Biomedical subjects

M Aviram

Publications and source records attributed to M Aviram.

At least 55 records · Page 3Linked to original sources

PubMed1999

Angiotensin II atherogenicity in apolipoprotein E deficient mice is associated with increased cellular cholesterol biosynthesis.

Angiotensin II (Ang II) was shown to be an important risk factor for accelerated atherosclerosis. Inhibition of Ang II action on the arterial wall by blocking its production with angiotensin converting enzyme (ACE) inhibitors, or by blocking binding to its receptors on cells with antagonists was shown to attenuate atherogenesis in animal model of atherosclerosis. We questioned whether Ang II atherogenicity is related to a stimulatory effect of Ang II on macrophage cholesterol biosynthesis. Angiotensin II injected intraperitoneally once a day (0.1 ml of 10(-7) M per mouse) for a period of 30 days, to the apolipoprotein E deficient mice increased the atherosclerotic lesion area by 95% (P < 0.01 vs. control), compared to placebo-injected mice, with no significant effect on blood pressure or on plasma cholesterol levels. On using mouse peritoneal macrophages (MPMs) that were harvested after intraperitoneally injection of Ang II, an increased rate of cellular cholesterol biosynthesis (measured as incorporation of [3H]acetate into cholesterol) by up to 90% (P < 0.01 vs. control) was observed. In mice treated with the ACE inhibitor, Fosinopril (25 mg/kg per day) a reduction in their MPM's cholesterol synthesis by up to 70% (P < 0.01 vs. control) was obtained. In vitro studies in human monocyte-derived macrophages (HMDM), in MPMs from control BALB/c mice, and in J-774 A.1 macrophage-like cell line demonstrated up to 44, 34 and 30% stimulation of macrophage cholesterol biosynthesis, respectively, following cell incubation with 10(-7) M Ang II for 18 h at 37 degrees C. The stimulatory effect of Ang II on macrophage cholesterol biosynthesis could be related to its interaction with the macrophage AT1 receptor, as Losartan (10(-5) M), an AT1 blocker, but not PD 123319 (10(-5) M), an AT2 blocker, prevented the stimulatory effect on macrophage cholesterol synthesis. Furthermore, in cells that lack the AT1 receptor (RAW macrophages), Ang II did not increase cellular cholesterol synthesis. Ang II increased macrophage 3-hydroxy-3-methyl glutaryl CoA (HMG CoA) reductase mRNA levels in a dose dependent manner in J-774 A.1 macrophages and in MPM. Losartan, the AT1 receptor antagonist clearly attenuated this mRNA induction. We thus conclude that Ang II stimulation of macrophage cholesterol biosynthesis is related to its interaction with the AT1 receptor, followed by stimulation of macrophage HMG CoA reductase gene expression, which leads to increased cellular cholesterol biosynthesis, and can possibly result in macrophage cholesterol accumulation and foam cell formation.

Angiotensin II↗

PubMed1999

Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants.

Human serum paraoxonase (PON1) can protect low density lipoprotein (LDL) from oxidation induced by either copper ion or by the free radical generator azo bis amidinopropane hydrochloride (AAPH). During LDL oxidation in both of these systems, a time-dependent inactivation of PON arylesterase activity was observed. Oxidized LDL (Ox-LDL) produced by lipoprotein incubation with either copper ion or with AAPH, indeed inactivated PON arylesterase activity by up to 47% or 58%, respectively. Three possible mechanisms for PON inactivation during LDL oxidation were considered and investigated: copper ion binding to PON, free radical attack on PON, and/or the effect of lipoprotein-associated peroxides on the enzyme. As both residual copper ion and AAPH are present in the Ox-LDL preparations and could independently inactivate the enzyme, the effect of minimally oxidized (Ox-LDL produced by LDL storage in the air) on PON activity was also examined. Oxidized LDL, as well as oxidized palmitoyl arachidonoyl phosphatidylcholine (PAPC), lysophosphatidylcholine (LPC, which is produced during LDL oxidation by phospholipase A2-like activity), and oxidized cholesteryl arachidonate (Ox-CA), were all potent inactivators of PON arylesterase activity (PON activity was inhibited by 35%-61%). PON treatment with Ox-LDL (but not with native LDL), or with oxidized lipids, inhibited its arylesterase activity and also reduced the ability of the enzyme to protect LDL against oxidation. PON Arylesterase activity however was not inhibited when PON was pretreated with the sulfhydryl blocking agent, p-hydroxymercurybenzoate (PHMB). Similarly, on using recombinant PON in which the enzyme's only free sulfhydryl group at the position of cysteine-284 was mutated, no inactivation of the enzyme arylesterase activity by Ox-LDL could be shown. These results suggest that Ox-LDL inactivation of PON involves the interaction of oxidized lipids in Ox-LDL with the PON's free sulfhydryl group. Antioxidants such as the flavonoids glabridin or quercetin, when present during LDL oxidation in the presence of PON, reduced the amount of lipoprotein-associated lipid peroxides and preserved PON activities, including its ability to hydrolyze Ox-LDL cholesteryl linoleate hydroperoxides. We conclude that PON's ability to protect LDL against oxidation is accompanied by inactivation of the enzyme. PON inactivation results from an interaction between the enzyme free sulfhydryl group and oxidized lipids such as oxidized phospholipids, oxidized cholesteryl ester or lysophosphatidylcholine, which are formed during LDL oxidation. The action of antioxidants and PON on LDL during its oxidation can be of special benefit against atherosclerosis since these agents reduce the accumulation of Ox-LDL by a dual effect: i.e. prevention of its formation, and removal of Ox-LDL associated oxidized lipids which are generated during LDL oxidation.

Amidines↗

PubMed1999

Expression, purification, and biochemical characterization of SAG, a ring finger redox-sensitive protein.

We recently reported the cloning and characterization of SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, that is redox responsive and protects mammalian cells from apoptosis. Here we report the expression, purification, and biochemical characterization of SAG. Bacterially expressed SAG is brown in color and dithiothreitol (DTT)-sensitive. SAG forms large oligomers without DTT that can be reduced into a monomer in the presence of DTT. These features help us to purify SAG using the chromatography with or without DTT. Likewise, purified SAG is redox sensitive. Upon H2O2 exposure, SAG forms oligomers as well as monomer doublets due to the formation of the inter- or intramolecular disulfide bonds, respectively. This process can be reversed by DTT or prevented by pretreatment with the alkylating reagent, N-ethylmaleimide (NEM). Although SAG contains two putative heme-binding sites and a RING finger domain, the protein appears not to bind with heme and to lack transcription factor activity as determined in a Gal4-fusion/transactivation assay. Wildtype, but not RING finger domain-disrupted SAG mutants, prevents copper-induced lipid peroxidation. These results, along with our previous observations, suggest that SAG is an intracellular antioxidant molecule that may act as a redox sensor to buffer oxidative-stress induced damage.

Copper↗

PubMed1999

Does paraoxonase play a role in susceptibility to cardiovascular disease?

Human serum paraoxonase (PON1) is an esterase that is bound to high-density lipoproteins (HDLs). It can hydrolyze organophosphates and its activity is inversely related to atherosclerosis. Some studies also suggest that a relationship exists between polymorphisms of the gene that encodes paraoxonase and coronary heart disease (CHD), whereas other studies, in different populations, have not found such an association. One mechanism by which certain PON1 allozymes might protect against atherosclerosis is by inhibition of the oxidation of HDL and low-density lipoprotein (LDL). Experimental studies suggest that this protection is associated with the ability of PON1 to hydrolyze specific lipid peroxides in oxidized lipoproteins. Interventions that preserve or enhance PON1 activity, as well as manipulations of PON1 polymorphisms, might help delay the onset of CHD.

Animals↗

PubMed1999

Macrophage foam cell formation during early atherogenesis is determined by the balance between pro-oxidants and anti-oxidants in arterial cells and blood lipoproteins.

Atherosclerosis is a multifactorial disease, where more than one mechanism, along more than one step, contributes to macrophage cholesterol accumulation and foam cell formation, the hallmark of early atherogenesis. Arterial macrophages take up oxidized low-density lipoproteins (Ox-LDL), leading to cellular accumulation of cholesterol and oxysterols. Atherogenic modifications of LDL include, in addition to oxidation, retention and aggregation. Intervention to inhibit LDL oxidation can affect the above additional LDL modifications. Indeed, we have demonstrated in the atherosclerotic apolipoprotein E-deficient mice that consumption of vitamin E or of flavonoids from red wine or licorice decreased LDL oxidation, LDL retention, and LDL aggregation and attenuated macrophage foam cell formation and atherosclerosis. The balance between pro-oxidants and anti-oxidants in the LDL particle (such as cholesteryl ester vs. vitamin E), as well as in arterial wall macrophages (such as NADPH oxidase vs. glutathione), determines the extent of LDL oxidation. Antioxidants can protect LDL from oxidation not only by their binding to the lipoprotein, but also following their accumulation in cells of the arterial wall. Whereas antioxidants can prevent the formation of Ox-LDL, human serum paraoxonase (PON 1), an HDL-associated esterase that hydrolyzes organophosphates, can eliminate oxidized LDL (by hydrolysis of its lipid peroxides), which is formed when antioxidant protection is not sufficient. Ox-LDL, in turn, can inactivate paraoxonase activity. Thus, the combination of antioxidants together with active paraoxonase decreases the formation of Ox-LDL and preserves PON1's ability to hydrolyze this atherogenic lipoprotein and hence, to attenuate atherosclerosis.

Antioxidants↗

PubMed1999

SAG, a novel zinc RING finger protein that protects cells from apoptosis induced by redox agents.

SAG (sensitive to apoptosis gene) was cloned as an inducible gene by 1,10-phenanthroline (OP), a redox-sensitive compound and an apoptosis inducer. SAG encodes a novel zinc RING finger protein that consists of 113 amino acids with a calculated molecular mass of 12.6 kDa. SAG is highly conserved during evolution, with identities of 70% between human and Caenorhabditis elegans sequences and 55% between human and yeast sequences. In human tissues, SAG is ubiquitously expressed at high levels in skeletal muscles, heart, and testis. SAG is localized in both the cytoplasm and the nucleus of cells, and its gene was mapped to chromosome 3q22-24. Bacterially expressed and purified human SAG binds to zinc and copper metal ions and prevents lipid peroxidation induced by copper or a free radical generator. When overexpressed in several human cell lines, SAG protects cells from apoptosis induced by redox agents (the metal chelator OP and zinc or copper metal ions). Mechanistically, SAG appears to inhibit and/or delay metal ion-induced cytochrome c release and caspase activation. Thus, SAG is a cellular protective molecule that appears to act as an antioxidant to inhibit apoptosis induced by metal ions and reactive oxygen species.

Amino Acid Sequence↗

PubMed1999

9-cis retinoic acid induces monocyte chemoattractant protein-1 secretion in human monocytic THP-1 cells.

Monocyte migration and activation are regulated by monocyte chemoattractant protein-1 (MCP-1). Prior studies have shown MCP-1 expression is modulated by a variety of ligands that act through extracellular receptors. In the current study, we show 9-cis retinoic acid (RA), a ligand for the nuclear hormone receptor retinoid X receptor (RXR) and retinoic acid receptor (RAR), markedly induces the expression of MCP-1. In human THP-1 monocytic leukemia cells cultured with RA (0.05 to 500 nmol/L), MCP-1 expression was induced rapidly, significantly, and dose-dependently by as much as 165-fold. MCP-1 RNA level was also increased in RA-treated cells. Expression of PPARgamma, a heterodimer partner of RXR, is also markedly induced by RA in THP-1 cells. However, BRL49653, a PPARgamma ligand, failed to induce MCP-1 secretion either alone or to modify the expression level induced by RA. In contrast, BRL49653 significantly increased MCP-1 (biotinylated MCP-1) binding to THP-1 cells, whereas RA had no effect. Other peroxisome proliferator activated receptor (PPAR) ligands, 15d-PGJ(2) and troglitazone (PPARgamma), Wy14,643 (PPARalpha), and PD195599 (PPARbeta) inhibited the induction of MCP-1 by RA. RA's effect on MCP-1 expression in human elutriated monocytes were similar to that observed in the THP-1 cells. These studies identify RA as a nuclear signal for MCP-1 induction in undifferentiated human monocytic cells. These studies also suggest monocyte MCP-1 expression induced through RA may modulate cell migration.

Alitretinoin↗

PubMed1999

Human serum Paraoxonase/Arylesterase's retained hydrophobic N-terminal leader sequence associates with HDLs by binding phospholipids : apolipoprotein A-I stabilizes activity.

In serum, human paraoxonase/arylesterase (PON1) is found exclusively associated with high density lipoprotein (HDL) and contributes to its antiatherogenic properties by inhibiting low density lipoprotein (LDL) oxidation. Difficulties in purifying PON1 from apolipoprotein A-I (apoA-I) suggested that PON1's association with HDL may occur through a direct binding between these 2 proteins. An unusual property of PON1 is that the mature protein retains its hydrophobic N-terminal signal sequence. By expressing in vitro a mutant PON1 with a cleavable N-terminus, we demonstrate that PON1 associates with lipoproteins through its N-terminus by binding phospholipids directly rather than binding apoA-I. Nonetheless, apoA-I stabilized arylesterase activity more than did phospholipid alone, apoA-II, or apoE. Consequently, we studied the role of apoA-I in PON1 expression and HDL association in mice genetically deficient in apoA-I. Though present in HDL fractions at decreased levels, PON1 arylesterase activity was less stable than in control mice. Furthermore, PON1 could be competitively removed from HDL by phospholipids, suggesting that PON1's retained N-terminal peptide allows transfer of the enzyme between phospholipid surfaces. Thus, our data suggest that PON1 is stabilized by apoA-I, and its binding to HDL and physiological distribution are dependent on the direct binding of the retained hydrophobic N-terminus to phospholipids optimally presented in association with apoA-I.

Animals↗

PubMed1999

Oxidized low density lipoprotein: atherogenic and proinflammatory characteristics during macrophage foam cell formation. An inhibitory role for nutritional antioxidants and serum paraoxonase.

Oxidative stress and inflammatory processes are of major importance in atherogenesis because they stimulate oxidized LDL (Ox-LDL)-induced macrophage cholesterol accumulation and foam cell formation, the hallmark of early atherosclerosis. Under oxidative stress, both blood monocytes and plasma lipoproteins invade the arterial wall, where they are exposed to atherogenic modifications. Oxidative stress stimulates endothelial secretion of monocyte chemoattractant protein 1 (MCP-1) and of macrophage colony stimulating factor (M-CSF), leading to monocyte adhesion and differentiation, respectively. LDL binds to extracellular matrix (ECM secreted by endothelial cells, smooth muscle cells and macrophages) proteoglycans, in a process that contributes to the enhanced susceptibility of the lipoprotein to oxidation by arterial wall macrophages. ECM-retained Ox-LDL is taken up by activated macrophages via their scavenger receptors. This leads to cellular cholesterol accumulation and enhanced atherogenesis. Protection of LDL against oxidation by antioxidants that can act directly on the LDL, or indirectly on the cellular oxidative machinery, or conversion of Ox-LDL to a non-atherogenic particle by HDL-associated paraoxonase (PON-1), can contribute to attenuation of atherosclerosis.

Antioxidants↗

PubMed1999

Clinical and genetic risk factors for cystic fibrosis-related liver disease.

OBJECTIVE: The aim of this study was to define the role of possible risk factors for the development of cystic fibrosis (CF)-related liver disease and to analyze the association between liver disease and the different genotypes present in the Israeli CF patient population. PATIENTS AND METHODS: All patients followed at the seven CF centers in Israel were included in this study. Liver disease was determined by persistently elevated serum liver enzymes and/or bilirubin, and/or significant ultrasonographic changes suggestive of chronic liver disease. The following clinical parameters were evaluated: ethnic origin, age at assessment of liver function, sex, history of meconium ileus, pancreatic function, history of distal intestinal obstruction syndrome, pulmonary function, and cystic fibrosis transmembrane conductance regulator mutation analysis. RESULTS: Of the 288 patients screened, 80 (28%) had liver disease. Of the 256 patients with pancreatic insufficiency, 80 (31%) had liver disease compared with none of the 32 patients with pancreatic sufficiency. Genotype-phenotype correlation was performed on 207 patients carrying identified mutations that were previously classified according to phenotype severity. Liver disease was found in 56 (32%) of 173 patients carrying mutations associated with a severe phenotype and in 6 (38%) of 16 patients carrying at least one mutation associated with a variable genotype (G85E and/or 5T allele). None of the 18 patients carrying the 3849+10kb C->T mutation had liver disease. Prevalence of liver disease increased with age. No correlation was found between liver disease and severity of lung disease, nutritional status, history of meconium ileus, or distal intestinal obstruction syndrome. CONCLUSION: CF patients who have pancreatic insufficiency and carry mutations associated with a severe or a variable genotype are at increased risk to develop liver disease.

Adolescent↗

PubMed1999

Predicting deterioration in previously healthy infants hospitalized with respiratory syncytial virus infection.

OBJECTIVE: To estimate the incidence of clinical deterioration leading to intensive care unit transfer in previously healthy infants with respiratory syncytial virus (RSV) infection hospitalized on a general pediatric unit and, to assess the hypothesis that history, physical examination, oximetry, and chest radiographic findings at time of presentation can accurately identify these infants. STUDY DESIGN: A virology database was used to identify and determine the disposition of all children </=1 year of age admitted to the Children's Hospital at Strong (CHaS) with RSV infection during the 1985 to 1994 respiratory seasons. Index patients were all previously healthy, full-term infants admitted initially to the general inpatient services at CHaS or Rochester General Hospital, a second University of Rochester teaching hospital, whose clinical deterioration led to transfer to the pediatric intensive care unit (PICU). These infants were matched retrospectively (for year and date of infection, sex, chronologic age, and race) with two hospitalized controls who did not require PICU transfer. Chest radiographic findings, respiratory rate (RR), O(2) saturation, and presence of wheezing at time of presentation to the emergency department (ED) were compared. RESULTS: During the study years, 542 previously healthy, full-term infants were admitted to the general pediatric unit at CHaS with proven RSV infection. Ten (1.8%; 95% confidence interval, 0.9%, 3.4%) were transferred subsequently to the PICU, primarily for close monitoring of progressive respiratory distress. Data for these patients and 7 patients transferred from Rochester General Hospital to the PICU at the CHaS were compared with those for control patients. The mean RR in the ED (63 vs 50), and O(2) saturation in the ED (88% vs 93%) were modestly abnormal in cases compared with controls. Wheezing on examination at time of presentation and chest radiographic findings did not differ between the two groups. A RR >80 and an O(2) saturation <85% at time of presentation each had a specificity >97% for predicting subsequent deterioration. Each parameter, however, had a sensitivity </=30%. CONCLUSION: Clinical deterioration requiring PICU admission is an uncommon occurrence in previously healthy infants admitted to a general pediatric inpatient unit with RSV infection. Extreme tachypnea and hypoxemia were both associated with subsequent deterioration; however, only a small proportion of patients who clinically deteriorated presented in this way. The clinical usefulness of these parameters, therefore, is limited. respiratory syncytial virus, deterioration, healthy infants, prediction.

Case-Control Studies↗

PubMed1999

Increased plasma oxidizability and decreased erythrocyte and plasma antioxidative capacity in patients with NIDDM.

BACKGROUND: Atherosclerosis and microvascular complications in patients with non-insulin-dependent diabetes have been linked to increased oxidative stress. The glutathione redox cycle is a major determinant of the antioxidative capacity of plasma and its constituents. METHODS: We attempted to investigate plasma oxidation and plasma and erythrocyte glutathione and glutathione enzymes in 20 patients with NIDDM, compared with euglycemic matched controls. Plasma oxidation was analyzed both basally (without) and as induced by 2,2'-azobis,2-amidopropane hydrochloride measured by the generation of thiobarbituric acid reactive substances and lipid peroxides. RESULTS: There was a significant increase in oxidation both basally (without) and as induced by AAPH. Plasma glutathione was lowered by 50% (P < 0.01) and erythrocyte glutathione peroxidase, glutathione s-transferase and glutathione reductase activities were lower by 30%, 27% and 46%, respectively (P < 0.01) in the patients with NIDDM. CONCLUSIONS: Confronted by increased oxidation, patients with NIDDM show an abnormal plasma and erythrocyte antioxidative capacity, which may result in an accelerated rate of complications.

Adult↗

PubMed1998

The molecular basis of disease variability among cystic fibrosis patients carrying the 3849+10 kb C-->T mutation.

Disease severity varies among cystic fibrosis (CF) patients carrying the same CFTR genotype. Here we studied the mechanism underlying disease variability in individuals carrying a splicing CFTR mutation, 3849+10 kb C-->T. This mutation was shown to produce both correctly and aberrantly spliced CFTR transcripts containing an additional cryptic exon. Semiquantitative nondifferential RT-PCR showed considerable variability in the level (0-28%) of aberrantly spliced RNA transcribed from the 3849+10 kb C-->T mutation in nasal epithelium from 10 patients. A significant inverse correlation was found between the level of the aberrantly spliced CFTR transcripts and pulmonary function, expressed as FEV1 (r = 0.92, P < 0.0001). Patients with normal pulmonary function (FEV1 > 80% predicted) had lower levels of aberrantly spliced CFTR RNA (0 to 3%) than those with FEV1 < 80%, (9 to 28% aberrantly spliced RNA). Only aberrantly spliced CFTR RNA was detected in the lung of a patient with severe lung disease who underwent lung transplantation. Our results show that the severity of CF lung disease correlates with insufficiency of normal CFTR RNA. Thus, the regulation of alternative splice site selection may be an important mechanism underlying partial penetrance in CF. Further understanding of this regulation will contribute to potential therapy for patients carrying splicing mutations in human disease genes.

Adolescent↗

PubMed1998

Induction of PPARgamma1 expression in human THP-1 monocytic leukemia cells by 9-cis-retinoic acid is associated with cellular growth suppression.

9-cis-Retinoic acid (RA) and peroxisome proliferator activated receptor gamma (PPARgamma) regulates cellular growth and differentiation. In THP-1 cells, a human monocytic leukemia cell line, RA markedly induced PPARgamma1 RNA, nuclear PPARgamma1 protein and suppressed cell growth. The PPARgamma ligand, BRL49653 enhanced RA's growth suppression ability. With BRL49653 alone, THP-1 cell growth was only marginally suppressed. Cell cycle analysis revealed the G1 phase cell population was significantly increased when cells were treated with both ligands. RA induced growth suppression did not differentiate the THP-1 cells to macrophages. Phorbol ester (PMA) induced differentiation of cells to macrophage also induced PPARgamma1 expression, however when RA is given either simultaneously or sequentially to these cells, no further increase in expression of the nuclear receptor was observed. Overall, these data suggest RA induction of PPARgamma1 may block cell growth and may have application for the treatment of proliferative diseases.

Alitretinoin↗

PubMed1998

Plasmalogen phospholipids are involved in HDL-mediated cholesterol efflux: insights from investigations with plasmalogen-deficient cells.

Plasmalogens are ether-glycerophospholipids that exist in all mammalian cells, but their physiological function remains thus far an enigma. It has been previously suggested that the association of high-density lipoprotein (HDL) with cellular phospholipid is a pre-requisite for the process of HDL-mediated cholesterol efflux (HDL-MCE). To investigate our hypothesis that plasmalogens might play a role in HDL-MCE, we used a model composed of plasmalogen-deficient cells including RAW mutant macrophages and fibroblasts from patients with rhizomelic chondrodysplasia punctata type II. In mutant macrophages, HDL-MCE was reduced by 57% compared to control macrophages, after 16 hours. A similar phenomenon was observed in plasmalogen-deficient patients fibroblasts. Incubation of plasmalogen-deficient fibroblasts with 1-0-hexadecyl-sn-glycerol, which restored plasmalogen levels to that of control cells, resulted in a 35% increase in HDL-MCE, compared to a 10% increment in controls. The novel finding that HDL-MCE is reduced in plasmalogen-deficient cells and increases following plasmalogen restoration leads us to suggest that plasmalogen has an important function in the mediation of cellular cholesterol efflux.

Animals↗

PubMed1998

Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.

HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.

Animals↗

PubMed1998

Macrophage glutathione content and glutathione peroxidase activity are inversely related to cell-mediated oxidation of LDL: in vitro and in vivo studies.

Macrophage-mediated oxidation of low-density lipoprotein (LDL) is thought to play a key role during early atherogenesis, and cellular oxygenases were shown to mediate this process. As macrophage antioxidants may also contribute to the extent of cell-mediated oxidation of LDL, we analyzed the role of cellular reduced glutathione (GSH) and glutathione peroxidase (GPx) in LDL oxidation. The present study examined the effect of the macrophage GSH-GPx status on the ability of the cells to oxidize LDL. Upon incubation of J-774 A.1 macrophages for 20 h at 37 degrees C with 50 microM of buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, cellular GSH content and GPx activity were reduced by 89 and 50%, respectively, and this effect was associated with a twofold elevation in macrophage-mediated oxidation of LDL. The BSO-treated cells contained high levels of peroxides, and released 32% more superoxide anions than nontreated cells in response to their stimulation with LDL in the presence of copper ions. To increase macrophage GSH content and GPx activity we have used L-2-oxothiazolidine-4-carboxylic acid (OTC), which delivers cysteine residues to the cells for GSH synthesis, and also selenium, which activates GPx and increases cellular glutathione synthesis. GSH content and GPx activity in J-774 A.1 macrophages were increased by 80 and 50%, respectively, following cells incubation with 2 mM OTC for 20 h at 37 degrees C, and this was paralleled by a 47% inhibition in LDL oxidation by these cells. An inverse correlation was found between the extent of macrophage-mediated oxidation of LDL and cellular GSH content (r = .97), or GPx activity (r = .95). Upon incubation of J-774 A.1 macrophages with selenomethionine (10 ng/ml) for 1 week, cellular GSH content and GPx activity were increased by about twofold compared to control cells, and this effect was associated with a 30% reduction in cell-mediated oxidation of LDL. Dietary selenium supplementation (1 microg/d/mouse) to the atherosclerotic apolipoprotein E-deficient mice for a 6-month period, increased GSH content and GPx activity in the mice peritoneal macrophages by 36 and 30%, respectively, and this effect was associated with a 46% reduction in cell-mediated oxidation of LDL. Finally, the atherosclerotic lesion area in the aortas derived from these mice after selenium supplementation was found to be reduced by 30% compared to the lesion area found in nontreated mice. Our results demonstrate an inverse relationship between macrophage GSH content/GPx activity and cell-mediated oxidation of LDL. Intervention means to enhance the macrophage GSH-GPx status may thus contribute to attenuation of the atherosclerotic process.

Animals↗

PubMed1998

The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation.

The effect of the consumption of glabridin, an isoflavan isolated from Glycyrrhiza glabra (licorice) root, on the susceptibility of low density lipoprotein (LDL) to oxidation was studied in atherosclerotic apolipoprotein E deficient (E[o] mice) and was compared with that of the known flavonoids, quercetin and catechin. Glabridin inhibitory activity on in vitro oxidation of human LDL was also investigated by determining the formation of lipid peroxides and oxysterols and the consumption of LDL-associated lipophilic antioxidants. Determination of the extent of LDL oxidation by measuring the formation of thiobabituric acid reactive substances (TBARS) after 2 h of LDL incubation with CuSO4 (10 microM) or 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) (5 mM), revealed that glabridin or quercetin consumption resulted in a 53 and 54% reduction in copper ion induced oxidation, respectively, and a 95 and 83% reduction in AAPH induced LDL oxidation, respectively. No inhibition was obtained with consumption of catechin. About 80% of glabridin was found to bind to the LDL human particle. In the in vitro oxidation of LDL induced by AAPH (5 mM), glabridin inhibited the formation of TBARS, lipid peroxides and cholesteryl linoleate hydroperoxide (CLOOH) at all the concentrations tested (5-60 microM), while in oxidation induced by copper ions (10 microM), glabridin exhibited a pro-oxidant activity at concentrations lower than 20 microM, and a clear antioxidant activity at concentrations greater than 20 microM. Glabridin (30 microM) inhibited the formation of cholest-5-ene-3,7-diol (7-hydroxycholesterol), cholest-5-ene-3-ol-7-one (7-ketocholesterol) and cholestan-5,6-epoxy-3-ol (5,6-epoxycholesterol) after 6 h of AAPH induced LDL oxidation, by 55, 80 and 40%, respectively, and after 6 h of copper ion induced LDL oxidation, by 73, 94 and 52%, respectively. Glabridin also inhibited the consumption of beta-carotene and lycopene by 38 and 52%, respectively, after 0.5 h of LDL oxidation with AAPH, but failed to protect vitamin E. The in vivo and in vitro reduction of the susceptibility of LDL to oxidation obtained with glabridin, may be related to the absorption or binding of glabridin to the LDL particle and subsequent protection of LDL from oxidation by inhibiting the formation of lipid peroxides and oxysterols, and by protecting LDL associated carotenoids.

Amidines↗