglycans (GAGs) in the cellular uptake of oxidized LDL (Ox-LDL). The aim of the present study was to identify the specific cell surface proteoglycans involved in this interaction. J-774 A.1 macrophage-like cell line plasma membrane proteoglycans were isolated by anion exchange chromatography from cells that were prelabeled with [35S]sodium sulfate. Using Sepharose 6B chromatography, cell surface major proteoglycans were identified as chondroitin sulfate (CS) proteoglycans (77%) and heparan sulfate (HS) proteoglycans (23%). Binding rates of these 35S-labeled proteoglycans to Ox-LDL and to native LDL were analyzed by their ability to bind lipoproteins coupled to a CnBr-activated Sepharose CL-4B chromatography. Of the total labeled cell surface proteoglycans added to the column, 57% were bound to the Sepharose-coupled Ox-LDL, whereas 73% of the cell surface proteoglycans were bound to the Sepharose-coupled native LDL. Binding of the plasma membrane macrophage 35S-labeled proteoglycans to Ox-LDL was inhibited by adding increasing concentrations of non-labeled chondroitin sulfate, or by pretreatment of the 35S-labeled proteoglycans fraction with chondroitinase ABC. In contrast, neither the addition of non-labeled heparan sulfate, nor pretreatment of the labeled proteoglycans fraction with heparinase III, had any significant effect on proteoglycan binding to Ox-LDL. These findings were further supported by using mutant cells characterized by specific glycosaminoglycan deficiencies. Ox-LDL binding and degradation by mutant 745 CHO cells which are characterized by a deficiency in both heparan sulfate and chondroitin sulfate, was decreased by 28 and 27% respectively, compared to the binding of Ox-LDL to the wild-type CHO cells. Ox-LDL binding and degradation by mutant 677 CHO cells, which lack heparan sulfate but have increased levels of chondroitin sulfate, however, was found to be increased by 29 and 19%, respectively, compared to Ox-LDL binding to the wild-type CHO cells. Finally, analysis of the cell surface proteoglycans in macrophages that were subjected to oxidative stress, by their preincubation with angiotensin II, exhibited a 51-59% increase in their cell surface proteoglycan content, with a major effect on chondroitin sulfate proteoglycans. The present study thus demonstrated that Ox-LDL can specifically bind to macrophage surface chondroitin sulfate proteoglycans, and the macrophage content of this proteoglycan is increased under oxidative stress. The interaction between macrophage chondroitin sulfate proteoglycans and Ox-LDL can contribute to enhanced uptake of Ox-LDL with the formation of cholesterol-loaded foam cells, and accelerated atherosclerosis.

Binding Sites↗
PubMed2000

Macrophage-released proteoglycans enhance LDL aggregation: studies in aorta from apolipoprotein E-deficient mice.

Aggregated low-density lipoprotein (LDL) was shown to be present in the atherosclerotic lesion, but the mechanism responsible for its formation in vivo is not known yet. To find out whether LDL aggregation occurs in the arterial wall during atherogenesis, LDLs were extracted from the aortas of apolipoprotein E-deficient (E(0)) mice during their aging (and the development of atherosclerosis), and were analyzed for their aggregation states, in comparison to LDLs isolated from aortas of control mice. LDL isolated from aortas of E(0) mice was already aggregated at 1 month of age and its aggregation state substantially increased with age, with 3-fold elevation at 6 months of age compared to younger, 1-month-old, mice. Only minimal aggregation could be detected in LDL derived from control mice. Electron microscopy examination revealed that LDL particles from aortas of the E(0) mice were heterogeneous in their size, ranging between 20 and 300 nm. The mouse aortic LDL contained proteoglycans (PGs) and their content increased with the age of the mice, with about 2-fold higher levels than those found in LDLs derived from aortas of control mice. Macrophage-released PGs were previously demonstrated to enhance LDL aggregation in vitro. However, their involvement in LDL aggregation in vivo has not been studied yet. Thus, we next studied the effect of arterial macrophage-released PGs on the susceptibility of plasma LDL to aggregation by Bacillus cereus sphingomyelinase (SMase). Foam cell macrophages were isolated from aortas of the atherosclerotic E(0) mice at 6 months of age and were found to be loaded with cholesterol and to contain oxidized lipids. To analyze the effect of macrophage-released PGs on LDL aggregation, PGs were prelabeled by cell incubation with [35S]sulfate, followed by incubation of macrophage-released PGs with E(0) mouse plasma LDL (200 microg protein/ml) for 1 h at 37 degrees C. [35S]Sulfated PGs were found to be LDL-associated and the susceptibility of PG-associated LDL to aggregation by SMase was increased by up to 45% in comparison to control LDL. Similar results demonstrating the involvement of PGs in LDL aggregation were obtained upon incubation of LDL with increasing concentrations of PGs that were isolated from the entire aorta of E(o) mice (rather than the isolated macrophages). The stimulatory effect of macrophage-released PGs on LDL aggregation was markedly reduced when the PGs were pretreated with the glycosaminoglycan-hydrolyzing enzymes, chondroitinase ABC or chondroitinase AC, and to a much lesser extent with heparinase. We thus conclude that macrophage-released chondroitin sulfate PG can contribute to the formation of atherogenic aggregated LDL in the arterial wall.

Aging↗
PubMed2000

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

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

Abietanes↗
PubMed2000

Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice.

BACKGROUND: Dietary supplementation with nutrients rich in antioxidants is associated with inhibition of atherogenic modifications to LDL, macrophage foam cell formation, and atherosclerosis. Pomegranates are a source of polyphenols and other antioxidants. OBJECTIVE: We analyzed, in healthy male volunteers and in atherosclerotic apolipoprotein E-deficient (E(0)) mice, the effect of pomegranate juice consumption on lipoprotein oxidation, aggregation, and retention; macrophage atherogenicity; platelet aggregation; and atherosclerosis. DESIGN: Potent antioxidative effects of pomegranate juice against lipid peroxidation in whole plasma and in isolated lipoproteins (HDL and LDL) were assessed in humans and in E(0) mice after pomegranate juice consumption for </=2 and 14 wk, respectively. RESULTS: In humans, pomegranate juice consumption decreased LDL susceptibility to aggregation and retention and increased the activity of serum paraoxonase (an HDL-associated esterase that can protect against lipid peroxidation) by 20%. In E(0) mice, oxidation of LDL by peritoneal macrophages was reduced by up to 90% after pomegranate juice consumption and this effect was associated with reduced cellular lipid peroxidation and superoxide release. The uptake of oxidized LDL and native LDL by mouse peritoneal macrophages obtained after pomegranate juice administration was reduced by 20%. Finally, pomegranate juice supplementation of E(0) mice reduced the size of their atherosclerotic lesions by 44% and also the number of foam cells compared with control E(0) mice supplemented with water. CONCLUSION: Pomegranate juice had potent antiatherogenic effects in healthy humans and in atherosclerotic mice that may be attributable to its antioxidative properties.

Adult↗
PubMed2000

Ginger extract consumption reduces plasma cholesterol, inhibits LDL oxidation and attenuates development of atherosclerosis in atherosclerotic, apolipoprotein E-deficient mice.

Oxidative modification of LDL is thought to play a key role in the pathogenesis of atherosclerosis. Consumption of nutrients rich in phenolic antioxidants has been shown to be associated with attenuation of development of atherosclerosis. This study was undertaken to investigate the ex vivo effect of standardized ginger extract on the development of atherosclerosis in apolipoprotein E-deficient (E(0)) mice, in relation to plasma cholesterol levels and the resistance of their LDL to oxidation and aggregation. E(0) mice (n = 60; 6-wk-old) were divided into three groups of 20 and fed for 10 wk via their drinking water with the following: group i) placebo (control group), 1.1% alcohol and water (11 mL of alcohol in 1 L of water); group ii) 25 microg of ginger extract/d in 1.1% alcohol and water and group iii) 250 microg of ginger extract/day in 1.1% alcohol and water. Aortic atherosclerotic lesion areas were reduced 44% (P<0.01) in mice that consumed 250 microg of ginger extract/day. Consumption of 250 microg of ginger extract/day resulted in reductions (P<0.01) in plasma triglycerides and cholesterol (by 27 and 29%, respectively), in VLDL (by 36 and 53%, respectively) and in LDL (by 58 and 33%, respectively). These results were associated with a 76% reduction in cellular cholesterol biosynthesis rate in peritoneal macrophages derived from the E(0) mice that consumed the high dose of ginger extract for 10 wk (P<0.01). Furthermore, peritoneal macrophages harvested from E(0) mice after consumption of 25 or 250 microg of ginger extract/day had a lower (P<0.01) capacity to oxidize LDL (by 45 and by 60%, respectively), and to take up and degrade oxidized LDL (by 43 and 47%, respectively). Consumption of 250 microg of ginger extract/day also reduced (P<0.01) the basal level of LDL-associated lipid peroxides by 62%. In parallel, a 33% inhibition (P<0.01) in LDL aggregation (induced by vortexing) was obtained in mice fed ginger extract. We conclude that dietary consumption of ginger extract by E(0) mice significantly attenuates the development of atherosclerotic lesions. This antiatherogenic effect is associated with a significant reduction in plasma and LDL cholesterol levels and a significant reduction in the LDL basal oxidative state, as well as their susceptibility to oxidation and aggregation.

Animals↗
PubMed2000

Dietary supplementation of a natural isomer mixture of beta-carotene inhibits oxidation of LDL derived from patients with diabetes mellitus.

BACKGROUND: Accelerated atherosclerosis is common in patients with diabetes mellitus which may be linked to increased lipid peroxidation. Therefore, we compared the oxidation of LDL derived from patients with diabetes to normoglycemic controls and followed-up the effect of dietary beta-carotene supplementation on LDL oxidation. METHODS: Twenty patients with long-standing non-insulin-dependent diabetes mellitus were studied in comparison with age- and sex-matched control subjects. Dunaliella bardawil-derived beta-carotene was supplemented to the patients for 3 weeks, 60 mg daily dose. LDL oxidation was analyzed by measuring malondialdehyde (MDA), lipid peroxides (PD), and conjugated dienes (CD) generation in response to CuSO(4)-induced oxidation. LDL lipid composition and the LDL associated vitamins A, E and carotenoids were also measured. RESULTS: LDL susceptibility to oxidation by CuSO(4) was increased in the patients by 40% with a 35% shorter lag time required for the initiation of LDL oxidation, i.e. 56 +/- 6 min in patients vs. 85 +/- 9 min in controls (p <0.01). Patients showed increased cholesterol/phospholipid and polyunsaturated/saturated ratios, as well as reduced content of LDL associated vitamins. Upon beta-carotene supplementation, there was a significant elevation in plasma and in LDL all-trans beta-carotene [from 0.296 +/- 0.020 to 0. 968 +/- 0.133 microg/mg LDL protein (p < 0.01)] paralleled by a significant reduction in LDL susceptibility to oxidation, as exhibited by increased lag time up to 115 +/- 10 min (p < 0.01) and reduction in MDA and PD generation (by 25 and 40%), respectively (p < 0.01). CONCLUSIONS: Increased susceptibility to oxidation of LDL derived from patients with diabetes mellitus is associated with abnormal LDL lipid composition and antioxidant content. Natural beta-carotene dietary supplementation normalizes the enhanced LDL oxidation and consequently may be of importance in delaying accelerated development of atherosclerosis in these patients.

Adult↗
PubMed2000

Review of human studies on oxidative damage and antioxidant protection related to cardiovascular diseases.

Under oxidative stress, which is associated with atherosclerosis, oxidative modifications of LDL take place. A major effect of antioxidants in the LDL environment is to prevent the formation of oxidized LDL during atherogenesis. The question that arises is what are the body's capabilities to inhibit LDL oxidation and to remove and/or to neutralize atherogenic Ox-LDL when formed. Strategies to reduce LDL oxidation and atherogenesis can involve the enrichment of the LDL and arterial cells with potent antioxidants that can prevent oxidative damage to the arterial wall. There seems to be a clear cause and effect relationship between LDL oxidation and atherosclerosis. Atherosclerosis is a multifactorial disease and LDL is oxidized by all major cells of the arterial wall during the development of atherosclerosis via more than one mechanism. The various LDL oxidation pathways produce several lipid peroxidation products such as isoprostanes from arachidonic, eicosapentaenoic and docosahexaenoic acids, oxysterols from unesterified and esterified cholesterol, hydroxy fatty acids, lipid peroxides and aldehydes. Thus, one single assay of lipid peroxidation is probably not sufficient to serve as a marker for cardiovascular risk and there is a need for measurements of several markers. The use of biomarkers provides a logical scientific basis for major intervention trials of antioxidants; such trials will, in turn, eventually validate or disprove the biomarker concept. Any intervention trial that does take place should be accompanied by measurements of one or more relevant biomarkers at intervals during the study. If the endpoint of the trial is disease incidence or mortality, such studies will help to validate or disprove the biomarker concept. They might also help to explore the possibility that in vivo levels of oxidative lipid damage are early predictors of subsequent development of cardiovascular disease. In addition, specific antioxidants in serum, as well as serum paraoxonase activity can provide very useful information on the risk for cardiovascular diseases. For vascular disease risk, in addition to the markers in use for lipid peroxidation, there is a need to include also markers for endothelial dysfunction, monocyte adhesion, macrophage uptake of lipoproteins, thrombotic, and inflammatory processes.

Antioxidants↗
PubMed1999

Properties of the retained N-terminal hydrophobic leader sequence in human serum paraoxonase/arylesterase.

Human serum paraoxonase/arylesterase (PON1) is HDL-associated and appears to protect low density lipoproteins (LDL) from oxidation. Mature PON1 retains its N-terminal hydrophobic signal sequence, which may be needed for binding to HDL. By site-directed mutagenesis, we created a mutant PON1 (A19A20) with a cleavable N-terminus to determine if this peptide mediated binding to lipoproteins. As a model system, we studied binding of mutant and wild type PON1s to lipoproteins in fetal bovine serum-containing expression medium and found that the wild type recombinant enzyme associated with lipoproteins whereas the A19A20 mutant did not. These results show that the N-terminus is required for binding to either apolipoproteins or phospholipids. Furthermore, we showed that wild type enzyme can bind to phospholipids directly without apolipoproteins. To determine if lipid binding is a requirement for PON1's protection against LDL oxidation, we used a copper ion-induced oxidation system and found that the wild type enzyme and A19A20 mutant showed similar reductions in both peroxide and aldehyde formation. We conclude that PON1 depends upon its N-terminal hydrophobic peptide for its association with serum lipoproteins.

Animals↗
PubMed1999

On the physiological role(s) of the paraoxonases.

In recent years several lines of evidence have indicated that serum paraoxonase (PON1), and perhaps other mammalian paraoxonases, act as important guardians against cellular damage from toxic agents, such as organophosphates, oxidized lipids in the plasma low density lipoproteins (LDL), and against bacterial endotoxins. For some of these protective activities but not all, PON1 requires calcium ion. The catalyzed chemical reactions generally seem to be hydrolytic, but for some types of protection this may not be so. Several other metals have very high affinity for PON1 and may displace calcium. Replacement or substitution of calcium by other metals could extend the range of catalytic properties and the substrate specificity of the paraoxonases, as it does for the mammalian DFPases. Although this Third International Meeting on Esterases Reacting with Organophosphorus Compounds focuses on the organophosphatase activities of paraoxonase and related enzymes, it is important to also briefly review some of the current directions in several laboratories searching for additional functions of the paraoxonases to extend our understanding of the properties of this family of enzymes which now seem to have both physiological and toxicological importance.

Amino Acid Sequence↗
PubMed1999

Macrophage enrichment with the isoflavan glabridin inhibits NADPH oxidase-induced cell-mediated oxidation of low density lipoprotein. A possible role for protein kinase C.

Macrophage-mediated oxidation of low density lipoprotein (LDL) is considered to be of major importance in early atherogenesis; therefore, intervention means to inhibit this process are being extensively studied. In the present study, we questioned the ability of the isoflavan glabridin (from licorice) to accumulate in macrophages and to affect cell-mediated oxidation of LDL. We first performed in vitro studies, using mouse peritoneal macrophages (MPMs) and the J-774 A.1 macrophage-like cell line. Both cells accumulated up to 1.5 micrograms of glabridin/mg of cell protein after 2 h of incubation, and this process was time- and glabridin dose-dependent. In parallel, in glabridin-enriched cells, macrophage-mediated oxidation of LDL was inhibited by up to 80% in comparison with control cells. Glabridin inhibited superoxide release from MPMs in response to phorbol 12-myristate 13-acetate, or to LDL when added together with copper ions, by up to 60%. Translocation of P-47, a cytosolic component of NADPH oxidase to the plasma membrane was substantially inhibited. In glabridin-enriched macrophages, protein kinase C activity reduced by approximately 70%. All of the above effects of glabridin required the presence of the two hydroxyl groups on the flavonoid's B phenol ring. In order to assess the physiological significance of these results, we next performed in vivo studies, using the atherosclerotic apolipoprotein E-deficient (E0) mice. MPMs harvested from glabridin-treated E0 mice (20 micrograms/mouse/day for a period of 6 weeks) demonstrated reduced capability to oxidize LDL by 80% in comparison with placebo-treated mice. This latter phenomenon was associated with a reduction in the lesion oxysterols and a 50% reduction in the aortic lesion size. We thus conclude that glabridin accumulation in macrophages is associated with reduced cell-mediated oxidation of LDL and decreased activation of the NADPH oxidase system. These phenomena could be responsible for the attenuation of atherosclerosis in E0 mice, induced by glabridin.

Animals↗
PubMed1999

Macrophage released proteoglycans are involved in cell-mediated aggregation of LDL.

Aggregated low density lipoprotein (LDL) is taken up by macrophages at enhanced rate, leading to macrophage cholesterol accumulation and foam cell formation. Since macrophages were shown to mediate self aggregation of modified forms of LDL, we sought to study the effect of macrophages on the susceptibility of native LDL to aggregation. Incubation of LDL (100 microg of protein/ml) with J-774A.1 macrophage-like cell line for 18 h at 37 degrees C, led to a 114 and 56% enhanced susceptibility of LDL to aggregation by vortexing and by Bacillus cereus SMase respectively. Macrophage conditioned media (MCMs) that were obtained from J-774A.1 cells also enhanced the susceptibility of LDL to aggregation by vortexing and SMase by 134 and 75% respectively, suggesting the involvement of macrophage secretory products in the enhanced aggregation of LDL. As proteoglycans were shown to be involved in lipoprotein aggregation, we analyzed the possible involvement of macrophage-released proteoglycans in LDL aggregation. Incubation of LDL (100 microg protein/ml) with 25 microg of proteoglycans that were isolated from MCM led to a dose-dependent enhanced susceptibility of LDL to aggregation by vortexing or by SMase by up to 62 and 77% respectively. The stimulatory effect of the MCMs on LDL aggregation was markedly reduced upon MCMs treatment with the glycosaminoglycan hydrolyzing enzyme chondroitinase ABC, chondroitinase AC, but not heparinase. On the contrary, incubation of LDL (100 microg of protein/ml) with increasing concentrations (up to 50 microg/ml) of chondroitin sulfate, or heparan sulfate enhanced the susceptibility of LDL to aggregation by up to 98 or by only 18% respectively, in comparison with non-treated LDL. Since macrophages under atherogenic conditions (cholesterol-loading, cellular lipid peroxidation and activation) demonstrate enhanced secretion of proteoglycans, we finally studied the effect of J-774A.1 macrophages on the susceptibility of native LDL to aggregation under the above atherogenic conditions. Incubation of LDL with cholesterol-loaded macrophages led to a 62% enhanced susceptibility of LDL to undergo aggregation by vortexing, in comparison with LDL that was incubated with non-loaded cells. Macrophage activation with phorbol myristate acetate (5 microM of PMA) also significantly increased cell-mediated aggregation of LDL by 50%, in comparison with non-activated cells. Lipid peroxidized macrophages obtained by cell treatment with either FeSO4 (50 microM), or angiotensin II (10(-7) M) enhanced the susceptibility of LDL to aggregation by 22 or by 39% respectively. These results suggest that under atherogenic conditions, macrophages release proteoglycans, and mainly chondroitin sulfate, which can contribute to cell-mediated formation of aggregated LDL, a potent inducer of macrophage foam cells which are the hallmark of early atherogenesis.

Animals↗
PubMed1999

Microsomal cytochromes P450 catalyze the oxidation of low density lipoprotein.

Low density lipoprotein (LDL) oxidation is a major contributor to foam cell formation during early atherogenesis. Several oxygenases have been implicated in the process of LDL oxidation in the arterial wall, where the environment is relatively low in antioxidants, but the exact mechanism for LDL oxidation in vivo is not known. In the present study we sought to determine the ability of cytochrome P450 2E1 (P450 2E1) and other P450s, located in the liver and in other tissues, to oxidize LDL. Upon incubation of LDL (0.1 mg of protein/ml) with purified, reconstituted rabbit P450 2E1 in the presence of NADPH and the NADPH-cytochrome P450 reductase, time- and P450 2E1 concentration-dependent LDL oxidation was observed, as analyzed by determining the formation of peroxides, thiobarbituric acid reactive substances (TBARS), and conjugated dienes. Within 1 h of initiating the reaction, almost maximal oxidation was observed. NADPH, and active P450 2E1 enzyme were required for LDL oxidation to occur. The rate of P450 2E1-induced LDL oxidation was also dependent on the lipoprotein concentration. P450 2E1 could also oxidize pure phospholipids and cholesteryl ester, the major lipids in LDL. In the presence of catalase or superoxide dismutase (SOD), LDL oxidation was completely blocked, suggesting that hydrogen peroxide and superoxide are involved in P450 2E1-induced LDL oxidation. The ability of P450 2E1 to oxidize LDL was not unique to this enzyme, and could be observed with some other purified, cytochromes P450 in the reconstituted system such as rat P450 2B1 and human P450 3A4. Finally, microsomal membranes obtained from rats that were induced to express high levels of P450s 2B1, 2E1, and 1A1/2 were able to oxidize LDL, whereas little oxidation was seen with microsomes that were induced to express 3A2. We thus conclude that LDL can be oxidized by some cytochrome P450s and, as some of these enzymes are present in liver and in arterial wall, they may have a physio/pathological relevance to LDL oxidation and atherogenesis.

Animals↗