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B Activation1 ,2
Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, CA 92350
3To whom correspondence should be addressed. E-mail: bLau{at}som.llu.edu.
ABSTRACT
Oxidative modification of LDL has been recognized as playing an
important role in the initiation and progression of atherosclerosis. In
this study, we determined the effects of aged garlic extract (AGE) and
its major compound, S-allylcysteine (SAC), on oxidized
LDL (Ox-LDL)–induced injury in endothelial cells (EC). Lactate
dehydrogenase (LDH) release as an index of membrane damage,
methylthiazol tetrazoium (MTT) assay for cell viability and
thiobarbituric acid reactive substances (TBARS) indicating lipid
peroxidation were measured. Ox-LDL caused an increase of LDH
release, loss of cell viability and TBARS formation. Both AGE and SAC
prevented all of these changes. To elucidate the mechanism, effects of
AGE or SAC on intracellular glutathione (GSH) level in EC, and release
of peroxide from EC and macrophages (M
) were determined. Ox-LDL
depleted intracellular GSH and increased release of peroxides. Both AGE
and SAC inhibited these changes. Effects of SAC on hydrogen peroxide
(H2O2) or tumor necrosis factor
(TNF)-
–induced nuclear factor (NF)-
B activation were determined.
Pretreatment of EC with SAC inhibited NF-B activation. We
demonstrated that both AGE and SAC can protect EC from Ox-LDL–induced
injury by preventing intracellular GSH depletion in EC and by
minimizing release of peroxides from EC and M. SAC also inhibited
H2O2- or TNF-–induced NF-B activation.
Our data suggest that AGE and its main compound, SAC, may be useful for
prevention of atherosclerosis.
KEY WORDS: • aged garlic extract • S-allylcysteine • oxidized LDL • endothelial cells • cytotoxicity
Cardiovascular disease is one of the most serious diseases among people
living a Western life style. Over the past two decades, a strong
association between elevated plasma LDL and the development of
atherosclerosis has been established (Kannel et al. 1971
). More recently, oxidation of LDL has been shown to
contribute to the initiation and progression of atherosclerosis
(Cox and Cohen 1996
, Steinberg et al. 1989
). LDL oxidation occurs when it is exposed to free radicals
released by surrounding cells such as smooth muscle cells or
monocytes/macrophages
(MÖ)4
(Cathcart et al. 1985
, Darley-Usmar et al. 1992
, Henriksen et al. 1981 and 1983
,
Rosenfeld et al. 1990
). Oxidized LDL (Ox-LDL) is taken
up by MÖ, resulting in the formation of cholesterol-loaded
foam cells and the fatty streak, a primary histologic feature of
incipient atherosclerosis (Gerrity 1981
). Ox-LDL
promotes vascular dysfunction by exerting direct cytotoxicity toward
endothelial cells (EC), by increasing monocyte chemotactic properties
and by decreasing motility of tissue MÖ (Kuzuya et al. 1991a
, Quinn et al. 1987
). Ox-LDL also
enhances the production and release of inflammatory mediators such as
reactive oxygen species, tumor necrosis factor (TNF)-, interleukin
(IL)-6, arachidonic acid metabolites and nitric oxide (NO)
(Durum and Oppenheim 1989
, Fu et al. 1990
, Marletta et al. 1988
). As second
messengers, these mediators stimulate cells to activate transcription
factors regulated by the intracellular redox state and promote the
development of inflammation leading to injury of surrounding cells and
tissues.
Nuclear factor (NF)-B is a well-known transcription factor
activated by oxidative stress. NF-B is a heterodimeric transcription
factor complex composed of two DNA-binding subunits, p50 and p65,
and it is associated with the regulation of numerous genes encoding
proteins in immune function, inflammation and cellular growth control
(Grimm and Baeuerle 1993
). Under stressed conditions in
EC, activation of NF-B leads to the expression of cell adhesion
factors such as vascular cell adhesion molecule-1 (VCAM-1) and
intercellular adhesion molecule-1 (ICAM-1) (Geng et al. 1997
, Sen and Packer 1996
). These events further
accelerate the formation of atherogenic lesions and cell death.
However, recent studies have shown that antioxidants can inhibit
oxidant-induced NF-B activation (Meyer et al. 1992
, Sen et al. 1996
, Schreck et al. 1992
), protect EC (Kuzuya et al. 1991b
,
Schmitt et al. 1995
) and normalize vascular functioning
in hypercholesterolemia and atherosclerosis (Anderson et al. 1995
, Keaney et al. 1994
, Stewart-Lee et al. 1994
).
Garlic (Allium sativum L.) is one of the oldest plants used
as a medicine; it has been considered a valuable healing agent by many
different cultures for thousands of years, particularly for treating
heart disease (Koch and Lawson 1996
). Aged garlic
extract (AGE) is a garlic preparation produced by a unique aging
process. Bioactivities of AGE and its major compound,
S-allylcysteine (SAC), whose structure is shown in
Figure 1
, include antioxidant (Imai et al. 1994
),
anticarcinogenic (Amagase and Milner 1993
, Hatono et al. 1996
), antiatherogenic (Steiner and Lin 1998
, Efendy et al. 1997
), immunostimulatory
(Abdullah et al. 1989
, Lau et al. 1991
),
liver protective (Nakagawa et al. 1986
) and antiaging
effects (Moriguchi et al. 1996
). Recent data from our
laboratory have shown that AGE and SAC protect vascular EC from
H2O2-induced injury
(Yamasaki et al. 1994
) and inhibit
Cu2+-induced LDL oxidation (Ide et al. 1997
). We have also demonstrated that AGE modulates the
glutathione (GSH) redox cycle (Geng and Lau 1997
), and
SAC inhibits NF-B activation in human T cells (Geng and Lau 1997
).
|
B
activation in EC.
MATERIALS AND METHODS
Chemicals.
AGE and SAC were provided by Wakunaga Pharmaceutical (Osaka, Japan).
n-Butanol, pyridine and endothelial cell growth
supplement (ECGS) were from Fisher Scientific (Fair Lawn, NJ).
Horseradish peroxidase and
2,2'-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) were
obtained from Boehringer Mannheim (Indianapolis, IN). Cupric sulfate
(CuSO4 · 5H2O) was from J. T. Baker
Chemical (Phillisburg, NJ). 2'7'-Dichlorofluorescin diacetate (DCFH-DA)
was purchased from Molecular Probes (Eugene, OR). The CytoTox96
Nonradioactive Cytotoxicity Assay and Gel Shift Assay System kits were
supplied by Promega (Madison, WI). Specific antibodies to NF-B
subunits p50 and p65 were purchased from Santa Cruz Biochemistry (Santa
Cruz, CA). [
-32P]ATP was obtained from ICN
Biochemicals (Irvine, CA). Dulbecco’s modification of Eagle’s medium
(DMEM), Eagle’s minimum essential medium (EMEM), trypsin-EDTA
solution and penicillin-streptomycin solution were from Mediatech
(Washington DC). Bovine calf serum (BCS) and fetal bovine serum (FBS)
were obtained from HyClone Laboratories (Logan, UT). All other
chemicals were purchased from Sigma Chemical (St. Louis, MO).
Aged garlic extract (AGE).
AGE manufactured under a license issued by the Ministry of Health and
Welfare of Japan was formulated using the following steps: sliced raw
garlic (Allium sativum L.) was dipped into aqueous
ethanol and extracted over 10 mo at room temperature. An analysis of
AGE showed the following (calculated as dry weight):
S-allylcysteine (1.6–2.4 mg/g), alliin (1.7 mg/g),
allicin (<0.01 mg/g) and ajoene (<0.01 mg/g) (Imai et al. 1994
, Moriguchi et al. 1996
).
Cell lines.
Bovine pulmonary artery EC (PAEC), human umbilical vein EC (HUVEC) and murine macrophage cell line (J774) were obtained from the American Type Culture Collection (Rockville, MD). PAEC, HUVEC and J774 were grown in EMEM with 20% BCS, in Ham’s F-12K with 0.1 g/L heparin, 0.04 g/L ECGS and 10% FBS, and in DMEM with 10% BCS, respectively. The media were supplemented with 200 U/mL penicillin, and 0.2 g/L streptomycin. Cells were incubated at 37°C in a humidified 5% CO2 atmosphere for 3–4 d before experimental use. Viability of cells used throughout the experiment was always >95% as determined by trypan blue exclusion.
Preparation of Ox-LDL.
LDL was dialyzed at 4°C for 48 h against 500 volumes of PBS to
remove EDTA. For preparation of Ox-LDL, LDL (5 g/L) was incubated
with 20 µmol/L CuSO4 at 37°C for 24 h and then dialyzed at 4°C for 48 h against 500 volumes of PBS
to remove Cu2+ (Kuzuya et al. 1991a
).
Ox-LDL was confirmed using agarose gel electropholesis
(Nobel 1968
). Protein content of Ox-LDL was
determined (Lowry et al. 1951
).
Lactate dehydrogenase (LDH) release.
PAEC (8 x 104 cells/well) in 24-well plates were preincubated with different concentrations of AGE (1, 2.5 or 5 g/L) or SAC (20, 10, 1 or 0.1 mmol/L) for 24 h, washed with Hank’s balanced salt solution (HBSS), and then incubated with 0.1 g/L Ox-LDL in HBSS for 24 h. LDH activity was measured by using CytoTox96 Nonradioactive Cytotoxicity Assay kit, following the manufacturer’s instruction. The absorbance was determined at 492 nm in an ELISA reader (400 AT EIA, Whittaker Bioproducts, Walkersville, MD). The percentage of LDH released from the cells was determined using the formula: % release = LDH activity in supernatant/(LDH activity in supernatant + LDH activity in cell lysate).
MTT assay for cell viability.
PAEC (8 x 103 cells/well) in 96-well plates were
preincubated with different concentrations of AGE or SAC for 24 h,
washed with HBSS and then incubated with 0.1 g/L Ox-LDL in HBSS for
24 h. Cell viability was determined as previously described
(Ide and Lau 1997
). The absorbance was measured at 620
nm using the ELISA reader.
Lipid peroxidation in EC.
PAEC (8 x 104 cells/well) in 24-well plates were
preincubated with different concentrations of AGE or SAC for 24 h,
washed with HBSS and then incubated with 0.1 g/L Ox-LDL in HBSS for
24 h. The extent of lipid peroxidation was determined by measuring
thiobarbituric acid reactive substances (TBARS) as previously described
(Ide and Lau 1997
). The fluorescence intensity was
measured with excitation of 515 nm and emission of 553 nm, using LS-3
Fluorescence Spectrophotometer (Perkin-Elmer, Norwalk, CT). The value
of fluorescence was calculated by comparing with standards prepared
from tetraethoxypropane.
Intracellular GSH.
Intracellular GSH was determined according to the method of
Sedlak and Lindsay (1968)
. PAEC (4 x 106 cells) in 25 cm2 flasks were preincubated
with different concentrations of AGE or SAC for 24 h, washed with
HBSS and then incubated with 0.1 g/L Ox-LDL in HBSS for 24 h.
The absorbance was then measured at 412 nm using the Spectronic 2000
spectrophotometer (Bausch & Lomb, Rochester, NY). The GSH level was
compared with that of EC without exposure to Ox-LDL and expressed
as a percentage of the control.
Peroxides released from EC treated with Ox-LDL.
Peroxides were measured by a fluorometric assay using DCFH-DA as a
probe (Wan et al. 1993
). DCFH-DA, a nonfluorescent
compound, is deacetylated by viable cells to highly fluorescent
2'7'-dichlorofluorescein (DCF) by hydrogen peroxide and lipid
peroxides. Confluent PAEC (8 x 104 cells/well) in
24-well plates were incubated with 0.2 mL of different concentrations
of AGE or SAC in HBSS, 0.1 g/L Ox-LDL and 10 µL of
0.5 mmol/L DCFH-DA. The fluorescence intensity (relative
fluorescence unit) was measured at 485 nm excitation and 530 nm
emission every 30 min for 3 h, using the 7620 Microplate
Fluorometer (Cambridge Technology, Watertown, MA).
Peroxides released from MÖ treated with Ox-LDL.
The assay is essentially the same as described for EC. Harvested J774 cells (2 x 105 cells/well) were incubated for 2 h in 96-well plates. After incubation, the media were removed, and cells were washed and incubated with 0.2 mL of AGE (1, 2.5 or 5 g/L) or SAC (20, 10, 1 or 0.1 mmol/L) in HBSS, 0.1 g/L Ox-LDL, and 10 µL of 0.5 mmol/L DCFH-DA. The fluorescence intensity (relative fluorescence unit) was measured at 485 nm excitation and 530 nm emission every 30 min for 3 h, using the 7620 Microplate Fluorometer (Cambridge Technology).
H2O2 scavenging assay.
The scavenging effects of AGE and SAC on H2O2
were determined according to the method of Okamoto et al. (1992)
; H2O2 (0.01 mL
of 50 nmol/L), 0.1 mL of different concentrations of samples, 0.6 mL of
10 U/mL peroxidase, and 0.6 mL of 0.1% ABTS were added to
1.8 mL of 0.1 mol/L phosphate buffer (pH 6.0). The solution was then
incubated at 37°C for 15 min. Absorbance at 414 nm was measured using
the Spectronic 2000 spectrophotometer (Bausch & Lomb).
Preparation of nuclear protein extracts.
HUVEC (1 x 107 cells) in 75 cm2 flasks
were preincubated with different concentrations of SAC for 24 h,
washed with HBSS and then incubated with 10 ng/mL TNF- or 50
µmol/L H2O2 for 2 h.
Cells were washed with HBSS, and nuclear protein extracts were prepared
as previously described (Geng et al. 1997
). The nuclear
protein extract was stored at -70°C until the experiments. Protein
concentration was determined by using the Bio-Rad protein assay
reagent.
Electrophoretic mobility shift assay (EMSA).
Labeling of NF-B oligonucleotide and EMSA were performed according
to the manufacturer’s instructions for the Gel Shift Assay System kit.
The DNA-protein complex was analyzed using 6% polyacrylamide gel
(16 x 18 cm) at room temperature in 0.5X TBE (pH 8.0) for 2–2.5
h at 150 V. Gels were vacuum dried and exposed to X-ray film
overnight at -70°C. Films were scanned by Desk Scan II program
(Hewlett-Packard, Boise, ID). Relative intensity of NF-B bands was
quantified by densitometry scanning of autoradiography using the Bio
Image Whole Band Analyzer, version 3.0 (Millipore Corporation, Ann
Arbor, MI).
Statistical analysis.
Data were analyzed using one-way ANOVA followed by Turkey’s multiple range test for significant difference; results were expressed as the mean ± SEM. A P-value of < 0.05 was considered significant. All statistical procedures were performed with Statgraphics software version 5.0 (STSC, Rockville, MD).
RESULTS
Effects of AGE and SAC on Ox-LDL–induced cell injury.
LDH is an intracellular enzyme that leaks into the culture medium when
cell membranes are damaged. Figure 2
shows the respective effects of AGE or SAC on LDH release from PAEC.
The exposure to Ox-LDL caused more than a fourfold increase in LDH
release compared with unexposed cells, indicating that Ox-LDL
induced cell damage. Pretreatment with AGE at 1, 2.5 and 5 g/L
inhibited LDH release by 11.3, 35.1 and 50.5%, respectively. SAC
exhibited a dose-dependent inhibition of LDH release ranging from
10.4 to 91.0%, and the inhibition observed at the higher dosages was
significant.
|
). When PAEC were treated with Ox-LDL
for 24 h, there was a significant decrease in cell viability
compared with untreated cells. Pretreatment with AGE or SAC resulted in
a dose-dependent increase in cell viability in Ox-LDL–treated
cells. The increase was significant with AGE at 2.5 and 5 g/L
(Fig. 3A
), and with SAC at all four concentrations (Fig. 3B
).
|
).
|
GSH is the most abundant low-molecular-weight thiol compound in cells,
and plays an important role in antioxidant defense and detoxification.
Figure 4
shows the effects of AGE or SAC on GSH level when cells were exposed to
0.1 g/L Ox-LDL. Ox-LDL caused a decrease of 60% in intracellular
GSH compared with cells not exposed to Ox-LDL. Pretreatment of EC
with AGE or SAC resulted in a dose-dependent inhibition of
intracellular GSH depletion.
|
Excess peroxides such as hydrogen peroxide and lipid peroxide change
cell function and interaction with surrounding cells, and lead to cell
dysfunction and death. In this study, Ox-LDL–induced peroxides in PAEC
were measured by a fluorometric method using DCFH-DA as a probe
(Wan et al. 1993
). DCFH-DA, a nonfluorescent
compound, is deacetylated by viable cells to DCF by hydrogen peroxide
and lipid peroxides. Figure 5
shows the effects of AGE or SAC on Ox-LDL–induced release of peroxides
in EC. Exposure of PAEC to Ox-LDL resulted in a significant release
of peroxides. Coincubation of PAEC with AGE and Ox-LDL exhibited a
dose-dependent inhibition of peroxide release.
|
Macrophages undergo an oxidative burst in response to phagocytic or
membrane stimuli, with production and release of a variety of reactive
oxygen metabolites such as superoxide anion, hydrogen peroxide,
hydroxyl radical and NO, leading to cell dysfunction and death
(Fantone and Ward 1982
). In this study, effects
of AGE and SAC on Ox-LDL–induced peroxides released from MÖ were
determined. Exposure of Ox-LDL to J774 cells caused a significant
release of peroxides. Coincubation of J774 cells with AGE or SAC and
Ox-LDL inhibited the release of peroxides in a dose-dependent
manner (Fig. 6
).
|
Table 2
shows the direct scavenging effects of AGE or SAC on hydrogen peroxide.
A decrease of hydrogen peroxide reflected scavenging by AGE or SAC with
significant activity observed at all concentrations except for 0.1
mmol/L of SAC.
|
–induced
NF-B activation.
NF-B is a heterodimeric transcription factor complex composed of two
DNA-binding subunits, p50 and p65, and it is associated with the
regulation of numerous genes encoding proteins in immune function,
inflammation and cellular growth control (Grimm and Baeuerle 1993
). Incubation of HUVEC with 10 ng/mL TNF- activated
NF-B expression. Preincubation of HUVEC with SAC inhibited the
activation (Fig. 7
). Incubation of HUVEC with 50 µg/mL H2O2 also activated
NF-B expression. Preincubation of HUVEC with SAC showed a trend
toward inhibited expression (data not shown).
|
DISCUSSION
It has been known for more than two decades that an elevated
plasma level of LDL is associated with the development of
atherosclerosis (Kannel et al. 1971
). More recently, LDL
oxidation has been recognized as playing an important role in the
initiation and progression of atherosclerosis (Berliner and Heinecke 1996
, Cox and Cohen 1996
,
Steinberg et al. 1989
). LDL has been shown to be
oxidized by cultured cells such as MÖ, endothelial and smooth
muscle cells with transition metals. Ox-LDL exerts several
biological effects that may contribute to the initiation and
progression of the atherosclerotic process, including such events as
chemotaxis for monocytes, inhibition of macrophage motility, formation
of foam cells, up-regulation of endothelial adhesion molecules,
stimulation of growth factors and chemokines, and proliferation of
smooth muscle cells (Holvoet and Collen 1995
).
Ox-LDL also appears to initiate vascular dysfunction by directly
exerting cytotoxicity. It can alter the composition and permeability of
the endothelial barrier (Guretzki et al. 1994
) and is
thus cytotoxic for EC (Kuzuya et al. 1989
,
Schmitt et al. 1995
).
In this study, the antioxidant effects of AGE and one of its major
compounds, SAC, on pulmonary artery EC were studied using a model of
oxidant injury induced by Ox-LDL. Ox-LDL caused significant cell
damage, as evidenced by the increase in LDH release. LDH is an
intracellular enzyme that leaks from cells when their membranes are
damaged. Data indicated that preincubation of PAEC with AGE or SAC
significantly inhibited the increase in LDH release induced by
Ox-LDL, showing the protective effects of these compounds on cell
membranes. The MTT assay was used to monitor cell viability. Because
MTT is cleaved only in active mitochondria (Mosmann 1983
), Ox-LDL–induced decrease of cell viability measured by
the MTT assay indicates that Ox-LDL may have damaged the
mitochondria of PAEC. Pretreatment of PAEC with AGE or SAC resulted in
a concentration-dependent increase in cell viability, suggesting a
protective effect on the mitochondria of EC. To elucidate the mechanism
of cell injury, TBARS, products of lipid peroxidation, were measured in
Ox-LDL–stressed PAEC. The exposure of PAEC to Ox-LDL resulted in a
significant increase in TBARS. Pretreatment of PAEC with AGE or SAC
inhibited TBARS formation, indicating protection against lipid
peroxidation. These data suggest that AGE and SAC can protect EC from
Ox-LDL–induced injury.
Ox-LDL has been shown to deplete intracellular GSH in cultured EC
(Schmitt et al. 1995
). Intracellular GSH depletion can
lead to increased endothelial cell susceptibility to injury caused by
Ox-LDL (Kuzuya et al. 1989
). GSH is the most
abundant low-molecular-weight thiol compound in cells, and plays an
important role in antioxidant defense and detoxification. GSH depletion
compromises cell defenses against oxidative damage and may lead to cell
death (Reed and Farris 1984
). Incubation of PAEC with
Ox-LDL for 24 h caused a 60% decrease in intracellular GSH.
Preincubation of PAEC with AGE or SAC prevented intracellular GSH
depletion. These data suggest that one of the mechanisms by which AGE
or SAC inhibits Ox-LDL–induced cell injury is by preventing
intracellular GSH depletion. It has previously been reported that AGE
can modulate the GSH redox cycle by maintaining intracellular GSH
levels (Geng and Lau 1997
), thus possibly protecting EC
from cytotoxicity.
Under oxidant-stressed conditions, peroxides such as hydrogen
peroxide and lipid peroxides change cell functions and their
interaction with surrounding cells. This can lead to cell dysfunction
and death. For instance, MÖ undergo an oxidative burst in
response to phagocytic or membrane stimuli, with production and release
of a variety of reactive oxygen metabolites (Fantone and Ward 1982
). Lysophosphatidylcholine, which is a lipid
peroxide–composed Ox-LDL, can also trigger inflammation and lead
to the generation of inflammatory mediators such as reactive oxygen
species, cytokines (e.g., TNF- and IL-6), arachidonic acid
metabolites and NO (Durum and Oppenheim 1989
, Fu et al. 1990
, Marletta et al. 1988
). In this
study, Ox-LDL–induced peroxides in MÖ were measured by a
fluorometric method using DCFH-DA as a probe. Incubation of
MÖ with Ox-LDL caused an increase of peroxides, and AGE or
SAC inhibited it in a dose-dependent manner. These data suggest
that AGE or SAC may have suppressed the release of peroxides and/or
prevented their uptake by MÖ.
The effects of AGE and SAC on Ox-LDL–induced release of peroxides from PAEC were also determined. Incubation of PAEC with Ox-LDL caused an increase of peroxides, and AGE or SAC inhibited it in a dose-dependent manner. These data suggest that AGE or SAC either removed peroxide, such as hydrogen peroxide and lipid peroxides from the EC or inhibited their release. Thus, AGE and SAC can minimize intracellular oxidative stress not only by modulating intracellular GSH level in EC, but also by either removing peroxides or preventing their formation in EC and MÖ.
It has been reported previously that Ox-LDL and inflammatory
mediators such as TNF- and hydrogen peroxide serve as important
second messengers in the activation of transcription factor NF-B.
NF-B is associated with expression of cell adhesion factors, VCAM-1
and ICAM-1 (Geng et al. 1997
, Sen and Packer 1996
). In this study, the effect of SAC on
H2O2- or TNF-–induced
NF-B activation was studied. Incubation of HUVEC with
H2O2 or TNF- for 2 h activated NF-B. SAC inhibited this activation. These data suggest
that SAC can minimize intracellular oxidative stress; furthermore, it
can inhibit NF-B activation by modulating intracellular signal
transduction.
Garlic (Allium sativum L.) has long been considered a
valuable healing agent by many different cultures. Even today it is
commonly used by much of the world, especially eastern Europe and Asia,
for its medical benefits. Aged garlic extract (AGE) is an original
garlic preparation produced by a 10-mo aging process. Numerous
bioactivities and efficacies have been reported since its inception in
1955 with >200 scientific publications. AGE has been shown to lower
blood cholesterol and triglycerides in human subjects (Lau et al. 1987
). More recently, it showed a trend toward reduced LDL
oxidation susceptibility in an ex vivo study (Steiner and Lin 1998
). Further, it reduces fatty streak development, vessel
wall cholesterol accumulation and the development of fibro-fatty
plaques in neointimas of cholesterol-fed rabbits (Efendy et al. 1997
).
SAC is one of the major water-soluble compounds derived from AGE.
Numerous biological activities have been reported for SAC
(Amagase and Milner 1993
, Geng et al. 1997
, Hatono et al. 1996
, Sumiyoshi and Wargovich 1990
, Welch et al. 1992
). Further, its
bioavailability has been well established in animals. It has been shown
to be evenly absorbed and distributed systemically (Nagae et al. 1994
). These reports demonstrate that SAC is a useful compound
with many biological activities and high bioavailability as well as an
original marker compound in AGE.
In conclusion, we demonstrated that AGE and SAC can protect EC from
Ox-LDL–induced injury by preventing the depletion of intracellular GSH
and by removing peroxides. SAC also inhibited
H2O2- or TNF-–induced
NF-B activation. These data suggest that AGE and SAC can be
protective agents against cytotoxicity associated with Ox-LDL, and
may be useful for the prevention of atherosclerosis.
FOOTNOTES
1 Presented at the conference "Recent Advances
on the Nutritional Benefits Accompanying the Use of Garlic as a
Supplement" held November 15–17, 1998 in Newport Beach, CA. The
conference was supported by educational grants from Pennsylvania State
University, Wakunaga of America, Ltd. and the National Cancer
Institute. The proceedings of this conference are published as a
supplement to The Journal of Nutrition. Guest editors:
John Milner, The Pennsylvania State University, University Park, PA and
Richard Rivlin, Weill Medical College of Cornell University and
Memorial Sloan-Kettering Cancer Center, New York, NY. 
2 Supported by the Chan Shun International
Foundation, San Francisco, CA and Wakunaga Pharmaceutical, Osaka,
Japan.
4 Abbreviations used: ABTS,
2,2'-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid]; AGE, aged
garlic extract; BCS, bovine calf serum; DCF, 2'7'-dichlorofluorescein;
DCFH-DA, 2'7'-dichlorofluorescin diacetate; DMEM, Dulbecco’s
modification of Eagle’s medium; EC, endothelial cells; ECGS,
endothelial cell growth supplement; EMEM, Eagle’s minimum essential
medium; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine
serum; GSH, glutathione; HUVEC, human umbilical vein endothelial cells;
ICAM-1, intercellular adhesion molecule-1; IL-6, interleukin 6; LDH,
lactate dehydrogenase; MÖ, macrophages; MTT, methylthiazol
tetrazoium; NF-B, nuclear factor-B; Ox-LDL, oxidized LDL;
PAEC, pulmonary artery endothelial cells; SAC,
S-allylcysteine; TBARS, thiobarbituric acid reactive
substances; TNF-, tumor necrosis factor-; VCAM-1, vascular cell
adhesion molecule-1.
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