Macrophage Enrichment with the Isoflavan Glabridin Inhibits
NADPH Oxidase-induced Cell-mediated Oxidation of Low Density
Lipoprotein
A POSSIBLE ROLE FOR PROTEIN KINASE C*
Mira
Rosenblat
,
Paula
Belinky§,
Jacob
Vaya§,
Rachel
Levy¶,
Tony
Hayek
,
Raymond
Coleman
,
Shoshana
Merchav**, and
Michael
Aviram

From the
Lipid Research Laboratory, the
** Haemopoiesis Unit, Department of Anatomy and Cell Biology, and the
Division of Morphological Sciences, Technion Faculty of
Medicine, The Rappaport Family Institute for Research in the Medical
Sciences and Rambam Medical Center, Haifa, Israel, the
§ Laboratory of Natural Compounds for Medical Use, Migal,
Galilee Technological Center, Kiryat Shmona, Israel, the
¶ Laboratory of Infectious Diseases and Clinical Biochemistry
Department, Faculty of Health Sciences, Soroka Medical Center, Ben
Gurion University of the Negev, Beer-Sheva, Israel
 |
ABSTRACT |
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 µg 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 ~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 µg/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.
 |
INTRODUCTION |
The LDL1 oxidation
hypothesis of atherosclerosis is supported by evidence for the
accumulation of oxidized LDL in the atherosclerotic lesion, by
increased LDL oxidizability in patients with increased risk for
atherosclerosis, and by the anti-atherogenicity of several potent
antioxidants against LDL oxidation (1-4). Extensive investigation is
being made to identify natural food products that can offer antioxidant
defense against LDL oxidation. Flavonoids are polyphenolic compounds
naturally present in fruits and vegetables and are an integral part of
the human diet (5). Consumption of flavonoids in the diet was shown to
be inversely associated with morbidity and mortality from coronary
heart disease (6, 7). Flavonoids are powerful antioxidants and their
antioxidative capacity is related to their chemical structure (8, 9).
We have recently shown that dietary supplementation of healthy human
volunteers with flavonoid-rich nutrients such as olive oil, red wine,
or licorice root, resulted in a reduction in LDL oxidizability
(10-12). Furthermore, consumption of red wine or its flavonol
quercetin or of licorice extract by atherosclerotic apolipoprotein
E-deficient (E0) mice resulted in a significant reduction
in the development of atherosclerotic lesions, along with a reduction
in their LDL oxidation (12, 13). We have also demonstrated that the
isoflavan glabridin binds to the LDL particle and substantially
inhibits its oxidation (12, 14-16). The hydroxyl groups on the
glabridin B ring were found to be most important for its antioxidative
properties (16). In vivo, antioxidants can bind to, and
accumulate in, cells, including arterial wall macrophages.
Macrophage-mediated oxidation of LDL is considered to be of major
importance during early atherogenesis, and the formation of oxidized
LDL can then induce cellular cholesterol accumulation and foam cell
formation (17). Macrophage-mediated oxidation of LDL is affected by the balance between cellular oxygenases (18, 19) and antioxidants, such as
the glutathione system (20). We have shown activation of the macrophage
NADPH oxidase following binding of LDL to its receptor under oxidative
stress (21), leading to LDL oxidation. Recently, this enzyme was shown
to be also present in the arterial wall adventitia (22). Several
studies have shown that flavonoids attenuate cell-mediated oxidation of
LDL when added to the extracellular medium (23-26). Under these
conditions, it is not clear whether the antioxidants exert their effect
within the lipoprotein particle, in the medium, or at the cellular
level. Only few studies examined the capability of macrophages enriched
with vitamin E to oxidize LDL (27, 28). In one study, it has been
reported that loading J-774 A.1 macrophages with high concentrations of
vitamin E can protect LDL against macrophage-mediated oxidation (27).
In another study, when MPMs or human monocytes were incubated with
vitamin E at a more physiological concentration, no effect on
cell-mediated oxidation of LDL was obtained (28). Flavonoids not only
can act as antioxidants, but they can also inhibit some cellular
enzymes (29-35). Since some of these enzymes are involved in
macrophage-mediated oxidation of LDL, the present study analyzed the
effect (and mechanisms) of macrophage enrichment with the isoflavan
glabridin on the capability of these cells to oxidize LDL.
 |
EXPERIMENTAL PROCEDURES |
Materials
EGTA, ATP, phenylmethanesulfonyl fluoride, leupeptin, cytochrome
c from horse heart, phorbol 12-myristate 13-acetate (PMA), FAD,
-NADPH, superoxide dismutase from bovine erythrocytes, SDS, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7),
calphostin C, staurosporine, and
N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride
were all purchased from Sigma.
Cells and Cell Fractionation
The J-774 A.1 murine macrophage-like cell line was purchased
from the American Type Culture Collection (ATCC, Rockville, MD). The
macrophages were plated at 1 × 106 cells/35-mm dish
in Dulbecco's modified Eagle's medium supplemented with 5%
heat-inactivated fetal calf serum. The cells were fed every 3 days
(36). Mouse peritoneal macrophages (MPM) were harvested from the
peritoneal fluid 4 days after intraperitoneal injection of 3 ml of
thioglycolate in saline (24 g/mouse) (37).
The cells (10-20 × 106/mouse) were washed and
centrifuged three times with phosphate-buffered saline (PBS) at
1000 × g for 10 min. Then the cells were resuspended
to 106/ml in Dulbecco's modified Eagle's medium
containing 10% horse serum (heat-inactivated at 56 °C of 30 min),
100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM
of glutamine. The cell suspension was dispensed into 35-mm plastic
Petri dishes and incubated in a humidified incubator (5%
CO2, 95% air) for 2 h. The dishes were washed once
with 5 ml of Dulbecco's modified Eagle's medium to remove nonadherent
cells, and the monolayer was further incubated under similar conditions
for 18 h prior to the beginning of the experiment. In some
experiments, lactic dehydrogenase release to the incubation medium was
measured in order to determine cell viability. Lactic dehydrogenase
release into the medium was quantitated over time by spectrophotometric
measurement of the consumption of NADH, using a commercially available
kit (Merck LDH, Merck, Darmstadt, Germany).
Membrane and cytosol fractions were prepared as described previously
(38). Cells were suspended at 108 cells/ml in relaxation
buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, 1 mM ATP, 10 mM Hepes, pH 7.4) containing 1 mM phenylmethanesulfonyl fluoride and 100 µM
leupeptin at 4 °C, following sonication for 3 × 10 s,
resulting in about 95% of cell breakage. Nuclei, granules, and
unbroken cells were removed by centrifugation (2 min, 15,600 × g), and the postnuclear supernatant was made in 5 mM EDTA, 1 mM Na3VO4
and 5 mM NaF. The supernatant was centrifuged in a Beckman
Airfuge (30 min, 134,000 × g) to obtain a cell
membrane pellet and a cytosolic supernatant. Membranes were suspended
at 109 cell equivalents/ml in 0.34 M
sucrose/half-strength relaxation buffer containing 1 mM
dithiothreitol. Solubilized membrane and cytosolic fractions were
stored at
70 °C.
Lipoproteins
Plasma LDL was derived from fasted normolipidemic volunteers.
LDL was prepared by discontinuous density gradient ultracentrifugation as described previously (39). The lipoprotein was washed at d = 1.063 g/ml and dialyzed against 150 mM
NaCI, 1 mM EDTA (pH 7.4) at 4 °C. LDL was then
sterilized by filtration (0.45 µm), kept at 3-6 mg of protein/ml
under nitrogen in the dark at 4 °C and used within 2 weeks. Prior to
the oxidation studies, LDL was dialyzed against PBS, EDTA-free
solution, pH 7.4, under nitrogen at 4 °C. The lipoprotein was found
to be free of lipopolysaccaride contamination as analyzed by the
Limulus Amebocyte Lysate assay (Associated of Cape Cod Inc., Woods
Hole, MA). The LDL used for all experiments was from different batches.
The variation in LDL oxidizability among batches did not exceeded
10%.
LDL Oxidation by Macrophages
Cells (1 × 106/35-mm dish) were incubated with
LDL (100 µg of protein/ml) in Ham's F-10 medium in the presence of 2 µM CuSO4. LDL was also incubated under
similar conditions in the absence of cells. The extent of LDL oxidation
was measured directly in the medium (after centrifugation at 1000 × g for 10 min), by the thiobarbituric acid-reactive
substances assay, using malondialdehyde for the standard curve
preparation (40). Macrophage-mediated oxidation of LDL was calculated
by subtraction of the oxidation rate in the absence of cells from that
obtained in the presence of macrophages.
Glabridin and 2',4'-O-Dimethylglabridin: Preparation and
Cellular Content
Preparation--
Glabridin was isolated from the acetone extract
of the roots of Glycyrrhiza glabra (the licorice plant) and
purified on silica gel column chromatography (14). Glabridin (100 mg,
0.31 mmol) was dissolved in acetone (4 ml) in a round bottom flask, and
methyl iodide (240 µl, 2.64 mmol) and K2CO3
(384 mg, 2.76 mmol) were added. The reaction mixture was heated to
50 °C with stirring and, after 8 h, was filtered, and the
solvent was evaporated. The residue was chromatographed on a silica gel
column, using CH2Cl2/hexane (4:1, v/v) and then
CH2Cl2 alone as eluents, to afford
2',4'-O-dimethylglabridin (70.6 mg, 65% yield), which was identified as described before (16). No attempt was made to optimize
the yield. The purity of the isolated and synthesized compounds used in
this study was above 98%, as determined by high performance liquid
chromatography (HPLC) using RP-18 column (Merck, Darmstadt, Germany;
25-cm length, 0.4-cm diameter, 5-µm particle size). The analysis was
performed by detecting the absorbance of the compounds at 230 nm, using
acetonitrile/water as eluents (16).
Glabridin Cellular Content Determination--
Glabridin and
2',4'-O-dimethylglabridin were dissolved in 100% ethanol to
stock solutions of 10 mM. Glabridin and
2',4'-O-dimethylglabridin were incubated with the cells in
Dulbecco's modified Eagle's medium containing 5% fetal calf serum.
The ethanol concentration added to the cells did not exceed 0.2%. At
the end of the incubation period, all preparation steps were performed
at 4 °C. The macrophages were washed three times with PBS, and then
the cells were scraped from the dish into 1 ml of PBS, followed by
sonication twice for 20 s each time at 80 watts. Cellular protein
content was determined using the method of Lowry et al.
(41). The glabridin and 2',4'-O-dimethylglabridin were
extracted from the sonicat with hexane/isopropyl alcohol (3:2, v/v).
The upper hexane phase from triplicate samples was collected and dried
under nitrogen. The amounts of glabridin and 2',4'-O-dimethylglabridin in the dried samples were analyzed
by HPLC using the appropriate standards (16).
Superoxide Generation by Macrophages
The production of the superoxide anion (O
2) by mouse
peritoneal macrophages was measured as the superoxide
dismutase-inhibitable reduction of acetyl ferricytochrome C by the
microtiter plate technique as described previously (42). Cells (2 × 105/well) were suspended in 100 µl of Hanks' balanced
salts solution containing acetyl ferricytochrome c (150 µM). Superoxide production by the cells was stimulated by
the addition of PMA (50 ng/ml) or LDL (100 µg of protein/ml) in the
presence of 2 µM CuSO4. The reduction of
acetyl ferricytochrome c was followed by the change in
absorbance at 550 nm between 2- and 5-min intervals on a Thermomax Microplate Reader (Molecular Devices, Menlo Park, CA). The maximal rates of superoxide generation were determined and expressed as nmol of
superoxides/106 cells/10 min using the extinction
coefficient of E550 = 21 mmol/liter
1 cm
1.
Cell Free Superoxide Generation Assay--
Superoxide generation
was measured as described previously (43). Membranes and cytosol were
prepared from control MPM or from MPM that were incubated for 2 h
with 20 µM of glabridin. Membranes were solubilized in a
buffer containing sodium deoxycholate (1.16%), glycerol (50%),
NaN3 (1 mM), CaCl2 (1.2 mM), and sodium glycine (20 mM), pH 7.0. The
final concentration of solubilized membranes was adjusted to 4 × 108 cell equivalents/ml. 0.5 µl of the membrane solution
(2 × 105 cell equivalents) and 10 µl of cytosol
(2 × 105 cell equivalents), at a final volume of 100 µl of reaction mixture, were added to wells of a chilled microtiter
plate. The reaction solution contained the following: acetyl
ferricytochrome c (150 µM), MgCl2
(4 mM), FAD (10 µM), EGTA (1 mM),
NADPH (200 µM), and SDS (100 µM) in 75 mM potassium phosphate, pH 7.0. Control wells also
contained superoxide dismutase (2.5 mg). The reaction was monitored at
22 °C. Light absorbance at 550 nm was determined at 2-min intervals.
Translocation of the NADPH Oxidase P-47 Cytosolic Component
to the Plasma Membrane
Immunoblot detection of cytosolic NADPH oxidase components was
performed as described (42). Samples were solubilized in 2× sample
buffer (12% SDS, 8 M urea, 250 mM Tris base, 8 mM EDTA, 0.2 mM leupeptin, 2 mM
phenylmethanesulfonyl fluoride, pH 6.9). The amount of protein in each
sample was quantified by the Pierce BCA protein assay with bovine serum
albumin as a standard. Cytosols or membranes were analyzed by
polyacrylamide gel electrophoresis. The resolved proteins were
electrophoretically transferred to nitrocellulose, which was stained
with Fast Green to detect protein banding, and then blocked with 5%
(w/v) nonfat dry milk in Tris-buffered saline (pH 7.4). The blots were
incubated for 1 h at room temperature in Tris-buffered saline,
0.01% gelatin containing goat antiserum to P-47 diluted 1:200 (a
generous gift of Dr. Leto, National Institutes of Health, Bethesda,
MD). Immunoblots were incubated with 1 µg/ml peroxidase-conjugated
rabbit anti-goat serum (Sigma) and developed by the ECL method.
Protein Kinase C and Protein Kinase A Activities
The activities of protein kinase C (PKC) and protein kinase A
were measured in the cytosolic or membrane fractions obtained from
control and from glabridin-enriched macrophages using the MESACUP
protein kinase assay kit, (PanVera Corp.).
Mouse Studies
Apolipoprotein E-deficient mice were generously provided to us
by Dr. Jan Breslow (Rockefeller University, New York). Gene targeting
in mouse embryonic stem cells was used to create these mice that lack
apolipoprotein E (44). The mice (15 mice in each group) were fed with
glabridin or 2',4'-O-dimethylglabridin in their drinking
water (20 µg/day/mouse). At the end of glabridin feeding, MPM were
harvested from the peritoneal fluid (after thioglycolate injection) for
analyses of cellular glabridin content and MPM-mediated oxidation of
LDL. Then the mice were sacrificed, and their hearts and entire aortas
were dissected and fixed in 3% glutaraldehyde in 0.1 M
sodium cacodylate buffer, pH 7.4, with 0.01% calcium chloride.
Following overnight fixation, the aortic arch was dissected free from
the surrounding fatty tissue under a binocular stereomicroscope, and
the first 4 mm of the ascending aorta (beginning with the aortic
valves) was removed and cut transversely with razor blades into four
blocks of approximately 1 mm each. The samples were then rinsed and
stored in 0.1 M sodium cacodylate buffer containing 7.5%
(w/v) sucrose prior to treatment with an unbuffered 1% aqueous solution of osmium tetroxide for 2 h. This was followed by a
cacodylate rinse and dehydration in ascending ethanols, prior to
propylene oxide and embedding in epoxy resin (Eponate 12; Pelco
International, Redding, CA). The blocks were orientated so that
transverse sections of the aorta could be cut. After heat
polymerization (18 h) at 60 °C, the blocks were trimmed, and 1-µm
transverse sections cut with glass or diamond knives on an LKB Nova
ultramicrotome (LKB, Bromma, Sweden). The sections were mounted on
glass slides and stained with 0.1% toluidine blue in 1% borax (sodium
tetraborate). In these toluidine blue-stained sections, the lipid
deposits appear a green color and are easily visualized in the
sections. When sufficient semithin sections were sampled from all of
the blocks, the remainder of the blocks were then cut into much thicker
sections (150-200 µm) for more macroscopic observation. The lipid
content of the lesions of these thicker sections was stained in intense black from the prolonged osmium treatment and permitted lesion areas to
be easily determined histomorphometrically.
Only the area of the aortic arch was examined as previous and on-going
studies by us and other groups have shown that this area is especially
prone to atherosclerosis in the ApoE gene knockout mice, and the area
is well defined with a clear starting point (aortic valves).
Histology and Histomorphometry--
Histomorphometric
determinations of lesion size were performed using an Olympus Cue-2
image analysis system with appropriate morphometry software (Olympus
Corp., Lake Success, NY). The system consists of a Zeiss Universal R
photomicroscope (× 10 objective) fitted with a Panasonic WV-CD50
camera with the video image seen on a Sony 14-inch color monitor and an
IBM-compatible PC. Measurements were made in standardized "windows"
(fields) with an area of 176,758 µm2.
Lesion Oxysterol Content--
Oxysterols were extracted from the
aortas derived from the mice after their homogenization, by diethyl
ether (3:1, v/v). The amount of total oxysterols (esterified and
unesterified) was analyzed by gas chromatography-mass spectrometry,
using the appropriate standards (15).
Statistical Analyses
Student's t test was used to analyze the
significance of the results in all comparisons of treated
versus untreated cells. Results are given as means ± S.D.
 |
RESULTS |
We have recently shown that the licorice-derived isoflavan
glabridin binds to LDL and substantially inhibits the oxidation of the
lipoprotein, secondary to its ability to scavenge free radicals (12,
14-16). Consumption of flavonoids can affect LDL oxidation not only by
a direct interaction of the flavonoids with the lipoprotein, but also
secondary to their accumulation in cells such as arterial wall
macrophages. In the present study, we analyzed the macrophage
capability to take up glabridin and studied the mechanisms by which
cellular accumulation of glabridin can affect macrophage-mediated
oxidation of LDL.
Macrophage Enrichment with Glabridin and Cell-mediated Oxidation of
LDL--
Upon incubation of macrophages (J-774 A.1 cell line or MPMs)
with glabridin (20 µM) for 20 h at 37 °C,
cellular glabridin content increased from 0 to 1.8 ± 0.2 µg of
glabridin/mg of cell protein (n = 3).
Incubation of MPMs or J-774 A.1 macrophages with LDL in the presence of
2 µM CuSO4 for 5 h at 37 °C resulted
in a 90% or 82% inhibition of LDL oxidation, respectively, by the
glabridin-enriched macrophages in comparison with the control cells
(Table I). Extension of the oxidation
period up to 20 h resulted in only 65% and 48% inhibition of LDL
oxidation by glabridin-enriched MPMs and J-774 A.1 cells, respectively,
in comparison with the control cells (Table I).
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Table I
The effect of macrophage enrichment with glabridin on cell-mediated
oxidation of LDL: time study of LDL incubation with macrophages
The J-774A.1 macrophage-like cell line or MPMs were plated (2 × 106 cells/35-mm dish) and incubated with glabridin (20 µM in ethanol, with glabridin) or with 0.2% ethanol
(control cells) for 20 h at 37 °C. The cells were then washed
(three times) in PBS and further incubated with LDL (100 µg of
protein/ml) in the presence of 2 µM CuSO4 in
Ham's F-10 medium for the indicated time periods (5 or 20 h). LDL
was similarly incubated in a cell-free system in order to obtain
cell-mediated oxidation of LDL as described under "Experimental
Procedures." The extent of LDL oxidation was measured by the
thiobarbituric acid-reactive substances assay. Results represent
mean ± S.D. (n = 5).
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In all of the following experiments, we have used 5 h for the
studies of LDL oxidation by macrophages. To find out the time required
for cell incubation with glabridin in order to reach a substantial cell
enrichment with this isoflavan (which can result in a significant
inhibition of macrophage-mediated oxidation of LDL), incubation time
and glabridin concentration studies were carried out. Fig.
1 demonstrates a
time-dependent increment in cellular accumulation of
glabridin by both MPMs and J-774 A.1 macrophages with a major effect
obtained already after 30 min of incubation (Fig. 1A). In
parallel, cell-mediated oxidation of LDL was substantially inhibited by
~80% at this time period (Fig. 1B). After 2 h of
incubation, there was about 90% inhibition in macrophage-mediated
oxidation of LDL in both cell types (Fig. 1B). Glabridin was
not cytotoxic to the cells, since no significant effect on cell count
or on the release of lactic dehydrogenase could be shown (25 ± 1 unit/liter in control cells and 27 ± 2 units/liter in
glabridin-treated cells). Cell fractionation revealed that about 60%
of the glabridin was localized in the macrophage membrane, and the rest
was in the cytosol (data not shown). Next, we analyzed the effect of
glabridin concentrations (0-20 µM) on macrophage-mediated oxidation of LDL after 2 h of cell incubation with glabridin at 37 °C, followed by 5 h of cell incubation
with LDL in the presence of copper ions. A glabridin
dose-dependent stimulatory effect on cellular glabridin
content was found, demonstrating a linearity between 5 and 20 µM of glabridin (Fig. 1C). In parallel, macrophage-mediated oxidation of LDL was reduced up to 90%, in comparison with control cells (Fig. 1D), and this effect was
dependent on cellular glabridin content.

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Fig. 1.
The effect of macrophage incubation with
glabridin on its cellular accumulation and on cell-mediated oxidation
of LDL: time and glabridin concentration studies. J-774 A.1
macrophages or MPMs were incubated with 20 µM of
glabridin for 10, 30, or 120 min (A and B) or
with increasing concentrations of glabridin (0-20 µM)
for 2 h at 37 °C (C and D). A
and C, at the end of the incubation period, the cells were
washed, and cellular glabridin content was analyzed by HPLC, as
described under "Experimental Procedures." B and
D, the cells were washed and further incubated with LDL (100 µg of protein/ml) in the presence of 2 µM
CuSO4 for 5 h at 37 °C. The extent of LDL oxidation
was measured in the incubation medium by the thiobarbituric
acid-reactive substances assay. Results represent mean ± S.D.
(n = 3).
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To find out the structural requirements for glabridin to act as a
potent inhibitor of cell-mediated oxidation of LDL, we compared the
effect of native glabridin to that of
2',4'-O-dimethylglabridin where the glabridin ring B
hydroxyl residues were blocked by methyl groups (Fig.
2A). Recently, we have shown
that unlike the potent inhibitory effect of glabridin on LDL oxidation,
the above modified glabridin completely lost its antioxidant activity
(16). Upon incubation of MPM with 20 µM of native
glabridin or with 2',4'-O-dimethylglabridin for 2 h at
37 °C, the macrophages accumulated similar amounts of these
compounds (Fig. 2B). Analysis of macrophage-mediated oxidation of LDL, after 5 h of incubation, revealed that in
comparison with a 90% inhibition of LDL oxidation by the
glabridin-enriched cells, no inhibition of cell-mediated oxidation of
LDL was obtained by the modified glabridin-enriched cells (Fig.
2C).

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Fig. 2.
Effect of the glabridin B ring hydroxyl
groups on its cellular accumulation and on cell-mediated oxidation of
LDL by mouse peritoneal macrophages. MPM were incubated with 0.2%
ethanol (Control), with 20 µM of glabridin, or
with 20 µM of 2',4'-O-dimethylglabridin for
2 h at 37 °C. A, molecular structures of glabridin
and 2',4'-O-dimethylglabridin. B, at the end of
the incubation period, the cells were washed, and cellular content of
glabridin and of 2',4'-O-dimethylglabridin were determined
by HPLC. C, the cells were washed and further incubated with
LDL (100 µg of protein/ml) in the presence of 2 µM
CuSO4 for 5 h at 37 °C. Cell-mediated oxidation of
LDL was determined by the thiobarbituric acid-reactive substances
assay. Results represent mean ± S.D. (n = 5). *,
p < 0.01 (versus control).
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We have previously shown that glabridin binds to the LDL particle and,
hence, inhibits its oxidation (12, 15). Thus, we next examined the
possibility that glabridin can be released from the cells to the
incubation medium and directly inhibit LDL oxidation by its binding to
the LDL particle in the medium. For this purpose, MPMs were incubated
with 20 µM of glabridin for 2 h at 37 °C. The
cells were washed and further incubated in a fresh medium for 2 h
at 37 °C. Medium samples were taken every 30 min for HPLC analysis
of glabridin content. After 2 h of incubation, the amount of
glabridin in the cells was 0.72 ± 0.10 µg/mg of cell protein, and the medium glabridin content (obtained from 1 mg of cell protein) was as high as 0.52 ± 0.05 µg (n = 3). When
this medium was then incubated with LDL and copper ions in a cell-free
system, only 10% inhibition in LDL oxidation was obtained (data not
shown) Thus, the substantial (~90%) inhibition of LDL oxidation by
glabridin-enriched cells, in comparison with control cells, is mainly
due to the effect of glabridin on the cellular oxidative state rather
than a direct effect of glabridin on the lipoprotein.
Mechanisms for the Inhibition of LDL Oxidation by
Glabridin-enriched Macrophages--
NADPH oxidase, which is found in
arterial wall cells (including macrophages), can convert native LDL
into oxidized LDL (19, 22). We have previously demonstrated that under
oxidative stress, induced by PMA or by the addition to the cells of LDL
in the presence of copper ions, the macrophage NADPH oxidase was
activated (19). The activated NADPH oxidase complex in the plasma
membrane is responsible for the production and release of superoxide
anions, which can then lead to cell-mediated oxidation of LDL. Since
some flavonoids were found to inhibit NADPH oxidase-induced superoxide release (33), we next analyzed the effect of cellular accumulation of
glabridin on superoxide production and release. Upon incubation of MPM
with increasing glabridin concentrations (0-20 µM) for 2 h at 37 °C, the release of superoxide anions from the cells to the medium was determined following 30 min of cell stimulation with
either LDL (100 µg of protein/ml) and 2 µM
CuSO4 or with the NADPH oxidase activator PMA (50 ng/ml). A
glabridin dose-dependent inhibitory effect on the release
of superoxides was found, with up to 61% or 56% inhibition in
response to PMA or to LDL plus CuSO4, respectively (Fig.
3, A and B). The
modified form of glabridin (2',4',-O-dimethylglabridin) had
no significant effect on macrophage release of superoxides (Fig. 3,
A and B). Kinetic analysis also demonstrated that
glabridin accumulation in macrophages reduced superoxides release (Fig.
3, C and D). In both systems (PMA and LDL plus
CuSO4), glabridin-enriched macrophages showed a
time-dependent reduction in the release of superoxides in
comparison with control cells, with up to 75% and 80% inhibition
observed in the PMA and in the LDL plus CuSO4-treated
cells, respectively (Fig. 3, C and D).

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Fig. 3.
The effect of glabridin on macrophage
released superoxide anions: glabridin concentration study and kinetic
analysis. A and B, MPMs were incubated with
increasing concentrations (0-20 µM) of glabridin ( )
or 2',4'-O-dimethylglabridin ( ) for 2 h at 37 °C.
At the end of the incubation period, the amount of superoxide anions
released to the medium in response to 50 ng/ml of PMA (A) or
to LDL (100 µg of protein/ml) plus 2 µM
CuSO4 (LDL + CuSO4) (B) was
determined as described under "Experimental Procedures." Results
represent the mean ± S.D. of three different experiments.
C and D, MPM were incubated with 0.2% ethanol
(Control) or with 20 µM of glabridin for
2 h at 37 °C. The amount of superoxide released to the medium
in response to 50 ng/ml PMA or to LDL (100 µg of protein/ml) in the
presence of 2 µM CuSO4 was then kinetically
monitored as described under "Experimental Procedures." Results
represent one of three similar experiments.
|
|
Superoxide production by cellular NADPH oxidase involves translocation
of the enzyme cytosolic components (P-47, P-67, and Ras-related
GTP-binding protein) to the plasma membrane, where they interact with
cytochrome b559 to form an active complex that can convert oxygen into superoxides (46, 47). To find out whether
glabridin directly inhibits the active NADPH oxidase complex or the
translocation of the cytosolic components to the plasma membrane, where
it can form the assembled enzyme, we assayed the production of
superoxide in a cell-free system by reconstitution of macrophage
membrane and cytosol fractions. MPMs (108 cells) were
incubated with 20 µM glabridin for 2 h at 37 °C. Control cells were incubated with 0.2% ethanol. The cells were then
washed, and membrane and cytosol fractions were prepared.
Upon incubating cytosol with membranes (2 × 105 cell
equivalents), superoxide production was identical in the two
preparations obtained from control cells or from glabridin-enriched
macrophages (Fig. 4).

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Fig. 4.
Superoxide production in reconstituted
macrophages. Cytosol (2 × 105 cell equivalents)
and membranes (2 × 105 cell equivalents) were
obtained from 0.2% ethanol-treated MPMs (Control) or MPMs
that were enriched with glabridin by cell incubation with 20 µM of glabridin (+Glabridin) for 2 h at
37 °C. Cell fractions were mixed, and the amount of superoxides
released in response to 100 µM SDS was kinetically
monitored as described under "Experimental Procedures." Results are
given as mean ± S.D. of three different experiments.
|
|
These results suggest that glabridin has no direct inhibitory effect on
the active NADPH oxidase complex, but rather an inhibitory effect on
cellular processes that lead to NADPH oxidase activation. Thus, we next
examined the effect of cellular glabridin on the translocation of P-47
from the cytosol to the plasma membrane, by Western blot analysis,
using a specific antibody against P-47. Translocation of P-47 from the
cytosol to the plasma membrane occurs upon cell incubation with 50 ng/ml of PMA or with LDL (100 µg of protein/ml) plus 2 µM CuSO4 (Fig.
5). P-47 translocation in response to LDL
plus copper ions was substantially inhibited in the glabridin-enriched
MPMs but not in the 2',4'-O-dimethylglabridin-enriched MPMs,
in comparison with control MPMs (Fig. 5 and Table
II). Glabridin caused a partial
inhibition of P-47 translocation induced with PMA (Fig. 5 and Table
II), in accordance with the partial inhibition of superoxide production
in this system (see Fig. 3).

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Fig. 5.
The effect of macrophage enrichment with
glabridin or with 2'4'-O-dimethylglabridin on the
translocation of the NADPH oxidase cytosolic component P-47 to the
plasma membrane. Results represent SDS-polyacrylamide gel
electrophoresis immunoblot analysis of P-47 in MPM cytosol and membrane
fractions. Results are shown for resting cells, control cells,
glabridin (20 µM)-treated cells, and
2',4'-O-dimethylglabridin (20 µM)-treated
cells, following cell stimulation with 50 ng/ml PMA or with LDL (100 µg of protein/ml) in the presence of 2 µM
CuSO4. A, resting cells; B, control
cells plus LDL plus Cu2+; C, control cells plus
PMA; D, 2',4'-O-dimethylglabridin-treated cells
plus LDL plus Cu2+; E,
2',4'-O-dimethylglabridin-treated cells plus PMA;
F, glabridin-treated cells plus LDL plus copper;
G, glabridin-treated cells plus PMA.
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Table II
The effect of macrophage enrichment with glabridin or with
2',4'-O-dimethylglabridin on the translocation of the NADPH oxidase
cytosolic component P-47 to the plasma membrane: densitometric analysis
of membrane immunoblots
The experiment is described in the legend to Fig. 5. The determination
by densitometry of the relative amounts of P-47 in the macrophage
membrane is given as mean ± S.D. from three different
experiments. Arbitrary units of density are presented, with the higher
numbers representing darker bands on the immunoblot.
|
|
The results so far suggest that macrophage accumulation of glabridin
caused inhibition of macrophage-mediated oxidation of LDL via an
inhibitory effect of glabridin on the translocation of the NADPH
oxidase P-47 cytosolic component to the plasma membrane and, hence, on
macrophage superoxide production.
Since PKC was shown to be required for LDL oxidation by activated human
monocytes (48) and since phosphorylation of the NADPH oxidase cytosolic
component is involved in the enzyme activation (47, 49), we next
questioned whether cellular accumulated glabridin could affect PKC
activity. For this purpose, we used the PKC inhibitors calphostin C,
staurosporine, and H-7. Calphostin C has been reported to compete with
phorbol ester for binding to the regulatory domain of PKC, and it has
been shown to be more potent in inhibiting PKC then any other protein
kinase, by at least 3 orders of magnitude (50). Hidaka et
al. (51) found that H-7 was 7-fold more potent as an inhibitor of
PKC than of protein kinase A. H-7 is thought to act competitively at
the ATP-binding site (51), whereas staurosporine has been reported to
have both competitive and noncompetitive effects with respect to ATP
(52). We have previously demonstrated that PKC inhibitors inhibited PMA-stimulated NADPH oxidase activity, as well as phosphorylation and
translocation of P-47, in a dose-dependent manner (49), and
these results are in accordance with other studies (53, 54). The
addition to J-774 A.1 macrophages of PKC inhibitors (such as 1 µM calphostin C, 40 ng/ml staurosporine, or 100 µM H-7) inhibited cell-mediated oxidation of LDL in
comparison with its oxidation by macrophages in the absence of the
inhibitors by 65%, 90%, and 60% (from 5.8 ± 0.2 to 2.0 ± 0.1, 0.6 ± 0.1, and 1.7 ± 0.2 nmol of malondialdehyde/mg of
LDL protein, respectively). The addition of 50-100 µM
HA1004 (protein kinase A inhibitor) to J-774 A.1 macrophages in the
presence of LDL plus copper ions had no effect on cell-mediated
oxidation of LDL (data not shown), suggesting the involvement of PKC,
but not of protein kinase A, in LDL oxidation by macrophages. The
inhibitors used had no effect on cell viability as analyzed by trypan
blue assay (data not shown). The activities of protein kinases were
next directly measured in the cytosolic fractions obtained from
glabridin-enriched MPMs or from
2',4'-O-dimethylglabridin-enriched MPMs in comparison with
control cells, following their activation with either PMA (50 ng/ml) or
LDL (100 µg of protein/ml) in the presence of copper ions. Macrophage
PKC activity was reduced by ~70% (Fig.
6A), whereas protein kinase A
activity was not significantly affected (Fig. 6B) in the
glabridin-enriched MPMs, in comparison with control cells (Fig. 6). In
2',4'-O-dimethylglabridin-enriched MPMs, however, both PKC
and protein kinase A activities were not affected (Fig. 6). These
results further indicate the importance of the glabridin hydroxyl
groups on its flavonoid B ring for the inhibition of PKC and NADPH
oxidase activation and, hence, for the attenuation of
macrophage-mediated oxidation of LDL.

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Fig. 6.
Protein kinase C and A activities in control
MPMs, glabridin-enriched MPMs, and
2'4'-O-dimethylglabridin-enriched MPMs. MPMs
(2 × 107 cells) were treated with 0.2% ethanol
(Control), with 20 µM glabridin, or with 20 µM 2'4'-O-dimethylglabridin for 2 h at
37 °C. The cells were then washed and stimulated with 50 ng/ml PMA
or with LDL (100 µg of protein/ml) in the presence of 2 µM CuSO4 for 2 min at 37 °C. The cytosolic
fractions were obtained as described under "Experimental
Procedures." PKC and protein kinase A (PKA) activities
were then measured in the cytosol. Results represent mean ± S.D.
(n = 3).
|
|
In order to examine the possibility that the reduced PKC activity in
the cytosol obtained from glabridin-enriched MPMs is associated with
its increased translocation to the plasma membrane, we measured PKC
activity in membrane fractions isolated from control cells or from
glabridin-treated cells, before or after their stimulation with 50 ng/ml PMA (Table III). Total PKC activity
(cytosol plus membrane) was reduced by 50% in glabridin-treated
unstimulated cells and by 70% in glabridin-treated cells that were
prestimulated with 50 ng/ml PMA (Table III). Finally, we questioned
whether glabridin has a direct inhibitory effect on macrophage PKC
activity. For this purpose, we measured PKC activity in unstimulated
MPMs in the absence or presence of increasing concentrations of
glabridin (0-20 µM) during an in vitro PKC
assay (Table IV). Under these conditions,
glabridin directly inhibited PKC activity in a
dose-dependent fashion by up to 80% at a glabridin
concentration of 20 µM (Table IV).
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Table III
PKC activity in cytosolic and membrane fractions obtained from control
MPMs or from glabridin-treated MPMs
PKC activity was measured in unstimulated cells and in cells stimulated
with 50 ng/ml PMA. PKC activity was measured in the cytosolic and
membrane fractions that were obtained from 2 × 107 MPMs
following their incubation with 0.2% ethanol (control cells), or with
20 µM glabridin for 2 h at 37 °C. Results are
given as the mean ± S.D. of three different experiments.
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Table IV
The effect of glabridin on PKC activity in unstimulated MPMs: glabridin
concentration study
Cytosol was prepared from 2 × 107 unstimulated mouse
peritoneal macrophages. Increasing concentrations of glabridin (0-20
µM) were added to the cytosol prior to the addition of
ATP. PKC activity was then measured as described under "Experimental
Procedures." Results are given as mean ± S.D. of three
different experiments.
|
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The Effect of Dietary Glabridin Supplementation to
E0 Mice on the Ability of Their Peritoneal Macrophages
to Oxidize LDL and on Their Atherosclerotic Lesion Size--
To assess the
in vivo role of macrophage enrichment with glabridin on
cell-mediated oxidation of LDL, we have used the atherosclerotic, apolipoprotein E-deficient mice. In these mice we have previously shown
increased LDL oxidation (45). The mice received glabridin or
2',4'-O-dimethylglabridin (20 µg/day/mouse) in their
drinking water for a period of 6 weeks, and then their peritoneal
macrophages (MPMs) were harvested.
We found 1.6 ± 0.3 µg of glabridin/mg of cell protein
(n = 10) in MPMs derived from the glabridin-treated
mice. To our surprise, MPMs derived from
2',4'-O-dimethylglabridin-treated mice contained glabridin
but not 2',4'-O-dimethylglabridin, indicating that the 2',4'-O-dimethylglabridin undergoes demethylation
in vivo.
After 5 h of LDL (100 µg of protein/ml) incubation with the
MPMs, in the presence of 2 µM CuSO4,
cell-mediated oxidation of LDL by the glabridin-enriched macrophages
was reduced by 88% compared with cell-mediated oxidation by control
macrophages obtained from placebo-treated mice (0.6 ± 0.2 and
5.6 ± 0.2 nmol of malondialdehyde equivalents/mg of LDL protein, respectively).
No effect on serum cholesterol levels was obtained by glabridin
consumption, in comparison with the placebo-treated mice (1011 ± 116 versus 1066 ± 179 mg/dl, respectively,
n = 10). To examine the effect of glabridin
supplementation to the mice on lesion oxysterols and on the size of the
aortic atherosclerotic lesions, the mice were sacrificed after 6 weeks
of glabridin consumption, and their aortas were dissected for
determination of oxysterol content and for lesion size analysis.
Fig. 7 demonstrates detailed analysis of
the lesion oxysterol derivatives. In lesions from the glabridin-treated
mice, reduced content of 7
-hydroxycholesterol,
7
-hydroxycholesterol,
-epoxycholesterol, and 7-ketocholesterol by
54%, 58%, 51%, and 56%, respectively, was noted in comparison with
the oxysterol content in lesions from the control mice (Fig. 7).
However, glabridin had little effect on lesion
-epoxycholesterol
content (Fig. 7).

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Fig. 7.
Lesion oxysterol content in control
mice and in glabridin-treated mice. E0 mice were
supplemented with 20 µg/mouse/day of glabridin or placebo
(Control) for 6 weeks. The mice aortas were dissected, and
the oxysterol content was measured as described under "Experimental
Procedures." 7 -OH, 7 -hydroxycholesterol;
7 -OH, 7 -hydroxycholesterol;
-epoxy, -epoxycholesterol; -epoxy,
-epoxycholesterol; 7-keto, 7-ketocholesterol. Results
represent means ± S.D. (n = 3).
|
|
Glabridin supplementation to the atherosclerotic mice resulted in about
50% reduction in the lesion area compared with the lesion areas in
aortas from placebo-treated mice (52 ± 8 versus 106 ± 11 µm2 × 103, respectively; *,
p < 0.01, n = 14). The
photomicrographs of the lesions (Fig. 8)
demonstrate only few foam cells in the glabridin-treated mice compared
with the placebo-treated mice (Fig. 8).

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Fig. 8.
Photomicrographs of a typical atherosclerotic
lesion of the aortic arch from E0 mice after treatment with
placebo (control mice, A), or glabridin
(B). All micrographs are at the same
magnification (30 µm).
|
|
These results demonstrated that in vivo, glabridin
accumulates in the mice peritoneal macrophages, and these
glabridin-enriched macrophages possess reduced capability to oxidize
LDL. The above characteristics were associated with a remarkable
attenuation in macrophage foam cells and in the development of the
atherosclerotic lesions in the apolipoprotein E-deficient mice.
 |
DISCUSSION |
In the present study, we have demonstrated for the first time that
glabridin, a lipophilic isoflavan isolated from licorice root, can be
taken up by and accumulated in macrophages. Glabridin cellular
accumulation substantially inhibited macrophage-mediated oxidation of
LDL, secondary to the inhibition of NADPH oxidase.
We have previously demonstrated the potent inhibitory effect of
glabridin on LDL oxidation (12, 14-16). When consumed, glabridin is
absorbed and binds to plasma lipoproteins (12, 15). Absorbed glabridin,
however, can also be taken up by cells, including arterial wall
macrophages and, hence, can affect the cellular oxidative state.
Indeed, the present study demonstrated that glabridin was taken up by
macrophages, accumulated in the cells' plasma membrane, and exerted a
potent inhibitory effect on macrophage-mediated oxidation of LDL. This
effect was time- and dose-dependent and required similar
structural elements (the hydroxyl groups on the isoflavan B ring), as
previously shown for the antioxidative effect of glabridin against LDL
oxidation (16). This last similar requirement suggests that the
inhibitory effect of glabridin on the macrophage machinery, which is
required for LDL oxidation, may be related to the antioxidative
properties of this isoflavan. We have demonstrated, however, that the
inhibitory effect of glabridin, which accumulated in the cells, on LDL
oxidation was mainly related to its effects on the cells rather than a
phenomenon that could be related to the release of some glabridin from
the cells, followed by its interaction with the LDL particle and
scavenging of reactive oxygen species. In addition, the inhibitory
effect of glabridin on cell-mediated oxidation of LDL was still a
substantial one, even after 20 h of LDL oxidation, suggesting that
the glabridin in the cells was stable and preserved its activity along
this relatively long time of incubation.
Cellular release of superoxide anions by arterial wall cells was
suggested as a possible mechanism for LDL oxidation (19, 20, 55-58).
Several flavonoids were shown to inhibit the release of reactive oxygen
species by stimulated human leukocytes or neutrophils (33, 59, 60).
This inhibitory effect was attributed to the structure of the
flavonoid, to its hydrophobicity, and to the number and/or position of
the hydroxyl groups on the flavonoid B ring (59). The flavonoid's free
radical scavenging capability (61, 62) may have a role in the
prevention of cellular production of free radicals and, thus, in the
inhibition of the formation of oxidized LDL, but glabridin enrichment
of macrophages reduced superoxide release from these cells and
inhibited macrophage-mediated oxidation of LDL, also via a direct
effect on NADPH oxidase activation. We have previously shown that
activation of the macrophage NADPH oxidase can lead to a substantial
LDL oxidation (19). The inhibitory effect of glabridin on superoxide
release was detected only when it was added to intact cells and not
when present in a cell-free system, indicating that this agent does not
directly affect the NADPH oxidase complex, but rather it affects the
signaling that leads to NADPH oxidase activation. An important role for
PKC was shown in the phosphorylation and translocation of P-47 from the cytosol to the plasma membrane to form an active NADPH oxidase complex
(49, 63-66). Continuous phosphorylation by PKC and translocation of
P-47 is necessary in order to maintain NADPH oxidase in an activated
state (53, 66). The results of the present study demonstrated that PKC
activity induced by either PMA or by LDL plus copper ions was inhibited
by cellular glabridin. Thus, inhibition of the macrophage PKC activity
inhibited the translocation of cytosolic P-47 to the plasma membrane to
form the assembled active NADPH oxidase.
These effects of glabridin were found to be specific to the glabridin
hydroxyl groups on the isoflavan B ring, since 2',4'-O- dimethylglabridin was not active anymore.
It was previously shown that methylation of the hydroxyl group at the
4'-position of the phenyl ring of hesperetin also contributes to the
loss of its PKC-inhibitory activity (34). We have demonstrated direct
inhibitory effect of glabridin on PKC activity in unstimulated cells.
This inhibitory effect of glabridin occurred only when glabridin was
added prior to the addition of ATP, suggesting that glabridin is a
competitive inhibitor with respect to ATP binding. Flavonoids that
inhibited PKC activity were indeed found to be competitive inhibitors
with respect to ATP binding (34).
Dimethylglabridin, in which the hydroxyl groups on the isoflavan B ring
were protected, had no inhibitory effect on PKC activity, on P-47
translocation, on cellular superoxide release, and on macrophage-mediated oxidation of LDL. These results point to the importance of the glabridin unique structure in the inhibition of the
sequence of events that leads to cell-mediated oxidation of LDL. In
order to assess the physiological significance of our in
vitro results, we questioned the relationship between macrophage glabridin content, cell-mediated oxidation of LDL, and the extent of
atherosclerosis by using the atherosclerotic apolipoprotein E-deficient
mice model. These mice demonstrate increased LDL oxidation and develop
extensive atherosclerosis within a few months of age. The present study
clearly demonstrated a significant reduction in MPM-mediated oxidation
of LDL, in lesion oxysterol content, and in the atherosclerotic lesion
area in the glabridin-treated mice, in comparison with placebo-treated
mice. These results indicate that inhibition of atherosclerosis in
these mice by glabridin consumption is related to reduced
macrophage-mediated oxidation of LDL. Glabridin may possess this
property not only because of its binding to LDL (12) but also by its
accumulation in cells such as macrophages, where it reduces cellular
oxidative stress by inhibiting protein kinase C and, hence, reducing
NADPH oxidase activation, an important inducer of macrophage-mediated
oxidation of LDL (19).
Flavonoids, as potent antioxidants against LDL oxidation, as well as
inhibitors of macrophage oxygenases, may be used as important intervention means to inhibit cell-mediated oxidation and to attenuate atherosclerosis (67-69).
 |
ACKNOWLEDGEMENT |
We thank Ilana Cohen for typing the manuscript.
 |
FOOTNOTES |
*
This study was supported by a grant from the Rappaport
Family Institute for Research in Medical Sciences, Haifa Israel.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.

To whom correspondence should be addressed: The Lipid Research
Laboratory, Rambam Medical Center, Haifa, Israel. Tel.: 972-4-8542970; Fax: 972-4-8542130; E-mail: aviram{at}tx.technion.ac.il.
 |
ABBREVIATIONS |
The abbreviations used are:
LDL, low density
lipoprotein;
H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine
dihydrochloride;
MPM, mouse peritoneal macrophage;
PBS, phosphate-buffered saline;
HPLC, high performance liquid
chromatography;
PMA, phorbol 12-myristate 13-acetate;
PKC, protein
kinase C.
 |
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