© 1995 American Heart Association, Inc.
Articles |
Reduction of Serum Cholesterol Levels Alters Lesional Composition of Atherosclerotic Plaques
Effect of Pravastatin Sodium on Atherosclerosis in Mature WHHL Rabbits
From the Institute for Experimental Animals, Kobe University School of Medicine (M.S., T.I., T.Y., Y.W.); the Second Department of Internal Medicine, Sanraku Hospital (T.T.); and the Pharmacology and Molecular Biology Research Laboratories (Y.T.), Biological Research Laboratories (M.F., J.F., T.H.), and the New Drug Development Department (A.T.), Sankyo Co, Ltd, Tokyo, Japan.
Correspondence to Masashi Shiomi, PhD, Institute for Experimental Animals, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
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Abstract |
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Abstract We examined whether serum cholesterol reduction alters the lesional composition of atherosclerotic plaques. To reduce serum cholesterol levels, we gave pravastatin sodium, a 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor, to mature Watanabe heritable hyperlipidemic rabbits, an LDL receptor–deficient animal model, for 48 weeks. Atherosclerotic lesions were immunohistochemically and conventionally stained and each lesional component area was measured by a color image analyzer. Compared with those of a placebo group, serum LDL cholesterol levels were reduced by 22% (P<.05). Data for atherosclerosis indicated a significant decrease in percent of surface lesion area (26% reduction) and in intimal thickening (30% reduction) in the abdominal aorta, as well as in coronary stenosis (29% reduction). Data for lesional composition indicated a significant decrease in the percent area of macrophage plus extracellular lipid deposits in aortic lesions (32% reduction) and coronary lesions (45% reduction). A significant increase was observed in the percent area of collagen in aortic lesions and in the percent area of smooth muscle cells in coronary lesions. The plaques seemed to become stable lesions as a result of pravastatin treatment. In conclusion, a long-term reduction of serum LDL cholesterol reduced lipid-related lesional components, in addition to suppressing the progression of established atherosclerosis.
Key Words: cholesterol reduction • macrophages • smooth muscle cells • atherosclerosis • WHHL rabbit
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Introduction |
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It is well recognized that a high plasma LDL level is one of the most important risk factors for atherosclerosis. A large amount of peroxidized LDL was observed in atherosclerotic lesions by immunohistochemical studies that used several types of monoclonal antibodies.1 2 3 These peroxidized LDLs are considered to be derived from LDLs infiltrated from the circulation. Therefore, reduction of the serum cholesterol level is one of the most effective strategies for the prevention of coronary heart diseases.
We have studied the effects of serum cholesterol reduction on atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of LDL receptor deficiency,4 5 6 treated with pravastatin sodium (pravastatin), a competitive inhibitor for 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase.7 Treatment with pravastatin reduced serum cholesterol levels in WHHL rabbits by about 30%,7 8 9 10 and suppressed the progression of coronary atherosclerosis in young rabbits.8 Next, mature WHHL rabbits with established atherosclerosis were administered pravastatin in combination with cholestyramine, a resin of bile acid sequestrant, for 8 months. Serum cholesterol levels were reduced by 60%, and established atherosclerosis in the aorta and coronary arteries was significantly suppressed.11 In these studies, we observed that the lesional composition in atherosclerotic lesions changed as a result of the drug treatments.
Several years ago, Tsukada et al obtained monoclonal antibodies: HHF35, specific for muscle actin,12 and RAM-11, specific for rabbit macrophages.13 It is well known that macrophages and smooth muscle cells play an important role in atherogenesis. In atherosclerotic lesions of the aorta in WHHL rabbits, macrophages and smooth muscle cells were recognized by HHF35 and RAM-11.13 14 15 16 17 Recently we quantitatively evaluated the components of the atherosclerotic lesions in WHHL rabbits stained with these monoclonal antibodies, using a color image analyzer.16 17 These monoclonal antibodies are useful in the analysis of lesional components of atherosclerosis.
In studies of human atherosclerosis, reduction of serum cholesterol levels by treatment with inhibitors of HMG-CoA reductase was shown to decrease the development of coronary events18 19 20 or prevent the progression of coronary atherosclerosis.21 22 However, histopathological changes of the atherosclerotic lesions have not been sufficiently examined. Recently Brown et al23 proposed that the reduction of LDL in the circulation would lead to a decrease in plaque rupture. However, there are no data that support their hypothesis. Therefore, it is important to confirm changes in lesional composition of the plaques.
We examined whether serum cholesterol reduction can change lesional composition of atherosclerotic plaques in WHHL rabbits. In this study, each lesional component in atherosclerotic lesions was immunohistochemically and conventionally stained and was quantitatively analyzed with a color image analyzer.
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Methods |
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Animals
The WHHL rabbits used in this study were bred at Kobe University. These rabbits have a high incidence of severe coronary atherosclerosis.24 Ten-month-old rabbits, 25 in total, were selected on the basis of their serum cholesterol levels, which ranged from 13.0 to 18.1 mmol/L, to make the degrees of atherosclerosis equal at the start of the experiment. These rabbits were divided into three groups to make their serum cholesterol levels uniform: eight rabbits (six males and two females) in a control group, nine rabbits (six males and three females) in a placebo group, and eight rabbits (eight males) in a treatment group. We used animals of both sexes because there are no sex-related differences in atherosclerosis in WHHL rabbits.8 11 24 The rabbits in the treatment group were given pravastatin orally in aqueous solution at a dose of 50 mg · kg-1 · d-110 for 48 weeks. In the placebo group, rabbits were given distilled water orally instead of pravastatin solution. For examination of a lesion progression from the start of the experiments, rabbits in the control group were killed at the age of 10 months. The suppressive effect of pravastatin on the progression of atherosclerosis was estimated by comparison of the lesions in the placebo and treatment groups. During the experiment the rabbits were housed individually in metal cages in a room maintained at a constant temperature and humidity, and they were fed 120 g standard rabbit chow (CR-3, Clea Japan Inc) per day. All animal experiments were conducted according to the guidelines for animal experimentation at the Kobe University School of Medicine.
Preparation of Histological Sections
Rabbits were anesthetized with intravenous
injection of sodium pentobarbital (25 mg/kg) and perfused with lactated
Ringer's solution and then Bouin's fixative by use of a perfusion
apparatus at a constant pressure of 100 mm Hg. After
perfusion-fixation, the aortas and hearts were excised and then
immersed in Bouin's fixative for at least 24 hours. After
immersion-fixation, cross-sections of aortas and
coronary arteries were prepared according to the method
described previously.11 24 In brief, aortic
atherosclerosis was examined at five locations: the
aortic arch, the proximal and distal parts of the thoracic aorta, and
the proximal and distal parts of the abdominal aorta. The segments that
showed macroscopically the most severe lesions in each portion were
embedded in paraffin, and 10 sections, 4 µm in thickness, were cut
serially from each segment. Coronary
atherosclerosis was examined in the main trunk of the
left coronary artery, the origin portion of the right
coronary artery, the left anterior descending artery, the left
circumflex artery, and the right coronary artery. The hearts were
divided into six blocks and the blocks were embedded in paraffin. The
blocks containing the main trunk of the left coronary artery
and the origin portion of the right coronary artery were
sectioned at 200-µm intervals and the other blocks were sectioned at
500-µm intervals. In total, about 50 segments from each heart were
examined, and 10 sections, 4 µm in thickness, were cut serially from
each segment with atherosclerotic lesions.
Serial sections from each segment were immunohistochemically or conventionally stained. Immunostaining with monoclonal antibodies HHF35,12 a smooth muscle–specific antibody, and RAM-11,13 a macrophage-specific antibody, was performed by use of the method reported by Tsukada et al.13 In brief, each monoclonal antibody was applied to sections in a dilution of 1:1000 and immunocytochemical staining was carried out with an avidin–biotinylated enzyme complex procedure by use of a Vectastain ABC-PO kit (Vector Laboratories Inc). The sections were also stained with elastic van Gieson's, Azan-Mallory's, and von Kossa stains.
Quantitative Analysis of Atherosclerotic
Lesions
All parameters for atherosclerotic lesions were
measured by a color image analyzer, the SP-500 (Olympus). The
severity of aortic atherosclerosis was estimated as a
ratio of surface area of lesion to the intimal surface area of
aorta8 and an average intimal thickening,11
and the severity of coronary atherosclerosis
was estimated as the most severe coronary stenosis
(lesion area/[lesion area+luminal area]) at each
artery24 according to the method previously described.
Intimal lesion area was estimated by use of sections treated with
elastic van Gieson's stain. In addition to estimating of
coronary stenosis by using the most severe lesions, we
also analyzed all coronary lesions using the following
scores: no lesion, 0 points; stenosis of .20 or less, 1 point;
stenosis of .20 to .40, 2 points; stenosis of .40 to
.60, 3 points; stenosis of .60 to .80, 4 points; and
stenosis of over .80, 5 points. These points were summed for
each artery, and the point total for each rabbit was also counted.
Lesional components were quantitatively evaluated according to the method described previously.16 17 In brief, each section was observed under the color image analyzer at a magnification of x56 to x280, and the area of each lesional component was measured for estimation of the quality of the lesions. We defined cells with black reaction products after HHF35 staining as smooth muscle cells, and cells with black reaction products after RAM-11 staining as macrophages. In sections treated with Azan-Mallory's stain, fibers with three prime color elements (red, green, and blue) in the color image analyzer were identified as being elastin, whereas fibers having only a blue element were considered to be collagen. Extracellular vacuoles and lacunae with Azan-Mallory's stain were defined as extracellular lipid deposits. We did not perform lipid-specific staining in order to exclude intracellular lipids involved in foam cells and small lipid droplets associated with fibers.
Measurement of Serum Cholesterol Levels and
Fractionation of Lipoproteins
Blood samples were withdrawn for measurement of serum
cholesterol levels, after overnight fasting, every 4 weeks.
Total cholesterol levels were measured enzymatically.
Before and at the end of pravastatin treatment, lipoprotein
was fractionated by ultracentrifugation as
described previously.7
Statistical Analysis
Data were represented as mean±SEM. Statistical
analysis was carried out by use of the Mann-Whitney
U test for the atherosclerotic lesions and Welch's
t test or Student's t test for the lipid
levels.
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Results |
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Serum and Lipoprotein Cholesterol Levels
Table 1
shows total serum and lipoprotein
cholesterol levels in the three groups. At the start of the
experiment, serum cholesterol levels in each group were
similar: 15.0±0.4 mmol/L in the control group, 13.8±0.7 mmol/L in the
placebo group, and 14.2±0.9 mmol/L in the pravastatin
group. Compared with the placebo group, serum cholesterol
levels in the pravastatin group were decreased by 15%
(11.5±0.5 versus 13.4±0.4 mmol/L, P<.05) at 4 weeks after
drug administration was begun and by 17% (9.2±0.6 versus 11.1±0.5
mmol/L, P<.05) at the end of drug administration. LDL
cholesterol levels in the pravastatin group
were also decreased, by 22%, compared with those in the placebo group
(7.6±0.7 versus 9.8±0.4 mmol/L, P<.05).
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Severity of Aortic Atherosclerosis
Table 2 shows the severity of aortic
atherosclerosis in each group. Data for thoracic and
abdominal lesions were estimated as the average of the data for the
proximal and distal portions. The atherosclerotic lesions progressed
with aging at each portion. At the abdominal aorta, however, the ratio
of surface lesion area to the intimal surface area in the
pravastatin group was decreased by 26% compared with that
in the placebo group (.34±.03 versus .46±.04, P<.05).
Although average intimal thickening in the placebo group also
significantly progressed with aging, there was no significant
difference in the total of intimal thickening in the aorta between the
control and pravastatin groups. Average intimal thickening
of the abdominal aorta in the pravastatin group was
decreased by 30% compared with that in the placebo group (139±18
versus 199±26 µm, P<.05).
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Histopathological Analysis of Aortic Lesions
Table 3 shows the area ratio of lesional components
to the atherosclerotic lesions on the aortic sections in each group,
and Fig 1 shows photomicrographs of typical aortic
atherosclerosis in the thoracic aorta. In a comparison
of the control group with the placebo group, a significant increase was
observed with aging in the ratio of collagen area to the lesional area
in sections, whereas a significant decrease in the area ratio of
lesional components was observed in smooth muscle cells,
macrophages, and macrophages plus extracellular lipid
deposits. In a comparison of the pravastatin group with the
placebo group, the area ratio of collagen to the lesion was increased
(.41±.03 versus .31±.02, P<.01), whereas a significant
decrease was observed in the area ratio of extracellular lipid deposits
(.04±.005 versus .07±.008, P<.05) and the area ratio of
macrophage plus extracellular lipid deposits (.07±.01% versus
.11±.01, P<.01). There were no significant differences in
the frequency of calcification, deposits of cholesterol
crystals, and fragmentation or disappearance of internal elastic lamina
between the placebo and pravastatin groups (data not
shown).
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Severity of Coronary
Atherosclerosis
Table 4 shows the severity of coronary
atherosclerosis in each group. Coronary
stenosis progressed significantly with aging in the main trunk
of the left coronary artery and the left circumflex artery, and
in the average of all arteries, in the control group compared with the
placebo group. In the pravastatin group, coronary
stenoses were similar to those in the control group except in
the main trunk of the left coronary artery. In comparison with
the placebo group, coronary stenosis in the
pravastatin group was decreased by 50% in the left
circumflex artery (.30±.10 versus .61±.05, P<.05) and by
29% in the average of all arteries (.39±.06 versus .54±.05,
P<.05). In addition, the score of coronary lesions
was also decreased in the left circumflex artery (12±5 versus 26±6,
P<.05), as was the total score (37±5 versus 58±8,
P<.05), in the pravastatin group compared with
the placebo group. The incidence of atherosclerosis was
similar among the three groups.
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Histopathological Analysis of Coronary
Lesions
Table 5 shows the area ratio of lesional components
to the coronary lesion in each group, and Fig 2
shows photomicrographs of typical coronary
atherosclerosis in the left coronary artery. In
a comparison of the control group with the placebo group, a significant
decrease was observed with aging in the area ratio of smooth muscle
cells, whereas a significant increase in the area ratio of lesional
components was observed in collagen, extracellular lipid deposits, and
macrophages plus extracellular lipid deposits. In a comparison
of the pravastatin group with the placebo group, the area
ratio of smooth muscle was increased (.08±.02 versus .04±.007,
P<.05), whereas the area ratio of macrophages plus
extracellular lipid deposits was decreased (.05±.007 versus
.08±.02%, P<.05). In addition, the ratio of collagen area
to the extracellular lipid deposits area of the pravastatin
group was about 1.5 times higher than that of the placebo group (8.33
versus 5.52). There were no significant differences in the frequency of
calcification, deposits of cholesterol crystals, and
fragmentation or disappearance of internal elastic lamina between the
placebo and treatment groups (data not shown).
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Smooth Muscle Cell Content of Media
Table 6 shows the smooth muscle cell content in
media without plaque regions. In aortic media, there were no
significant differences in the media area, the smooth muscle cell
area, and the ratio of smooth muscle cell area to media area between
10-month-old control rabbits and 24-month-old placebo rabbits.
In the pravastatin group, however, a significant increase
was observed in the smooth muscle cell area and in the ratio of smooth
muscle cell area to media area compared with those in both the control
and placebo groups. In the coronary media, although the ratio
of smooth muscle cell area to media area in the placebo group was
significantly decreased compared with that in the control group, the
ratio in the pravastatin group was not decreased.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Many studies have demonstrated that progression of atherosclerosis can be suppressed by reduction of serum cholesterol levels. In these studies, however, histopathological changes of the atherosclerotic lesions have not been sufficiently examined. Results of our study show that a reduction of serum cholesterol levels due to pravastatin treatment altered the lesional composition of atherosclerotic plaques in WHHL rabbits.
In the present study we showed that a long-term reduction of
serum cholesterol levels led to the following
histopathological changes in the atherosclerotic plaques: (1) a
decrease in the area of macrophages plus extracellular lipid
deposits, (2) an increase in collagen area, (3) a suppression of the
decrease in smooth muscle cell area with lesion progress, and (4) an
increase in the ratio of collagen area to the area of extracellular
lipid deposits (Tables 3
and 5).
In recent human studies, several groups reported on acute clinical coronary events. Becker and coworkers (van der Wal et al25 ) observed that the site of plaque rupture is invariably associated with the occurrence of large numbers of macrophages and extracellular lipid deposits, as well as a lack of smooth muscle cells and collagen fibers. Similar findings were observed by many groups.26 27 28 29 In addition, Libby30 concluded that impaired collagen synthesis and accelerated collagen degradation due to inflammatory stimuli provided by modified lipoproteins or secreted by macrophages probably contribute to weakening of the fibrous cap. In our study with WHHL rabbits, reduction of serum cholesterol levels resulted not only in a decrease in macrophages and extracellular lipid deposits but also in an increase in collagen fibers and smooth muscle cells in the atherosclerotic plaques. Therefore, we considered that serum cholesterol reduction may prevent plaque rupture, if findings similar to those of our study were observed in human atherosclerotic plaques treated with an inhibitor of HMG-CoA reductase.
We observed that the smooth muscle cell content in the
arterial media decreased in the placebo group but not in
the pravastatin group. We measured the area of cells
stained with HHF35 in the medial regions without plaque (Figs 1 and 2).
Although the medial areas were similar among the three groups, the area
of HHF35-positive cells in the aortic media was significantly higher in
the pravastatin group than in the other groups. The area
ratio of smooth muscle cells to media in the pravastatin
group was increased about 1.4-fold in the aortas and about 1.3-fold in
the coronary arteries compared with the placebo group. Similar
findings were confirmed by observation of sections treated with
Azan-Mallory's stain. We consider that reduction of
macrophages and oxidized LDLs in the arterial wall
probably contributed to preservation of medial smooth muscle cells in
the pravastatin group. Previously we observed a decrease in
smooth muscle cells that were neighboring macrophages
infiltrated into the media.17 This finding suggests that
macrophages affect medial smooth muscle cells by their
secretion of cytokines and toxic metabolites. In addition, in
studies in vitro, Morel et al31 and Jimi et
al32 demonstrated that oxidized LDLs injured vascular
smooth muscle cells. Therefore, preservation of smooth muscle cell
content in the media of the pravastatin group may be
related to a reduction of macrophages and oxidized LDLs in the
arterial wall due to reduction of the serum
cholesterol levels.
In the present study we showed that a long-term reduction of
serum cholesterol levels by pravastatin
treatment (Table 1) led to a decrease in the area of
macrophages plus extracellular lipid deposits in both aortic
and coronary atherosclerosis in WHHL rabbits
(Tables 3 and 5). It is well known that LDL infiltrates atherosclerotic
lesions. In immunohistochemical studies, a large amount of oxidized LDL
was observed in atherosclerotic lesions in the aortas of WHHL
rabbits.1 2 3 Kume et al33 reported that
lysophosphatidylcholine, a component of oxidized LDL, stimulates
appearance of adhesion molecules for monocytes on the
endothelial cells. In addition, in vitro studies
demonstrate that macrophages take up these oxidized LDLs and
are then transformed into foam cells.34 35 36 These
observations suggest that increased LDL levels in the circulation are
closely associated with atherogenesis. Therefore, we considered that
pravastatin treatment decreased in macrophages plus
extracellular lipid deposits in plaques in WHHL rabbits as a result of
the serum cholesterol reduction, and then suppressed
progression of the atherosclerosis.
In conclusion, a long-term mild reduction of LDL cholesterol levels in the circulation by pravastatin treatment in WHHL rabbits can cause a decrease in the sum of macrophages plus extracellular lipid deposits and an increase in the collagen content of the plaques, in addition to suppressing the progression of established atherosclerosis.
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Acknowledgments |
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We wish to thank T. Tamura, M. Shiraishi, and M. Tsutsumi-Okayama for the care of animals and the administration of drugs. We are also indebted to H. Shimazu and H. Mizuno for their excellent technical assistance.
Received February 19, 1995; accepted August 14, 1995.
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G. K. Sukhova, J. K. Williams, and P. Libby Statins Reduce Inflammation in Atheroma of Nonhuman Primates Independent of Effects on Serum Cholesterol Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1452 - 1458. [Abstract] [Full Text] [PDF]
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M. Crisby, G. Nordin-Fredriksson, P. K. Shah, J. Yano, J. Zhu, and J. Nilsson Pravastatin Treatment Increases Collagen Content and Decreases Lipid Content, Inflammation, Metalloproteinases, and Cell Death in Human Carotid Plaques : Implications for Plaque Stabilization Circulation, February 20, 2001; 103(7): 926 - 933. [Abstract] [Full Text] [PDF]
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Y. Fukumoto, P. Libby, E. Rabkin, C. C. Hill, M. Enomoto, Y. Hirouchi, M. Shiomi, and M. Aikawa Statins Alter Smooth Muscle Cell Accumulation and Collagen Content in Established Atheroma of Watanabe Heritable Hyperlipidemic Rabbits Circulation, February 20, 2001; 103(7): 993 - 999. [Abstract] [Full Text] [PDF]
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S. H. Hohnloser Prevention of recurrent life-threatening arrhythmias: will lipid-lowering therapy make a difference? J. Am. Coll. Cardiol., September 1, 2000; 36(3): 773 - 775. [Full Text] [PDF]
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P. M. Ridker, N. Rifai, M. A. Pfeffer, F. Sacks, and E. Braunwald Long-Term Effects of Pravastatin on Plasma Concentration of C-reactive Protein Circulation, July 20, 1999; 100(3): 230 - 235. [Abstract] [Full Text] [PDF]
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M. M. Kockx, G. R. Y. De Meyer, N. Buyssens, M. W. M. Knaapen, H. Bult, and A. G. Herman Cell Composition, Replication, and Apoptosis in Atherosclerotic Plaques After 6 Months of Cholesterol Withdrawal Circ. Res., August 24, 1998; 83(4): 378 - 387. [Abstract] [Full Text] [PDF]
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M. Aikawa, E. Rabkin, Y. Okada, S. J. Voglic, S. K. Clinton, C. E. Brinckerhoff, G. K. Sukhova, and P. Libby Lipid Lowering by Diet Reduces Matrix Metalloproteinase Activity and Increases Collagen Content of Rabbit Atheroma : A Potential Mechanism of Lesion Stabilization Circulation, June 23, 1998; 97(24): 2433 - 2444. [Abstract] [Full Text] [PDF]
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M. J. Davies Stability and Instability: Two Faces of Coronary Atherosclerosis: The Paul Dudley White Lecture 1995 Circulation, October 15, 1996; 94(8): 2013 - 2020. [Full Text]
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W. Ni, K. Egashira, C. Kataoka, S. Kitamoto, M. Koyanagi, S. Inoue, and A. Takeshita Antiinflammatory and Antiarteriosclerotic Actions of HMG-CoA Reductase Inhibitors in a Rat Model of Chronic Inhibition of Nitric Oxide Synthesis Circ. Res., August 31, 2001; 89(5): 415 - 421. [Abstract] [Full Text] [PDF]
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