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*
The Lipid Research Laboratory, the
Department of Anatomy and Cell Biology, Bruce Rappaport Faculty of Medicine, Technion, The Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, 31096, Israel and
**
Laboratory of Natural Compounds for Medical Use, Migal, Galilee Technological Center, Kyriat Shmona, 10200, Israel
2To whom correspondence should be addressed. E-mail: aviram{at}tx.technion.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • pomegranate • lipid peroxidation • LDL • HDL • macrophages • antioxidants • atherosclerosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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,2)
. Cholesterol
accumulation in macrophages can result from an unbalanced cellular
cholesterol flux, i.e., increased uptake of atherogenic lipoproteins
and/or decreased cholesterol efflux from the cells (3
4
5)
.
Oxidized
(Ox)3
-LDL are taken up by macrophages at an enhanced rate via scavenger
receptors (4)
, leading to the formation of lipid-laden
foam cells and accelerated atherosclerosis (3
,4
,6)
.
Moreover, oxidative stress has been shown to affect not only the LDL
lipids, but also cellular lipids, including those found in arterial
macrophages (7
,8)
. We have previously shown that such
lipid-peroxidized macrophages exhibit an increased ability to
oxidize LDL and to take up Ox-LDL (7
,8)
, thus leading
to cellular accumulation of cholesterol and oxidized lipids. Serum
paraoxonase is an HDL-associated esterase and it was shown to
protect from oxidation lipids in lipoproteins and in lesions by its
hydrolytic and peroxidative-like properties (9
10
11)
.
Because induction of oxidative stress on LDL and on arterial cells
plays an important role in atherogenesis, its inhibition by nutritional
antioxidant should slow the progression of the disease. Enrichment of
LDL, as well as of arterial cells with nutritional antioxidants such as
vitamin E or polyphenolic flavonoids, protects the LDL against lipid
peroxidation and attenuates the development of atherosclerosis
(12
13
14
15)
. Pomegranate juice (PJ), which is very rich in
flavonoids, was recently shown to be antiatherogenic, and this effect
is probably related to its potent antioxidative activity
(16)
.
PJ supplementation to atherosclerotic apolipoprotein
E-deficient (E0) mice before they develop
atherosclerosis significantly inhibited the progression of
atherosclerotic lesion (16)
. No studies, however, have
been conducted on the effect of nutritional antioxidants on macrophage
cholesterol accumulation in mice that already exhibit advanced
atherosclerotic lesions.
PJ contains 85% moisture, 10% sugar, 1.5% pectin, ascorbic acid and
polyphenolic flavonoids. The content of soluble polyphenols in PJ
varied between 0.2% and 1.0% and include mainly anthocyanins (such as
cyanidin-3-glucoside, cyanidin-3, 5-diglucoside and
delphindin-3-glucoside), catechins, ellagic tannins, gallic and ellagic
acids (17)
E0 mice are often used to study atherogenesis
because they develop severe hypercholesterolemia low cholesterol diet,
they exhibit high oxidative stress and develop extensive
atherosclerosis by the age of 4 mo (18
,19)
.
In the present study we examine the effect of PJ administration to E0 mice with advanced atherogenesis on their macrophage lipid peroxidation, cellular cholesterol flux and the development of atherosclerotic lesions. In addition, we analyzed the antiatherosclerotic properties of an isolated antioxidant fraction of PJ.
|
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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E0 mice were generously provided by Dr.
Jan Breslow (Rockefeller University, NY). Gene targeting in mouse
embryonic stem cells was used to create mice that lack apolipoprotein E
(18)
.
To analyze the effect of PJ on the development of the atherosclerotic lesions, 30 male E0 mice were divided into three groups of 10.
PJ group. A concentrated PJ, obtained by squeezing the entire fruit, was diluted in water (6.25 mL of concentrated PJ in 1 L of water). The PJ solution diluted in water was given to the mice at 4 mo of age for 2 mo, whereas only water was given to the placebo mice. Because mice drank an average of 5 mL/d, they effectively drank 31 µL of PJ/d, which is equivalent to 0.875 µmol of total polyphenols/d.
This dose of PJ was used based on results of our previous study
(16)
, showing that it substantially inhibited the
development of atherosclerotic lesion.
Placebo group. Mice received no addition to their drinking water from 4 to 6 mo of age.
Control group. Mice at 4 mo of age received no addition to their drinking water. This reference group was included to measure the state of the atherosclerotic lesions in E0 mice at the beginning of the treatment period.
At the end of the study (6 mo of age for the first two groups and 4 mo of age for the control group) blood, peritoneal macrophages and aortas were obtained.
Serum cholesterol, lipid peroxidation and paraoxonase
(arylesterase) activity.
Blood was collected from the retroorbital plexus of mice under
ether anesthesia. Serum cholesterol was determined using a commercial
kit (Ref 352-20; Sigma Diagnostics, St Louis, MO)
(20)
.
To determine plasma lipid peroxidation, plasma samples were incubated
in the absence or presence of 100 mmol/L of 2,2'-azobis
2-amidinopropane hydrochloride (AAPH; Wako Chemical Industries Ltd,
Osaka, Japan) for 2 h at 37°C. AAPH is a
water-soluble azo compound that thermally decomposes to produce
peroxyl radicals at a constant rate. Plasma lipid peroxide content was
determined by measuring their ability to convert iodide to iodine,
measured at 365 nm (21)
.
Serum paraoxonase is an enzyme with multiple activities. Its activity
is commonly determined by its arylesterase activity using phenylacetate
as the substrate. Initial rates of hydrolysis were determined
spectrophotometrically at 270 nm. The assay mixture included 5 µL of
serum, 1.0 mmol/L phenylacetate and 0.9 mmol/L CaCl2 in 20
mmol/L Tris HCl (pH 8.0). Nonenzymatic hydrolysis of phenylacetate was
subtracted from the total rate of hydrolysis. The E270 for
the reaction was 1310 (mol/L)-1
cm-1 (22)
.
Mouse peritoneal macrophages (MPM). MPM were harvested from the peritoneal fluid of E0 mice 4 d after intraperitoneal injection into each mouse of 3 mL of thioglycolate (24g/L) in saline. The cells (10–20 x 106/mouse) were washed and centrifuged three times with phosphate-buffered saline (PBS) at 1000 x g for 10 min, then resuspended at 109/L in DMEM containing 10% horse serum (heat-inactivated at 56°C for 30 min) and 1 x 105 U penicillin/L, 100 mg streptomycin/L and 2 mmol/L glutamine. The dishes were incubated in a humidified incubator (5% CO2, 95% air) for 2 h, washed with DMEM to remove nonadherent cells and the monolayer was incubated under similar conditions for 18 h.
The level of cellular lipid peroxidation in MPM was determined by lipid
peroxide determination in a sonicated cell preparation
(23)
.
Lipoprotein preparation.
LDL and HDL were prepared from human plasma (drawn into 1 mmol/L of
Na2 EDTA) from fasting normolipidemic volunteers. LDL (d
= 1.019–1.063 kg/L) and HDL (d = 1.064–1.21 kg/L) were
prepared by discontinuous density gradient ultracentrifugation
(24)
. The lipoproteins were then dialyzed against 150
mmol/L NaCl, 1 mmol/L Na2 EDTA (pH 7.4). LDL were
sterilized by filtration and was used within 2 wk. The protein content
of the lipoproteins was determined with the folin phenol reagent
(25)
. LDL was radioiodinated using the iodine monochloride
method (26)
modified for lipoproteins.
125I-Oxidized LDL were prepared by an overnight dialysis of
125I-labeled LDL (1 g of lipoprotein protein/L) against PBS
to remove any residual EDTA, followed by incubation with 10 µmol/L
CuSO4 for 18 h at 37°C. Oxidation was terminated by
refrigeration and the addition of 0.1 mmol/L Na2 EDTA. The
degree of LDL oxidation was determined by using the thiobarbituric acid
reactive substances assay (27)
, and it ranged from 18 to
25 nmol of mafondialdehyde (MDA) equivalents/mg lipoprotein
protein compared with 0.5–1.0 nmol of MDA equivalents/mg
lipoprotein protein in native LDL.
Macrophage cholesterol metabolism
Cellular uptake of lipoproteins by macrophages.
Uptake of lipoproteins by macrophages was assessed by measuring the
degradation of lipoproteins. 125I-Ox-LDL or native
125I-LDL at 10 mg of protein/L were incubated with the MPM
at 37°C for 5 h. Lipoprotein cellular degradation was measured
in the collected medium as the trichloroacetic acid-soluble,
nonlipid radioactivity, which was not due to free iodide
(28)
. Lipoprotein degradation in a cell-free system
measured under identical conditions was minimal (<10%) and was
subtracted from the total degradation. The remaining cells were washed
three times with cold PBS and dissolved in 0.1 mol/L NaOH for protein
determination.
Cellular cholesterol esterification.
Cellular cholesterol esterification was estimated by measurement of the
incorporation of [3H]oleate into cholesteryl ester.
Cells were incubated with medium in 10% fetal calf serum, for 18 h at 37°C. During the last 2 h of incubation,
[3H]-oleate in complex with albumin (2.7 mmol/L, 83 mmol
oleate/mg albumin, 10 Ci/L) was added to the medium. At the end of the
incubation, cellular lipids were extracted with hexane/isopropanol (3:2
v/v) and the cholesteryl ester was separated by thin layer
chromatography using hexane/ether/acetic acid (130:30:1.5 v/v/v),
scraped into vials containing scintillation fluid and counted in a
ß-counter (29)
.
Cellular cholesterol efflux.
MPM were incubated with [3H]-labeled cholesterol for
18 h at 37°C followed by cell wash in ice-cold PBS (three
times) and further incubation in the absence or presence of 100 mg of
human HDL protein/L for 3 h at 37°C. Cellular and medium
[3H]-label were quantitated and HDL-mediated
cholesterol efflux was calculated as the ratio of
[3H]-label in the medium/[3H]-label in the
medium+[3H]-label in cells (30)
.
PJ fractionation and characterization of antioxidant activities of an isolated fraction. Purification of active compounds from PJ was achieved by HPLC on reverse-phase Sep-Pak. PJ (8 g) was dissolved in a mixture of methanol:water (30 mL; 1:2 v:v), and diethyl ether was added (40 mL). The aqueous phase was separated and mixed with ethyl acetate (200 mL). After phase separation, the organic layer was evaporated. This residue was dissolved in 1 mL water, chromatographed on reverse-phase Sep-Pak column with water (3 x 2 mL) followed by mixtures of water:methanol (10–20%), and a pure fraction was collected.
This fraction was named compound 25 because it appears after 25 min of chromatography on the Sep-Pak column. Compound 25 was hydrophilic with medium solubility in ethyl acetate, suggesting that it is not a lipid or a protein. Furthermore, its maximal absorption was at 254 nm and, therefore, it is not a simple flavonoid. It was shown from infrared spectra to contain many hydroxyl groups. Nuclear magnetic resonance-spectra revealed the presence of quaternary carbons and a few hydrogen atoms, and, hence, compound 25 might be a complexed polyphenol, such as hydrolyzable tannin.
The antioxidant capacity of compound 25, which was isolated from PJ,
was tested by its ability to inhibit CuSO4-induced LDL
oxidation. LDL were oxidized in the absence or presence of increasing
concentrations of compound 25, by its incubation with 5 µmol/L of
CuSO4 for 2 h at 37°C. The degree of LDL oxidation
was determined by the thiobarbituric acid reactive substances assay
(27)
and the lipid peroxide content (21)
.
Mice at 2 mo old were administrated either compound 25 (20 µg/ d) or PJ [31 µL (equivalent to 0.875 µmol of total polyphenols)/d] for 2 mo and were analyzed for their atherosclerotic lesion size compared with age-matched, placebo-treated mice.
Histopathology of aortic atherosclerotic lesions. After PJ treatment to E0 mice for 2 mo, the heart and aorta were rapidly removed and immersion fixed in 3% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer with 0.1 g/L calcium chloride (pH 7.4) at room temperature. The aortic arch was then dissected free from the surrounding fatty tissue and the first 4 mm of the ascending aorta was removed and cut transversely with razor blades into blocks of 1 mm each. After overnight treatment with fixative, the samples were then rinsed and stored in 0.1 mol/L of sodium cacodylate buffer containing 75 g/L sucrose before treatment with an unbuffered 10 g/L aqueous solution of osmium tetroxide for 4 h. This was followed by dehydration in ascending ethanols, before propylene oxide and embedding in epoxy resin (Eponate 12; Pelco Int., Redding, CA). The blocks were then 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 sections cut with diamond knives on an LKB Nova ultramicrotome (LKB, Bromma, Sweden). When sufficient semithin sections were obtained from all the blocks, the remainder of the blocks was 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 an intense black from the osmium treatment and permits lesion areas to be easily determined histomorphometrically. Only the area of the aortic arch was examined because previous and on-going studies by us and other groups have shown that this area is especially prone to atherosclerosis in E0 mice and the areas well-defined with a clear starting point (aortic valves). Histomorphometric determinations of lesion size were performed using an Olympus Cue-2 image analysis system with appropriate morphometry software (Olympus Corporation, Lake Success, NY). The system consists of a Zeiss Universal R photomicroscope (x10 objective) fitted with a Panasonic WV-CD50 camera. Measurements were made in standardized windows (fields) with an area of 176,758 µm2.
Statistics. Student’s t-test was used in comparing two means, whereas ANOVA and post-hoc tests were used when more than two groups were compared. Results are given as means ± SEM. Differences were considered significant if P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Plasma cholesterol concentrations were not significantly affected by PJ consumption (621 ± 36, 593 ± 58 and 666 ± 47 mg/dL in the control, placebo-treated and PJ-treated mice, respectively). PJ consumption minimally reduced plasma lipid peroxide concentration produced by AAPH by 8% in comparison to control and placebo-treated mice, as shown by values of 547 ± 39, 545 ± 27 and 507 ± 32 mmol lipid peroxides/L in the plasma of control, placebo-treated and PJ-treated mice, respectively (P < 0.05).
A nonsignificant 12% reduction (P = 0.12) in serum
paraoxonase was noted in placebo-treated mice (6 mo old), in
comparison to the control, 4-mo-old mice (Fig. 1
). A significant 43% increase in serum paraoxonase activity was
obtained in mice that consumed PJ for 2 mo compared with
placebo-treated mice. Moreover, serum paraoxonase activity in
PJ-treated mice was 26% higher than in control mice (Fig. 1)
.
|
MPM lipid peroxides content in placebo-treated mice was 40% higher
than in control, 4-mo-old mice (Fig. 2
). PJ consumption for 2 mo reduced macrophage lipid peroxide content by
42% compared with placebo-treated mice (P < 0.05). Furthermore, macrophage lipid peroxide content in MPM harvested
from PJ-treated mice was 20% lower than in control, 4-mo-old mice
(Fig. 2)
.
|
Degradation of Ox-LDL by MPM harvested from 6-mo-old,
placebo-treated mice was 34% greater than in control, 4-mo-old
mice (Fig. 3
). Incubation of MPM, derived from E0 mice that
consumed PJ with 125I-labeled Ox-LDL (10 mg
of protein/L) significantly reduced cellular lipoprotein degradation,
by 31% and 10%, compared with Ox-LDL uptake by MPM harvested from
age-matched, placebo-treated mice or control mice, respectively
(Fig. 3)
. In contrast, the uptake of native LDL by MPM was not
significantly affected by age or PJ supplementation (Fig. 3)
.
|
MPM harvested from 6-mo-old, placebo-treated mice had a 110%
greater cholesterol esterification rate compared with control, 4-mo-old
mice (Fig. 4
). Cholesterol esterification rates in MPM isolated from PJ-treated
mice was 80% and 57% lower, respectively, compared with
age-matched, placebo-treated mice and control mice (Fig. 4)
.
|
MPM cholesterol efflux was 46% lower (P < 0.05) in
cells harvested from the 6-mo-old, placebo-treated mice than in
control, 4-mo-old mice (Fig. 5
). In contrast, PJ treatment significantly increased, by 39%,
macrophage cholesterol efflux from macrophages compared with
cholesterol efflux from MPM harvested from the placebo-treated mice
(Fig. 5)
.
|
As illustrated in the photomicrographs of the aortic lesions
(Fig. 6A
, and B
), lesions of the placebo-treated
E0 mice were progressively enlarged and became
more complex from 4 to 6 mo with increased numbers of lipid-laden
macrophage-derived foam cells. The PJ-supplemented mice, in
contrast, showed less advanced lesions with fewer foam cells (Fig. 6C
) compared with the age-matched, placebo-treated mice.
|
).
Although the atherosclerotic lesion area in the PJ-treated mice was
greater than in the younger control mice (Fig. 6D
), it was
17% (P < 0.01) smaller than in the lesion area in
aortas from age-matched, placebo-treated mice (Fig. 6D
). PJ isolated fraction on atherosclerotic lesion development.
We isolated an active antioxidant compound from PJ, referred to as
compound 25, which inhibited CuSO4-induced LDL
oxidation (Table 1
). Although this active compound was not fully identified, it has
characteristics of complexed tannins.
|
, we administered compound 25
and whole PJ to young, 2-mo-old E0 mice. In
comparison to control, nontreated E0 mice,
compound 25 and PJ possessed potent antiatherosclerotic characteristics
as shown by significant reductions of 54% and 44%, respectively, in
the lesion size (Fig. 7
, A
, B
, C
, D
).
|
In MPM from E0 mice that consumed compound 25 or whole PJ, cellular degradation rates of Ox-LDL (25 mg/L of Ox-LDL) were decreased by 34% and 25%, respectively, compared with Ox-LDL degradation by control E0 mice (from 2998 ± 238 ng/mg cell protein in MPM from control mice to 1969 ± 161 ng/mg and 2258 ± 277 nmol/mg in MPM from mice treated with compound 25 or PJ,respectively).
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. Major contributors to cholesterol accumulation in
arterial cells during atherogenesis include high plasma cholesterol
concentration (31)
, increased oxidative stress
(23
,32)
, reduced serum paraoxonase activity
(33)
, increased uptake of atherogenic lipoproteins by
arterial cells (34
35
36)
, enhanced macrophage cholesterol
esterification rate (37)
and decreased cholesterol efflux
from arterial cells (5
,38)
. Because oxidative stress is
believed to play an important role in early atherogenesis,
efforts should be made to reduce oxidative stress and its harmful
consequences (39
40
41
42
43)
. Antioxidants, such as those found
in some fruits and vegetables, which are rich in polyphenolic
flavonoids (red wine, grapes, licorice, ginger and pomegranate),
significantly reduce oxidative stress by inhibiting the formation of
Ox-LDL and macrophage lipid peroxidation. The capacity of these
antioxidants to reduce atherogenesis may be due to the reduction in the
oxidative stress (13
14
15
16
,44)
.
In this study PJ consumption by atherosclerotic mice significantly
reduced cholesterol accumulation and foam cell formation. PJ
supplementation to atherosclerotic mice reduced all the proatherogenic
variables determined compared with age-matched, placebo-treated
mice. A major difference between the present study and our previous
study (16)
on the effect of PJ supplementation to
E0 mice is that in the present study, PJ
consumption started in mice with already advanced atherosclerosis
(4-mo-old). Under such conditions of extensive atherosclerosis, the
lesions mature and become more resistant to the influence of drug or
nutrient interventions. When initiating antioxidant-rich nutrient
supplementation after the onset of atherosclerosis, inhibition of the
progression of atherogenesis is more difficult than intervention during
early stages of atherogenesis, but this condition may be a more
realistic reflection of common practice. By using two sets of controls,
i.e., the 4-mo-old, control mice and the placebo-treated, 6-
mo-old mice, we were able to measure the atherogenic variables in
similar mice before PJ supplementation.
In the present study, the reduction in the size of the atherosclerotic
lesion by PJ consumption was smaller (only 17%) compared with our
previous study (16)
, where up to a 33% reduction in the
lesion size was obtained. Whereas in the present study mice were
initially supplemented with PJ at 4 mo of age when they already had
extensive atherosclerosis, in our previous study (16)
treatment began as early as 5 wk of age when no atherosclerosis could
be observed. The present study, however, demonstrated that even in the
atherosclerotic mice, PJ consumption still showed antiatherogenic
activity and could still slow down the progression of atherosclerosis.
This may be related to the impressive inhibitory effect of PJ
consumption on macrophage cholesterol and cholesteryl ester
accumulation as evidenced by attenuation of cholesterol influx and
esterification and by an enhanced macrophage cholesterol efflux.
However, the relatively small reduction in the atherosclerotic lesion
size after PJ consumption, in comparison to the substantial reduction
in macrophage lipid peroxides content may indicate that in the advanced
atherosclerotic mice, factors in addition to oxidative stress may
affect lesion progression. Such factors may include growth factors and
cytokines produced and released by arterial cells at later stages of
atherogenesis.
As shown in previous studies, the antioxidative effect of PJ
consumption could be detected in plasma and in the harvested peritoneal
macrophages (MPM), as measured by analysis of their lipid peroxides
(16
,45)
. Due to the onset of PJ consumption by the mice
when they already presented advanced atherosclerosis, the antioxidant
effect of PJ in the plasma was rather small compared with our previous
study (16)
, which showed a substantial decrease in plasma
susceptibility to oxidation after PJ supplementation to young,
nonatherosclerotic E0 mice. Of interest is that
the increase with age (6 mo vs. 4 mo) in the E0
MPM lipid peroxide content was reduced by PJ consumption to levels that
were even lower than those observed in MPM from control, young
E0 mice.
Furthermore, serum paraoxonase activity was significantly increased by
PJ consumption. Because paraoxonase protects against LDL oxidation
(11)
and hydrolyzes and reduces lipid peroxides and
cholesteryl linoleate hydroperoxides in oxidized lipoproteins
(46)
and in atherosclerotic lesions (47)
, the
beneficial effect of PJ on serum paraoxonase may further contribute to
the reduction in oxidative state found in the PJ-treated
E0 mice both in their plasma and MPM. In addition
to its ability to hydrolyze specific oxidized lipids in oxidized
lipoproteins, paraoxonase itself is inactivated by lipid peroxides
(47)
. Thus, the increased serum paraoxonase
activity may have resulted from the reduced oxidative stress in the
serum of PJ-treated mice.
In the present study we have extended the search for possible
mechanisms related to the inhibitory effect of PJ consumption on
macrophage foam cell formation. Cellular cholesterol flux was analyzed
by determining macrophage cholesterol influx (uptake of atherogenic
Ox-LDL), as well as macrophage cholesterol efflux (release of cellular
cholesterol from MPM by HDL). The present study clearly demonstrated
the beneficial effect of PJ consumption by E0
mice on both fluxes, i.e., inhibition of Ox-LDL uptake by the
harvested MPM and stimulation of HDL-induced cholesterol release
from these cells. Both of these processes reduced macrophage
cholesterol content, foam cell formation and atherosclerosis
(1
,3
4
5
6)
. PJ reversed the increased macrophage uptake of
Ox-LDL, which was associated with aging and the development of
atherosclerosis, to levels of lipoprotein degradation rates by MPM that
were even lower than those observed in control young mice. Macrophage
cholesterol efflux was not affected with mice age (6 mo vs. 4 mo), but
PJ supplementation increased the efflux rate, and, thus, could
contribute to the reduction in macrophage cholesterol content.
Native LDL cellular uptake (via the LDL receptor), unlike macrophage
uptake of Ox-LDL (via the scavenger receptors), was not affected by
age (placebo-treated vs. control, young mice) or by PJ consumption.
This may be related to the inability of native LDL to cause macrophage
cholesterol accumulation, because the LDL receptor is tightly regulated
by the cellular cholesterol content (48)
.
In macrophage foam cells, substantial amounts of the cholesterol are in
the esterified form and acyl cholesterol acyltransferase activity is
increased during atherogenesis (37)
. Enhanced
esterification of the cholesterol leads to the accumulation of
cholesteryl ester droplets, a feature that resembles lesion’s
macrophage foam cells (1
,31
,49)
. The present study
demonstrated that macrophage cholesterol esterification rate
substantially increased with age (6 mo vs. 4 mo). PJ consumption,
however, inhibited macrophage cholesterol esterification rate to rates
that were slower than those in control mice. Macrophage cholesterol
esterification rate was measured using MPM that were harvested from
mice after PJ or placebo administration. The decreased cholesterol
esterification rate after PJ consumption, thus, may indicate an
inhibitory effect of PJ on acyl cholesterol acyltransferase activity,
which is in line with antiatherogenicity secondary to decreased
cholesteryl ester formation. However, because the MPM are incubated
with fetal calf serum that contains plasma lipoproteins, it is also
possible that the reduced macrophage cholesterol esterification rate
observed after PJ consumption is related to a decreased uptake of
atherogenic lipoprotein, and, hence, to a reduced macrophage
cholesterol content, which is reflected by a slower cholesterol
esterification rate because less substrate (lipoprotein-derived
unesterified cholesterol) is available for the added labeled fatty
acid.
We have also isolated an active antiatherosclerotic fraction (compound 25) from PJ with significant antioxidative capacities. Compound 25 consumption by E0 mice significantly reduced the atherosclerotic lesion size and this effect was associated with decreased plasma lipid peroxidation and attenuated MPM uptake of Ox-LDL. Because the anti-atherosclerotic effect of PJ was more pronounced when it was given to the young, nonatherosclerotic mice, we analyzed the effect of the isolated active PJ tannin fraction on lesion progression in the young E0 mice.
In conclusion, PJ supplementation to E0 mice possesses very impressive antiatherogenic properties, which could be related to its potent antioxidative activity and beneficial effect on macrophage cholesterol flux, which results in decreased macrophage cholesterol accumulation. The effect of PJ consumption on atherosclerosis was shown not only when supplementation of PJ to E0 mice started before they developed atherosclerotic lesions, but also in mice with extensive atherosclerosis. The above antiatherosclerotic properties of PJ could be related to the presence of a tannin fraction in PJ with potent antioxidative characteristics.
|
FOOTNOTES |
|---|
3 Abbreviations used: AAPH, 2,2'-azobis 2-amidinopropane hydrochloride; E0, apolipoprotein E-deficient; MPM, mouse peritoneal macrophage; Ox, oxidized; PBS, phosphate-buffered saline; PJ, pomegranate juice.
Manuscript received February 1, 2001. Initial review completed March 29, 2001. Revision accepted May 8, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
1.
Berliner J. A., Navab M., Fogelman A. M., Frank J. S., Demer L. L., Edwards P. A., Watson A. D., Lusis A. J. Atherosclerosis—basic mechanisms: oxidation, inflammation and genetics. Circulation 1995;91:2488-2496
2. Gerrity R. G. The role of monocytes in atherogenesis. Am. J. Pathol. 1981;103:181-190[Abstract]
3. Aviram M. Interaction of Ox-LDL interaction with macrophages in atherosclerosis, and the antiatherogenicity of antioxidants. Eur. J. Clin. Chem. Clin. Biochem. 1996;34:599-608[Medline]
4.
Steinberg D. LDL oxidation and its pathobiological significance. J. Biol. Chem. 1997;272:20963-20966
5. Krieger M. The best of cholesterols, the worst of cholesterols: a tale of two receptors. Proc. Natl. Acad. Sci. U.S.A. 1998;14:4077-4080
6. Witztum J. L., Steinberg D. Role of Ox-LDL in atherogenesis. J. Clin. Invest. 1991;88:1785-1792
7. Fuhrman B., Oiknine J., Aviram M. Iron induces lipid peroxidation in cultured macrophages increases their ability to oxidatively modify LDL and affects their secretory properties. Atherosclerosis 1994;111:65-78[Medline]
8. Fuhrman B., Oiknine J., Keidar S., Ben-Yaish L., Kaplan M., Aviram M. Increased uptake of LDL by oxidized macrophages is the result of an initial enhanced LDL receptor activity and a further progressive oxidation of LDL. Free Radic. Biol. Med. 1997;23:34-46[Medline]
9. La Du B. N. Structural and functional diversity of paraoxonases. Nat. Med. 1996;2:1186-1187[Medline]
10. Mackness M. I., Mackness B., Durrington P. N., Connelly P. W., Hegele R. A. Paraoxonases biochemistry, genetics and relationship to plasma lipoproteins. Curr. Opin. Lipidol. 1996;7:69-76[Medline]
11.
Aviram M., Billecke S., Sorenson R., Bisgaier C., Newton R., Rosenblat M., Erogul J., Dunlop C., La Du B. Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different than that required for its arylesterase/paraoxonase activities: selective action of human paraoxonase allozymes Q and R. Arterioscler. Thromb. Vasc. Biol. 1998;18:1617-1624
12.
Srimm E. B., Stampfer M. J., Asherio A., Giovannucci E., Colditz G. A., Willett W. C. Vitamin E consumption and the risk of coronary heart disease in men. N. Engl. J. Med. 1993;328:1450-1456
13. Aviram M., Fuhrman B. Polyphenolic flavonoids inhibit macrophage-mediated oxidation of LDL and attenuate atherogenesis. Atherosclerosis 1998;137(suppl.):S45-S50
14.
Hayek T., Fuhrman B., Vaya J., Rosenblat M., Belinky P., Coleman R., Elis A., Aviram M. Reduced progression of atherosclerosis in the apolipoprotein E-deficient mice following consumption of red wine, or its polyphenols quercetin, or catechin, is associated with reduced susceptibility of LDL to oxidation and aggregation. Arterioscler. Thromb. Vasc. Biol. 1997;17:2744-2752
15.
Fuhrman B., Buch S., Vaya J., Belinky P. A., Coleman R., Hayek T., Aviram M. Licorice extract and its major polyphenol glabridin protect LDL against lipid peroxidation: in vitro and ex-vivo studies in humans and in atherosclerotic E0 mice. Am. J. Clin. Nutr. 1997;66:267-275
16.
Aviram M., Dorenfeld L., Rosenblat M., Volkova N., Kaplan M., Hayek T., Presser D., Fuhrman B. Pomegranate juice consumption reduces oxidative stress, low density lipoproteins modifications and platelet aggregation: studies in the atherosclerotic E0 mice and in humans. Am. J. Clin. Nutr. 2000;71:1062-1076
17. Narr Ben C., Ayed N., Metche M. Quantitative determination of the polyphenolic content of pomegranate peel. Z Lebensm. Unters. Forsch. 1996;203:374-378[Medline]
18. Arnal J. F., Castano C., Maupas E., Mugniot A., Darblade B., Gourdy P., Michel J. B., Bayard F. Omapatrilat, a dual angiotensin-converting enzyme and neutral endopeptidase inhibitor, prevents fatty streak deposit in apolipoprotein E-deficient mice. Atherosclerosis 2001;155:291-295[Medline]
19.
von Der Thusen J. H., van Berkel T. J., Biessen E. A. Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Circulation 2001;103:1164-1170
20. Hayek T., Oiknine J., Brook J. G., Aviram M. Increased plasma and lipoprotein lipid peroxidation in apo E deficient mice. Biochem. Biophys. Res. Commun. 1994;201:1567-1574[Medline]
21. El-Saadani M., Esterbauer N., El-Sayed M., Goyer M., Nassar A. Y., Jurgens G. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J. Lipid Res. 1989;30:627-630[Abstract]
22. Gan K. N., Smolen A., Eckerson H. W., La Du B. N. Purification of human serum paraoxonase/arylesterase: evidence for one esterase catalyzing both activities. Drug Metab. Dispos 1991;19:100-106[Abstract]
23. Maor I., Kaplan M., Hayek T., Vaya Y., Hoffman A., Aviram M. Oxidized monocyte-derived macrophages in aortic atherosclerotic lesion from E0 mice and from human carotid artery contain lipid peroxides and oxysterols. Biochem. Biophys. Res. Commun. 2000;269:775-780[Medline]
24. Aviram M. Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipidemic patients. Biochem. Med. 1983;30:111-118[Medline]
25.
Lowry O. H., Rosenbrough N. J., Farr J. L., Randall R. I. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265-275
26. Bilheimer D. W., Eisenberg S., Levy R. I. The metabolism of VLDL: preliminary in vitro and in vivo observations. Biochim. Biophys. Acta 1972;260:212-221[Medline]
27. Buege J. A., Aust S. D. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302-310[Medline]
28.
Aviram M., Dankner G., Brook J. G. Platelet secretory products increase LDL oxidation enhance its uptake by macrophages and reduce its fluidity. Arteriosclerosis 1990;10:559-563
29. Keidar S., Kaplan M., Shapira C., Brook J. G., Aviram M. LDL from patients with essential hypertension exhibits increased propensity for oxidation and enhanced uptake by macrophages: a possible role for angiotensin II. Atherosclerosis 1994;107:71-84[Medline]
30. Keidar S., Aviram M., Maor I., Oiknine J., Brook J. G. Pravastatin inhibits cellular cholesterol synthesis and increases LDL receptor activity in macrophages: in vitro and in vivo studies. Br. J. Clin. Pharmacol. 1994;38:513-519[Medline]
31.
Ross R. Atherosclerosis-an inflammatory disease. N. Engl. J. Med. 1999;340:115-126
32. Aviram M., Maor I., Keidar S., Hayek T., Oiknine J., Bar-el Y., Adler Z., Kertzman V., Milo S. Lesioned LDL in atherosclerotic apolipoprotein E-deficient transgenic mice and in humans is oxidized and aggregated. Biohem. Biophys. Res. Commun. 1995;216:501-513[Medline]
33. MacKness B., Mackness M. I., Durrington P. N., Arrol S., Evans A. E., McMaster D., Ferrieres J., Ruidavets J. B., Williams N. R., Howard A. N. Paraoxonase activity in two healthy populations with differing rates of coronary heart disease. Eur. J. Clin. Invest. 2000;30:4-10[Medline]
34. Steinbrecher U. P., Zhang H., Laugheed M. Role of oxidatively modified LDL in atherosclerosis. Free Radic. Biol. Med. 1990;9:155-168[Medline]
35. Witztum J. L. The oxidation hypothesis of atherosclerosis. Lancet 1994;344:793-795[Medline]
36. Steinberg D., Parthasarathy S., Carew T. E., Khoo J. C., Witztum J. L. Beyond cholesterol: modifications of LDL that increase its atherogenecity. N. Engl. J. Med. 1989;320:915-924[Medline]
37. Tabas I. The stimulation of the cholesterol esterification pathway by atherogenic lipoproteins in macrophages. Curr. Opin. Lipidol. 1995;6:260-268[Medline]
38. Von Eckardstein A. Cholesterol efflux from macrophages and other cells. Curr. Opin. Lipidol. 1996;7:308-319[Medline]
39. Giugliano D. Dietary antioxidants for cardiovascular prevention. Nutr. Metab. Cardiovasc. Dis. 2000;10:38-44[Medline]
40. Stocker R. Dietary and pharmacological antioxidants in atherosclerosis. Curr. Opin. Lipidol. 1999;10:589-597[Medline]
41.
Parthasarathy S., Santanam N., Ramachandran S., Meilhac O. Oxidants and antioxidants in atherogenesis: an appraisal. J. Lipid Res. 1999;40:2143-2157
42. Aviram M., Fuhrman B. LDL oxidation by arterial wall macrophages depends on the oxidative status in the lipoprotein and in the cells: role of prooxidants vs. antioxidants. Mol. Cell. Biochem. 1998;188:149-159[Medline]
43. Maxwell S. R., Lip G. Y. Free radicals and antioxidants in cardiovascular disease. Br. J. Clin. Pharmacol. 1997;44:307-317[Medline]
44.
Fuhrman B., Rosenblat M., Hayek T., Coleman R., Aviram M. Ginger extract consumption reduces plasma cholesterol, inhibits LDL oxidation and attenuates development of atherosclerosis in atherosclerotic, apolipoprotein E-deficient mice. J. Nutr. 2000;130:1124-1131
45. Schubert S. Y., Lansky E. P., Neeman I. Antioxidant and eicosanoid enzyme inhibition properties of pomegranate seed oil and fermented juice flavonoids. J. Ethnopharmacol. 1999;66:11-17[Medline]
46. Aviram M., Rosenblat M., Bisgaier C. L., Newton R. S., Primo-Parmo S. L., La Du B. N. Paraoxonase inhibits HDL oxidation and preserves its functions: a possible peroxidative role for paraoxonase. J. Clin. Invest. 1998;101:1581-1590[Medline]
47. Aviram M., Hardak E., Vaya J., Mahmood S., Milo S., Hoffman A., Billicke S., Draganov D., Rosenblat M. Human serum paraoxonases Q and R selectively decrease lipid peroxides in human coronary and carotid atherosclerotic lesions: PON1 esterase and peroxidase-like activities. Circulation 2000;30:2510-2517
48. Goldstein J. L., Brown M. S. LDL pathways and its relation to atherosclerosis. Annu. Rev. Biochem. 1978;46:897-930
49. Maor I., Aviram M. Ox-LDL leads to macrophage accumulation of unesterified cholesterol as a result of lysosomal trapping of the lipoprotein hydrolyzed cholesterol ester. J. Lipid Res. 1994;35:803-819[Abstract]
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