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Centre for Research in Vascular Biology, Department of Anatomical Sciences, The University of Queensland, Brisbane, Queensland 4072 Australia
2To whom correspondence should be addressed. E-mail: julie.campbell{at}mailbox.uq.edu.au.
ABSTRACT
The aim of this study was to determine the mechanism by which the aged
garlic extract "Kyolic" has a protective effect against
atherosclerosis. Plasma cholesterol of rabbits fed a 1%
cholesterol-enriched diet for 6 wk was not reduced by
supplementation with 800 µL Kyolic/(kg body · d).
In spite of this, Kyolic reduced by 64% (P < 0.05) the surface area of the thoracic aorta covered by fatty streaks
and significantly reduced aortic arch cholesterol. Kyolic also
significantly inhibited by 
50% the development of thickened,
lipid-filled lesions in preformed neointimas produced by Fogarty 2F
balloon catheter injury of the right carotid artery in
cholesterol-fed rabbits. In vitro studies found that Kyolic
completely prevented vascular smooth muscle phenotypic change from the
contractile, high volume fraction of filament (Vvmyo)
state, and inhibited proliferation of smooth muscle cells in the
synthetic state with a 50% effective dose (ED50) of 0.2%.
Kyolic also slightly inhibited the accumulation of lipid in cultured
macrophages but not smooth muscle, and had no effect on the expression
of adhesion molecules on the surface of the endothelium or the
adherence of leukocytes. It is concluded that Kyolic exerts
antiatherogenic effects through inhibition of smooth muscle phenotypic
change and proliferation, and by another (unclarified) effect on lipid
accumulation in the artery wall.
KEY WORDS: • atherosclerosis • garlic • rabbits • smooth muscle • cholesterol
Extracts of garlic are known to reduce serum cholesterol levels in
humans, inhibit cholesterol biosynthesis, suppress LDL oxidation, lower
plasma fibrinogen and increase fibrinolytic activity (Bordia et al. 1977
, Harenberg et al. 1988
), and thus to
possess antiatherosclerotic properties (Lau et al. 1987
,
Phelps and Harris 1993
). However, atherosclerosis is a
complex disease, which can be described as an excessive inflammatory,
fibro-fatty, proliferative response to damage of the artery wall
involving several cell types, particularly smooth muscle cells
(SMC),3
monocyte-derived macrophages, T-lymphocytes and platelets
(Schwartz et al. 1993
). Many people believe it can be
induced from simple dysfunction of the endothelial lining as occurs
with hyperlipidemia, hypertension or cigarette smoke, causing imbalance
of angiotensin II and nitric oxide production in the artery wall
(Dusting et al. 1998
, Luscher and Noll 1995
). This initiates a cascade of events, which includes
expression of adhesion molecules on the surface of the endothelium, the
oxidation of lipoproteins, monocyte invasion of the vessel wall, foam
cell formation, smooth muscle phenotypic change, and proliferation and
platelet deposition (Ross 1993
).
In this study, we determined the effect of the aged garlic extract
(AGE) Kyolic (Wakunaga Company, Mission Viejo, CA) on the development
of atherosclerosis in a rabbit model of the disease as an extension of
our earlier report (Efendy et al. 1997
), and examined
how it might function at a cellular and molecular level using cell
culture techniques.
MATERIALS AND METHODS
In vivo
At time 0, New Zealand White rabbits (female) had their right
carotid artery de-endothelialized with a 2F Fogarty balloon
catheter to induce the formation of a myointimal thickening
(Efendy et al. 1997
). Two weeks after surgery the
rabbits were divided randomly into four groups (n = 6/group) as follows: Group 1, standard diet (Norco High Fiber pellets
mixed 1:1 with oaten and lucerne chaff); Group 2, standard diet + 800
µL Kyolic/(kg body · d); Group 3: 1%
cholesterol-enriched diet; Group 4: 1% cholesterol-enriched
diet + 800 µL Kyolic/(kg body · d). The 1%
cholesterol diet was prepared by dissolving 200 g cholesterol
(Sigma Chemical, St. Louis, MO) in 1 L diethyl ether, which was mixed
with 10-kg standard pellets. After overnight evaporation of the
solvent, the 2% cholesterol-impregnated pellets were mixed 1:1
with chaff to obtain a 1% cholesterol diet. Rabbits in Groups 2 and 4
were administered Kyolic via a 1-mL syringe inserted between their
front incisors and back molars each morning at the time of feeding.
After a further 6 wk, the rabbits were killed by a lethal dose of
sodium pentabarbitone and the vessels excised and prepared for
morphometric, histologic and biochemical examination.
At time 0, 2, 4, 6 and 8 wk (termination), all rabbits were bled
(2–2.5 mL) via their central ear vein for plasma cholesterol analysis
using a Cobas-Bio centrifugal autoanalyzer (Roche, Basel,
Switzerland) utilizing the commercial diagnostic kit Monotest
Cholesterol (Boehringer Mannheim, Mannheim, Germany). Cholesterol
concentration (mg/g tissue) of the washed and blotted aortic arch was
determined after lipid extraction and purification using a
spectrophotometric assay (Zlatkis et al. 1953
).
Whole lengths of thoracic aorta (3–4 cm) were cut longitudinally, rinsed, fixed in 10% buffered formal saline and stained for 40 min in a saturated solution of Oil-Red-O (Sigma Chemical) in isopropyl alcohol. The percentage of luminal area of the thoracic aorta covered by fatty streaks was then measured using the Mocha 227 Image analysis system (Jandel Scientific, San Raphael, CA). For morphometric analysis of the atherosclerotic lesions formed in the de-endothelialized right carotid artery, the vessel from each rabbit was fixed in 10% buffered formal saline, embedded in paraffin, and 7-µm cross sections were stained with toluidine blue and photographed under light microscopy. The area of intima as a percentage of wall area (intima plus media) was measured using the Mocha 227 Image analysis system.
In vitro
Culture procedure.
SMC from the aortic media of rabbits of mixed strains (Central Animal
Breeding House, University of Queensland, Queensland, Australia) were
dispersed into single cells and plated at 2 x 106
cells/90-mm dish or 4 x 104 cells/well in 24-well
plates in Medium 199 (M199) + 5% fetal calf serum (FCS)
(Campbell and Campbell 1993
). For subculture, cells were
passaged 3 times, then seeded at 4 x 104 cells/well
in 24-well plates.
The macrophage cell line J774 (American Type Culture Collection, from
BALB/C mice) was seeded at 3 x 104 cells/well in
Dulbecco’s modified Eagles medium (DMEM) + 10% FCS. Endothelial cells
and SMC were harvested from the rat thoracic aorta according to
standard procedure (Campbell and Campbell 1993
), plated
in primary culture and passaged 5 times before seeding at 3 x 104 cells/well on glass coverslips in RPMI + 10% FCS + 100
µg/mL heparin (Sigma Chemical) + 20
µg/mL endothelial cell growth supplement (Sigma
Chemical) (endothelial cells) or DMEM + 10% FCS (smooth muscle).
Antibody staining.
Rat endothelial and SMC on coverslips were incubated in 0, 0.1, 1 and
5% Kyolic for 24 h, fixed in methanol at -20°C for 10 min, and
blocked in 1% bovine serum albumin in PBS for 20 min at room
temperature. Antibody to the adhesive molecule ICAM-1 (Sekagaku, Tokyo,
Japan) was added for 2 h; the cells were then washed and incubated
with the fluoresceinated secondary antibody fluorescein isothiocyanate
(FITC) (Silenus, Victoria, Australia) for 1 h, washed and mounted.
Frozen sections of carotid artery were similarly stained with
antibodies to smooth muscle 
-actin (HHF-35, Dako Laboratories, San
Diego, CA).
Lipid uptake. Passaged rabbit aortic SMC and J774 macrophages were incubated for 24 h with DMEM + 0.5% FCS to maximize lipoprotein receptor number. Medium was replaced with DMEM + 5% hyperlipidemic serum (which had been exposed to J774 macrophages for 24 h to minimally oxidize the lipoproteins) plus 0, 0.01, 0.1, 1 and 5% Kyolic and incubated at 37°C for 24 h.
Cells on coverslips were fixed in 10% neutral buffered formalin for 30 min, washed, incubated for 10 min in Oil-Red-O (Sigma) (which renders lipid red), then differentiated in 60% isopropyl alcohol.
Separate wells were used to determine cellular cholesterol
biochemically. Cell pellets from 3 wells were dissolved in 5 mmol/L
NaOH with sonication. Sodium deoxycholate (20%) was added and
incubated for a minimum of 48 h. A fourth well was trypsinized and
the cells counted using a hemocytometer. Cholesterol per
104 cells was determined by spectrophotometric methods
[see Moran et al. (1994)
].
Smooth muscle cell phenotype.
SMC freshly dispersed from the rabbit aorta were seeded at 4.5 x 105 cells/30-mm dish. As soon as the cells had attached and
flattened, 1% Kyolic was added to half of the cultures. All dishes
were fixed in 1% glutaraldehyde on d 5 and prepared for electron
microscopy. For both control and Kyolic-treated cells, the
percentage area of cytoplasm occupied by myofilaments was measured in
60 cells and the mean volume fraction of myofilaments
(Vvmyo) calculated (Campbell et al. 1992
).
Smooth muscle proliferation. Both primary and passaged rabbit aortic SMC were seeded at 4 x 104cells/well. After 24 h, the M199 + 5% FCS was changed to M199 + 0.5% FCS for 24 h to synchronize the cells in G0-G1. The next day, the medium was changed to M199 5% FCS with 0, 0.01, 0.1, 1, 5 and 10% Kyolic; after a further 24 h, [3H-thymidine] at 1.1 MBq/well (Amersham, Little Chalfont, Buckinghamshire, UK) was added for 4 h before the cells were harvested and counted (ß-counter, Beckman Instruments, Fullerton, CA, USA).
Statistical analysis. All statistical analyses were performed using the statistical software package SigmaStat (Jandel Scientific). Comparison of data was carried out with the paired Student’s t test and the Kruskal-Wallis ANOVA on Ranks test where necessary. In all statistical analyses, a P-value of < 0.05 was considered significant. All data are expressed as means ± SEM.
RESULTS AND DISCUSSION
Due to the general unpalatability of the diet, the rabbits fed cholesterol-enriched pellets weighed 20% less than those provided the standard pellets over the 6-wk period, although this was not significant. In addition, with both the standard and cholesterol-enriched diet, the weight gain over 6 wk in the Kyolic-treated groups (Groups 2 and 4) vs. control (Groups 1 and 3, respectively) was slightly less (-16%), but again this was not significant.
Plasma cholesterol in the Group 3 cholesterol-fed rabbits rose significantly compared with the standard diet Group 1 rabbits (6.0 ± 0.6 vs. 1.3 ± 0.2 µmol/L, with no decrease induced by Kyolic administration in either case (Group 4: 6.2 ± 0.7 and Group 2: 1.3 ± 0.1 µmol/L). There was also no change in lipoprotein profile (VLDL, LDL and HDL) with Kyolic administration.
The rabbits fed a standard diet had no fatty lesions on the intimal
surface of their thoracic aortae in the presence or absence of Kyolic.
However, in the cholesterol-fed Group 3 rabbits, 70% of the
luminal aortic surface was covered by lipid-filled lesions, with
only 25% coverage in the Kyolic-treated Group 4 rabbits; this
represents a 64% reduction. Similarly, in Kyolic-treated rabbits
fed a cholesterol-enriched diet, the level of accumulated
cholesterol in the aortic arch was significantly reduced by 26% from
1.7 ± 0.1 to 1.27 ± 0.1 mg cholesterol/g tissue [see
Efendy et al. (1997)
].
There was a neointima 6–7 cell layers thick in the carotid arteries of
rabbits fed a standard diet in the presence and absence of Kyolic. This
was greatly increased in Group 3 rabbits fed a cholesterol-enriched
diet, with a concentric thickening of fibro-fatty plaque. In the
Kyolic-treated, cholesterol-fed Group 4, the neointima was only
half the size of the lesion in Group 3 rabbits (23.8 ± 2.3 vs.
42.6 ± 6.5%, P < 0.05). There were no
lipid-filled cells, and the size of the deep, smooth
muscle-rich region was greatly decreased (Efendy et al. 1997
). This was particularly evident when fresh frozen sections
were stained with FITC-labeled antibodies to smooth muscle
-actin.
We next examined the mechanism by which these in vivo effects might
have occurred. Because there were fewer fatty streaks and thus fewer
subendothelial lipid-laden, monocyte-derived macrophages, we
examined the effect of 0.1, 1 and 5% Kyolic on the adhesion of
leukocytes to the endothelium by scanning electron microscopy and by
the expression of the adhesion molecule ICAM-1 on cultured endothelial
cells. In both cases it was found that Kyolic had no effect (Fig. 1A
,B
). Also, the accumulation of cholesterol (both by spectrophometry and
staining with Oil-Red-O) in cultured macrophages exposed to 5%
hyperlipidemic serum (minimally oxidized) for 24 h at 37°C was
only slightly decreased by the addition of Kyolic at all concentrations
(20 vs. 12 µg/104 cells) (Fig. 1C
,D
), whereas there was no decreased
accumulation of cholesterol in SMC under the same conditions.
|
). In primary cell culture, this change in phenotypic expression from a
cell whose predominant function is contraction to one whose function is
proliferation, synthesis of matrix or accumulation of lipid usually
occurs during the first 5 d after enzyme dispersion into single
cells; this is due to disruption of the heparan sulfate–rich basal
lamina that surrounds each cell (Campbell et al. 1992
).
It is characterized by a significant decrease in the volume fraction of
the smooth muscle cytoplasm occupied by myofilaments, with a
concomitant increase in the volume fraction occupied by synthetic
organelles such as rough endoplasmic reticulum, Golgi and free
ribosomes.
|
), indicating a second, separate mechanism by which Kyolic may inhibit
the development of a proliferative vascular lesion.
|
Thus, in this rabbit model of atherosclerosis, administration of
garlic in the form of the AGE Kyolic causes a decrease in aortic tissue
cholesterol as determined biochemically, a decrease in fatty streak
formation and a decrease in the size of atherosclerotic plaque formed
in the balloon-injured carotid artery. There was no change in
plasma cholesterol level or lipoprotein fractions, only a slight
decrease in lipid accumulation in cultured macrophages, and no change
in ICAM-1 expression or leukocyte adhesion to the endothelium. This
atheroprotective effect, despite no decrease in plasma cholesterol
levels, is consistent with the hypothesis of Lau et al. (1987)
that Kyolic mobilizes accumulated lipid into the blood
stream, where it is cleared from the body. It would also provide an
explanation for the lower weight gain observed in rabbits given Kyolic.
The most significant cellular effect seen with Kyolic was the
maintenance of the contractile smooth muscle cell phenotype under
conditions such as injury, in which change to the proliferative,
synthetic state usually occurs. In the contractile phenotype, which is
the phenotype of smooth muscle in the mature, noninjured vessel wall,
the cells do not migrate, synthesize appreciable matrix or accumulate
lipid. They also do not respond to mitogens present in serum
(Campbell et al. 1992
). All of these functions are
crucial to the development of atherosclerosis. Inhibition of phenotypic
change to the state in which these functions can occur may be a major
mechanism whereby Kyolic exerts its antiatherogenic effect.
ACKNOWLEDGMENTS
The assistance of Barbara Rolfe for ICAM-1 staining is acknowledged, as is the assistance of Anita Thomas in preparation of the manuscript and figures.
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. 
3 Abbreviations used: AGE, aged garlic extract;
DMEM, Dulbecco’s modified Eagles medium; ED50, 50%
effective dose; FCS, fetal calf serum; FITC, fluorescein
isothiocyanate; M199, Medium 199; SMC, smooth muscle cells.
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