© 1995 American Heart Association, Inc.
Articles |
Inhibition of Hypercholesterolemia-Induced Atherosclerosis in the Nonhuman Primate by Probucol
II. Cellular Composition and Proliferation
From the Department of Medicine (M.Y.C., A.C.) and the Department of Pathology (E.W.R., R.R.), University of Washington, Seattle, and the Department of Pathology (M.S.), Shiga University of Medical Science, Otsu, Japan.
Correspondence to Russell Ross, PhD, Department of Pathology, University of Washington School of Medicine, Box 357470, Seattle, WA 98195-7470. E-mail rross@u.washington.edu.
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Abstract |
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Abstract In nonhuman primates (Macaca nemestrina) treated with the antioxidant probucol during diet-induced hypercholesterolemia, intimal lesion area in the thoracic aorta was decreased, with increased resistance of plasma LDL to oxidation. The cellular and molecular changes associated with the decrease in lesion size in the probucol-treated hypercholesterolemic animals are quantitatively evaluated in this study. Lesions from the probucol-treated animals appear less mature and have altered lipid distribution. Abundant lipid-laden smooth muscle cells are found in the intima and media of the probucol-treated animals, with fewer medial lipid-laden macrophages, compared with lesions at similar sites in the control hypercholesterolemic animals. In both the control and probucol-treated animals, macrophages are the predominant cells in most lesions, but the ratio of macrophages to smooth muscle cells is decreased in the lower thoracic and upper abdominal aortic sites in the probucol-treated animals. Lesions at all aortic sites in the probucol-treated animals have a 35% to 80% reduction in the percentage of cells in cell cycle traverse, as indicated by immunostaining for proliferating cell nuclear antigen (% PCNA-positive). In both groups, macrophages and smooth muscle cells are PCNA-positive, but the majority (>60%) are macrophages. No difference in % PCNA-positive cells is seen in the iliac arteries, where the most advanced lesions were present at the time probucol administration was initiated. Limited Northern analysis of growth-regulatory molecules possibly involved in the cellular changes associated with lesions shows a 30% to 50% decrease in mRNA levels of platelet-derived growth factor (PDGF) B-chain, PDGF ß-receptor, colony-stimulating factor type 1, and monocyte chemotactic protein 1. Thus, a potential role for an antioxidant such as probucol in the treatment of atherosclerosis may be to alter the early inflammatory fibroproliferative processes of the disease. Whether these effects are directly related to the antioxidant properties or some other activity of probucol is not yet known.
Key Words: LDL • antioxidant • atherosclerotic lesions • cellular composition • proliferating cell nuclear antigen
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Introduction |
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Studies have shown the presence of oxLDL and/or oxidation-specific epitopes in aortic lesions of the WHHL rabbit,1 2 3 4 5 in human lesions of atherosclerosis,6 and in human xanthomata.7 Evidence for the importance of LDL oxidation in atherogenesis was suggested by the ability of probucol, a powerful antioxidant,8 to reduce the rate of development of lesions of atherosclerosis in the WHHL rabbit9 10 and to reduce lesion size in the thoracic aorta after diet-induced hypercholesterolemia in the nonhuman primate Macaca nemestrina.11 Analysis of intimal lesion areas in the nonhuman primates demonstrated a trend toward an inverse correlation between intimal thickness in the abdominal aorta and iliac arteries and resistance of LDL to oxidation. This was under conditions in which plasma lipoprotein profiles were comparable. An earlier preliminary study in primates12 had demonstrated a 7% to 12% reduction in aortic surface lesion area with probucol treatment, but probucol treatment was also associated with a 27% decrease in plasma cholesterol levels. There is also considerable in vitro evidence that suggests a role for oxLDL in the cellular changes associated with the developing lesions of atherosclerosis.13 14
In addition to preventing lipoprotein oxidation, probucol is reported
to have other actions that may be important for its antiatherogenic
effects. Pretreatment of cultured vascular endothelial
cells with probucol protects the cells against injury from oxLDL and
organic hydroperoxides.15 
-Tocopherol,
another lipid-soluble antioxidant, has similar protective effects
against oxidative damage to cellular membranes.15 16
Lipopolysaccharide-stimulated resident peritoneal
macrophages from probucol-treated mice secrete 40% to 80%
less interleukin-1 than macrophages from control
mice,17 suggesting that probucol might have an effect on
this inflammatory component of atherosclerosis.
Probucol also has been shown to prevent lipid storage in a
macrophage-like cell line in vitro,18 which
may be related to its ability to induce regression of xanthomas in
patients with familial
hypercholesterolemia.7
To investigate the mechanisms for the decrease in intimal lesion size in the probucol-treated nonhuman primates, the present study has evaluated the cellular and molecular characteristics of the lesions. Specific parameters that were used to compare lesions from control and probucol-treated monkeys included (1) the amount and localization of native and oxLDL epitopes; (2) the cellular composition, including total cell density, macrophage density, and smooth muscle cell density; (3) an estimate of the percentage of proliferating cells by PCNA; and (4) the percentage of PCNA-positive cells that were smooth muscle cells and macrophages. Possible molecular mediators of these cellular changes were evaluated by Northern analysis of mRNA isolated from the aorta.
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Methods |
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Animals
The present study provides further analysis of the nonhuman primates examined by Sasahara et al.11 In that study, 16 Macaca nemestrina analyzed for intimal lesion size were fed a diet containing 0.5% cholesterol19 20 for a total of 11 months. After 3.5 months on the diet, probucol (60 mg/kg, suspended in 0.25% methylcellulose in water at 240 mg/dL) was administered to 8 randomly selected animals. The remaining 8 control animals received only the carrier, 0.25% methylcellulose in water and apple juice. Procedures conformed to the Guidelines for Care and Use of Laboratory Animals issued by the US Institute of Laboratory Resources.
In the present study, a subset of these monkeys was selected for detailed analysis of lesions at all aortic and iliac artery sites on the basis of previously determined lag times for conjugated diene formation,11 a measure of resistance of plasma LDL to ex vivo copper ion–induced oxidation.21 Three control monkeys whose plasma LDL had normal resistance to oxidation (lag times <400 minutes) and represented the lower quartile for the entire study group with respect to resistance to oxidation were selected from the control group, and 3 probucol-treated animals whose plasma LDL had significantly increased resistance to oxidation (lag times >960 minutes) and represented the upper quartile with respect to oxidation resistance were selected from the probucol-treated group for quantitative immunohistochemical analyses. To further evaluate how representative of the entire study group the selected groups were, two parameters of interest, the percentage of PCNA-positive cells and oxidation-specific epitope content, were examined in selected aortic sites from all 16 animals.
Tissue Fixation
Immediately after the animals were killed, the thoracic and
abdominal aorta and iliac arteries were rapidly removed, washed well
with sterile PBS, and cut into 16 sections: 6 from the thoracic aorta
(T1 through T6), 6 from the abdominal aorta (A1 through A6), and 2 each
from the left and right iliac arteries (Lil1, Lil2, Ril1, and Ril2), as
previously described.11 22 23 The tissue was
immersion-fixed overnight in methanol Carnoy's fixative (60%
methanol, 30% chloroform, 10% acetic acid), dehydrated through a
graded series of methanols, and paraffin-embedded for quantitative
immunohistochemistry. The fixative solution contained the antioxidant
BHT (25 µmol/L) to prevent ex vivo oxidation after death.
Immunohistochemistry
Serial 5-µm sections were cut from the paraffin-embedded
segments, incubated with specific antibodies for 1.5 hours at room
temperature, and developed with biotinylated second antibodies and
peroxidase-conjugated streptavidin (Jackson ImmunoResearch
Laboratories, Inc) with or without NiCl enhancement, as indicated. For
estimation of cell proliferation, sections were single-stained with
NiCl enhancement with a monoclonal antibody to PCNA (anti-PCNA,
6.25x10-6 mg/mL, American Biotechnology).24
Adjacent sections were double-stained with anti-PCNA (with NiCl)
followed by a cell type–specific antibody (without NiCl), either
the anti-macrophage antibody HAM-56 (1:4000 dilution of
ascites fluid, gift from Allen Gown, University of Washington, Seattle,
Wash)25 or an anti–-actin antibody
(2.5x10-4 mg/mL, Boehringer Mannheim
Biochemicals)26 to identify smooth muscle cells.
To evaluate lipoprotein localization, a separate series of 4 adjacent
sections were single-stained with anti–-actin, HAM-56, OX5,
and MB47. OX5 is a monoclonal antibody raised against
copper-oxidized LDL that recognizes oxidation-specific
epitopes7 ; MB47 (gift from Linda K. Curtiss, Scripps
Research Clinic, La Jolla, Calif) is a monoclonal antibody specific for
apo B.27 OX5- (3.4x10-4 mg/mL) and MB47-
(1:4000 dilution of ascites fluid) immunostained sections
were developed with an avidin-biotin alkaline phosphatase system
(Vector Laboratories); anti–-actin and HAM-56 staining of
adjacent sections were as described above.
Immunostaining with anti-PCNA, HAM-56, and
anti–-actin antibodies was performed on 18 sections per run;
these sections included 3 sites (eg, T1, T2, and T3) from each of the 3
control and 3 probucol-treated animals.
Immunostaining with OX5 and MB47 was performed on 12
sections (2 sites from each of the 3 control and 3 probucol-treated
animals) per run to closely monitor the alkaline phosphatase color
reaction. All working reagents were from the same lot or stock
solutions.
Quantitative Analyses
Intimal lesion areas were measured with the Optimas quantitative
image analysis system (Bioscan) interfaced with an Olympus BH-2
light microscope, an IBM-compatible computer, and a Sony image monitor.
Areas were measured by tracing the luminal surface and internal elastic
lamina on the RGB computer image of the aortic segment at x400
magnification and were calibrated in units of
millimeters.11
The total number of nuclei per section and the number of PCNA-positive nuclei were also quantified with the Optimas system from PCNA single-stained sections. Three random-sample regions of interest were selected for each sample to determine the threshold and optical-density parameters for each section. The total number of nuclei was determined by adjusting the threshold level to include all methyl green–counterstained nuclei and nuclei that showed weak and strong immunoreactivity for PCNA. The distinction between weak and strong immunoreactivity for PCNA was based on the inverse logarithm of the ILIGV/area, which is a measure of the optical density for a given area. Frequency distribution histograms of ILIGV/area were obtained for each of the three sample regions per section. A cutoff ILIGV/area ratio was determined such that all nuclei with a ratio below the cutoff were considered PCNA-negative (all nuclei that were green or only weakly immunoreactive for PCNA), and all nuclei with a ratio above the cutoff were considered PCNA-positive (all nuclei with strong immunoreactivity for PCNA).
OX5 and MB47 epitopes were quantified by measurement of total areas of deposition of the red alkaline phosphatase reaction product. Threshold levels corresponding to the red reaction product were also determined from three sample regions for each section. These sampling procedures were necessary to adjust for variations in staining intensity between runs.
Serial sections single-stained with HAM-56 or -actin
antibodies were manually counted by use of an ocular grid at x400
magnification to identify the primary cell types present in the
lesions. Sections double-stained with PCNA and either HAM-56 or
-actin antibodies were also manually counted to identify the
cell types displaying PCNA immunostaining.
All quantification was performed by the same operator (M.Y.C.).
Preparation of Total RNA and Northern Blot
Analysis
Two animals from the control group and 2 from the
probucol-treated group were randomly selected for isolation of
total RNA and Northern blot analysis. Aortas were divided into
thoracic and abdominal regions, dissected as described for preparation
of tissue for immunohistochemistry,23 and snap-frozen
for isolation and analysis of total RNA.28 RNA was
isolated as previously described.29 RNA (15 µg/lane) was
separated on a 1% agarose gel containing 0.2 mol/L MOPS and 1%
formaldehyde and transferred to nylon membranes (Nytran, Schleicher
& Schuell). Filters were hybridized with cDNA probes that had been
labeled with [-32P]dCTP (3000 Ci/mmol; DuPont–New
England Nuclear)30 by use of a random-primer labeling
system (Amersham Corp). The following cDNA probes were used: PDGF
A-chain (EcoRI fragment, 1.3 kb)31 ; PDGF
B-chain (BamHI fragment, 704 bp)32 ; PDGF
-receptor (EcoRI, 12 kb)33 ; PDGF
ß-receptor (BamHI–Sph I fragment, 1.3
kb)34 ; MCP-1 (Xho I–EcoRI, 350
bp)35 ; PCNA (BamHI fragment, 1.4
kb)36 ; CFS-1 receptor (c-fms) (Bgl I
fragment, 750 bp)37 ; HB-EGF (EcoRI fragment,
1.1 kb)38 ; TGF-ß (EcoRI fragment, 1.05
kb)39 ; and ß-actin (Pst I fragment, 1.33
kb).40 X-ray films were scanned and quantified with a
Molecular Dynamics Personal Densitometer and IMAGE QUANT
software with ß-actin hybridization as a reference for relative
mRNA load per lane.
Statistical Methods
Because of the small number of observations (n=3 at a given site
for each group), adjacent aortic sites were grouped (T1-T2, T3-T4, etc)
and all iliac artery sites were grouped (I). This increased the number
of observations within each group, although the adjacent sites were not
considered independent observations. Therefore, measurement of
HAM-56–positive cells/-actin–positive cells, areas of MB47
and OX5 staining, and % PCNA-positive cells are presented as
mean±SEM, with no additional statistical analyses. Because of
the small number of animals used for Northern blot analysis,
normalized mRNA values are presented as mean±SD, with no
additional statistical analyses.
Statistical analyses were performed on % PCNA-positive cells and area of OX5 staining in the two aortic sites (T2 and A1) from all of the animals included in the first phase of this study11 (n=8 at each site for both controls and probucol-treated animals). Values are expressed as mean±SEM. Differences between the two groups were assessed by Student's t test for unequal variances.
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Results |
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Selection of Animals Was Based on Ex Vivo Resistance of Plasma LDL to Oxidation
The 3 control and 3 probucol-treated animals selected for detailed immunohistochemical analyses are shown in Table 1
. A new series of sections was cut from the
previously prepared paraffin-embedded tissue11 for
this study. Therefore, total intimal areas of the thoracic and
abdominal aorta and iliac arteries were measured again to approximate
location within lesions. In the thoracic and abdominal aorta and iliac
arteries, probucol-treated animals whose LDL had significantly
increased resistance to oxidation had less total intimal thickening
than did the control animals whose LDL had normal resistance to
oxidation (Table 1). This was under conditions in which the lipoprotein
profiles of the probucol-treated and control monkeys were not
significantly different, as shown in our initial description of the
study.11
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Lesions Were Less Mature in the Probucol-Treated
Animals
Intimal lesions in both the control and probucol-treated
animals included early fatty streaks, intermediate fibrofatty lesions,
and advanced fibrous plaques. The intermediate and advanced lesions
from the probucol-treated animals (Fig 1a and 1c)
were less mature (ie, smaller [thinner]), less vacuolated (contained
less lipid), and less necrotic than lesions from similar sites in the
control animals (Fig 1b and 1d). Lesions from the control animals
contained lipid-laden macrophages that had invaded deep
into the media in regions in which the internal elastic lamina was
disrupted (Fig 1b, arrows). In contrast, lesions from the
probucol-treated animals contained fewer macrophages in the
media (Fig 1a). Abundant lipid-laden smooth muscle cells were
observed in the intima and media of the aorta and the iliac arteries of
the probucol-treated animals (Fig 1e, arrows). In contrast, fewer
intimal lipid-laden smooth muscle cells were observed in the
control animals, and medial lipid-laden smooth muscle cells were
seen primarily in regions in which the internal elastic lamina was
disrupted and the intimal lesion protruded into the media (not
shown).
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Ratio of Macrophages to Smooth Muscle Cells Was Decreased
in Lower Thoracic and Upper Abdominal Lesions of the
Probucol-Treated Animals
The number of cells per lesion was quantified by use of the
Optimas image analysis system. The decrease in the number of
cells per lesion was accompanied by a comparable decrease in lesion
area in the probucol-treated animals. Thus, lesion cell density
(number of cells per lesion normalized for lesion area) did not differ
in any region from that in the control animals (data not shown). Cell
densities were also compared on the basis of lesion type. Lesions were
classified as early, intermediate, and advanced.22 No
differences in total cell density were found between any types of
lesions (data not shown).
The numbers of macrophages and smooth muscle cells per lesion
were quantified separately. Because of the somewhat diffuse cytoplasmic
nature of the staining with HAM-56 for macrophages and of
-actin for smooth muscle cells, each cell type was counted
manually. Macrophages were the predominant cells in the
majority of lesions in both groups of animals. However, the proportions
of macrophages and smooth muscle cells varied with the site.
The ratio of macrophages to smooth muscle cells was decreased
in lesions of the lower thoracic and upper abdominal aorta of the
probucol-treated monkeys, resulting in relatively more smooth
muscle cells at these sites (Fig 2). A trend toward the
reverse, relatively more macrophages, was observed in the iliac
arteries of the probucol-treated animals.
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Apo B Distribution Differed in Localization and Relative
Accumulation, Whereas Oxidation-Specific Epitopes Were Decreased in the
Abdominal Aorta of the Probucol-Treated Animals
The aortic distribution and relative amount of apo B, the major
apolipoprotein of LDL, was evaluated by immunostaining
with the anti–apo B antibody MB47. In the probucol-treated
animals, MB47 localized primarily along the internal elastic lamina and
secondarily in a subendothelial region (Fig 3a). In the control animals, MB47
immunostaining was diffuse and extracellular and
localized primarily along the internal elastic lamina but was also
present throughout the thickened intima (Fig 3b). Very little
adventitial staining was observed in either group. The area of MB47
staining as a percent of the total intimal area was increased in levels
A3-A4 of the abdominal aorta and in the iliac arteries of the
probucol-treated animals; no differences in the area of MB47
staining were observed in the thoracic aorta or in other regions of the
abdominal aorta (Fig 4a).
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Oxidation-specific epitopes were evaluated by
immunostaining with the monoclonal antibody OX5. OX5 is
an antibody prepared by immunization with extensively
copper-oxidized LDL and recognizes LDL oxidized to various degrees
but does not recognize native or acetyl- or
malondialdehyde-modified LDL.7 In both groups of
animals, OX5 immunostaining was primarily intracellular
and cytoplasmic (Fig 3c
) and colocalized with many of the
lipid-laden macrophages, as indicated from serial sections
stained with HAM-56 (Fig 3d). Very little adventitial staining was
observed in either group. The areas of OX5 staining as a percentage of
the total intimal area were decreased in the abdominal aorta of the
probucol-treated animals relative to control animals but were
similar in the thoracic aorta and the iliac arteries (Fig 4b).
Percentage of PCNA-Positive Cells Was Decreased in All Lesion Types
Throughout the Aortas of the Probucol-Treated Animals
Cells in cell cycle traverse were estimated by use of an antibody
specific for PCNA. PCNA-positive cells generally were found in regions
close to the internal elastic lamina of early lesions at all levels of
the aorta in both groups of animals. In the more advanced lesions,
PCNA-positive cells were most concentrated deep in the core and in the
shoulders of plaques, with fewer PCNA-positive cells in the most
luminal area of the intima.
The percentage of PCNA-positive cells was decreased at all sites
in the thoracic and abdominal aortas of the probucol-treated
monkeys, but it was not different in the iliac arteries compared with
the controls (Fig 5). In the thoracic aortas of
the probucol-treated animals, there was a 35% to 60% reduction in
% PCNA-positive cells compared with control animals. In the
abdominal aortas, there was a 60% to 80% decrease in % PCNA-positive
cells in the probucol-treated animals. This was not dependent on
lesion severity, since early, intermediate, and advanced lesions in the
aortas of the probucol-treated animals all had decreased %
PCNA-positive cells (data not shown). In the iliac arteries, in which
the most advanced fibrous plaques were observed and the most advanced
lesions were present when probucol treatment was initiated, there
were no differences between the two groups of monkeys.
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To identify the primary cell types in cell cycle traverse, serial
sections were double-immunostained with anti-PCNA
antibody and either HAM-56 or anti– actin antibody to detect
macrophages and smooth muscle cells, respectively. In both
groups of animals, >90% of PCNA-positive cells could be identified as
either macrophages or smooth muscle cells. The majority were
macrophages (Fig 6a and 6b). In the control
animals, 
68% of PCNA-positive cells were macrophages and
32% were smooth muscle cells at all aortic sites. In the
probucol-treated animals, 80% of PCNA-positive cells were
macrophages and 20% were smooth muscle cells at all aortic
sites (data not shown).
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Three Animals Selected From Each Group for Detailed
Analyses Were Representative of Their Group of
Eight Animals
The two parameters in which significant differences
were observed between the 3 probucol-treated and 3 control animals,
% PCNA-positive cells and oxidation-specific epitope staining
area, were analyzed from two selected aortic sites, T2 and A1,
of all 16 animals included in the original study (Table 2). The % PCNA-positive cells was decreased from
8.50±1.40% in T2 of the 8 control animals to 2.50±0.77%
(P<.01) in T2 of the 8 probucol-treated animals and
from 5.44±1.05% in A1 of the 8 control animals to 1.44±0.73%
(P<.01) in A1 of the 8 probucol-treated animals. This
70% decrease in % PCNA-positive cells in T2 and A1 of the
probucol-treated animals was in the same range as the decreases
between the same regions of the two groups of 3 animals chosen for
detailed analyses (Fig 5). No difference in area of
oxidation-specific epitope staining was seen in T2 between the two
groups, but OX5 area was decreased 60% (P<.05) in A1 of
the 8 probucol-treated animals (Table 2). Again, this
analysis is consistent with the OX5 findings in the two
groups of 3 animals shown in Fig 4b.
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Expression of Several Growth-Regulatory Molecules Was Decreased in
Probucol-Treated Animals
Total RNA was isolated from the combined intimal and medial layers
of the thoracic and abdominal aortic segments of two control and two
probucol-treated animals and analyzed for expression of
growth factors and cytokines that might be involved in the
altered cellular response. mRNA levels of the smooth muscle cell
mitogen and chemoattractant PDGF B-chain and its receptor, PDGF
ß-receptor, were both decreased by 50% in the
probucol-treated animals, while no changes were observed in the
levels of PDGF A-chain or the PDGF -receptor (data not shown).
Levels of the monocyte chemoattractant and mitogen CSF-1 were decreased
by 50% in the probucol-treated animals, with no change in the
level of its receptor, c-fms. The monocyte chemoattractant
MCP-1 and PCNA mRNA levels also were somewhat decreased in these
animals. No differences in HB-EGF or TGF-ß mRNA levels were observed
(Fig 7).
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Discussion |
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Smaller Lesions in the Probucol-Treated Animals Were Less Mature and Relatively Enriched in Smooth Muscle Cells in Regions of the Thoracic and Abdominal Aorta
This study was designed to evaluate the cellular and molecular differences in the hypercholesterolemia-induced lesions of control and probucol-treated monkeys in a subset of the animals studied by Sasahara et al.11 Aortic lesions from the probucol-treated animals were smaller, contained less lipid, were less necrotic, and showed less invasion of macrophages into the medial layer of the aorta than lesions at the same sites in the control animals. The earlier observation of lipid-laden cells in the media of probucol-treated animals and their morphological identification as smooth muscle cells was confirmed in this study by immunohistochemistry using an anti–
-actin antibody. More
lipid-laden smooth muscle cells were present in the
probucol-treated animals than in the controls, and these
lipid-filled smooth muscle cells were located throughout the intima
as well as in the media. The greater presence of medial lipid-laden
smooth muscle cells in the probucol-treated animals suggests that
medial smooth muscle cells have become activated to accumulate
lipid, perhaps as a result of the relative decrease in
macrophages capable of ingesting lipid in these lesions.
However, since the decrease in the relative proportion of
macrophages was observed only in the lower thoracic and
abdominal aorta but not in the upper thoracic aorta, other factors may
be involved in this altered lipid metabolism. The apparent increase in intimal smooth muscle cells relative to macrophages at some aortic sites is consistent with the findings in probucol-treated WHHL rabbits.5 On the basis of our findings of decreased PCNA-positive cells in the aorta of the probucol-treated monkeys, this increase in smooth muscle cells is not likely to be due to increased proliferation of smooth muscle cells from the media. It may be due to decreased recruitment of monocytes or to increased egress of macrophages. This raises the possibility that probucol treatment may result in more fibrotic, less lipid-rich lesions, which would be predicted to have increased stability and to be less prone to intimal tearing and thrombus formation.41 42 However, it is unclear how the increased accumulation of lipid by smooth muscle cells might affect lesion stability.
Both PCNA-Positive Macrophages and Smooth Muscle Cells Were
Decreased Throughout the Aorta of the Probucol-Treated Monkeys but Not
in the Iliac Arteries
Cell proliferation has been proposed to play a key role in
atherosclerosis.43 44 45 Our finding of
20% PCNA-positive cells in lesions of control
hypercholesterolemic monkeys is comparable to that
reported in other animal models of
atherosclerosis.46 The % PCNA-positive
cells in the control monkeys after 11 months of diet-induced
hypercholesterolemia is greater than that
observed in the more advanced atherosclerotic lesions of human
coronary artery specimens.47 This may reflect a
species difference, may result from the more acute induction of the
disease process, or may indicate that cell proliferation is a
relatively early event of atherosclerosis, or all of
the above. The marked decrease in PCNA-positive cells in early,
intermediate, and advanced lesions in the thoracic and abdominal aorta
of probucol-treated monkeys suggests that the decrease in intimal
thickening reported previously in these animals11 may be
due to a reduced number of cells passing through the cell cycle.
In both the control and probucol-treated animals, the majority of
PCNA-positive cells were lipid-laden macrophages, in
agreement with other studies.47 In this study, >90% of
PCNA-positive cells could be accounted for as either
macrophages or smooth muscle cells, whereas <10% of cycling
cells were negative for either cell-specific marker. The identity
of these cells is unknown; they may be endothelial
cells,48 49 smooth muscle cells that no longer express the
smooth muscle cell–specific -actin
phenotype,50 or T lymphocytes.51 The
10% increase in the percentage of PCNA-positive macrophages
and the corresponding decrease in PCNA-positive smooth muscle cells is
not sufficient to explain the 35% to 60% reduction in %
PCNA-positive cells in the thoracic aorta and the 60% to 80%
reduction in the abdominal aorta of the probucol-treated animals.
Therefore, the significant reduction in % PCNA-positive cells reflects
decreases of both PCNA-positive macrophages and PCNA-positive
smooth muscle cells, rather than loss of a single cell type. The
greatest decrease in lesion size is in the thoracic aorta, the site at
which the most immature lesions are located, which suggests that a
major effect of probucol may be on the earliest stages of the chronic
inflammatory component of the lesion.
Probucol Treatment Has Little Effect on the Content of Apo B or
Oxidation-Specific Epitopes Within the Lesions
Analysis of apo B and oxidation-specific epitope
staining suggests that lipoprotein immunoreactivity within the lesions
was related to lesion size and was generally not altered by probucol
treatment, except for the decreased area of oxidation-specific
epitope staining in the upper abdominal aorta of the
probucol-treated animals. This immunostaining
reflects net accumulation throughout the intima over the entire
11-month experimental period. Clearance of tyramine
cellobiose–labeled LDL was also evaluated before the animals were
killed11 and revealed a decrease in the fractional
catabolic rate of LDL in all arterial sites. Fractional
catabolic rate analysis represents degradation over the
92- to 94-hour period before the animals were killed51
rather than accumulation, but it may also be more sensitive to changes
in LDL metabolism. These quantitative
oxidation-specific epitope staining results are consistent
with the qualitative findings observed in probucol-treated WHHL
rabbits.5 Thus, although the increased resistance of
plasma LDL to oxidation11 is indicative of intimal
thickness in the probucol-treated animals, it does not relate to
the apparent apo B content of the lesions.
How Does Probucol Exert Its Effects?
It is unknown whether the decreased lesion maturity, altered lipid
distribution, and regulation of cell proliferation and growth factor
expression observed in this study are directly related to the
antioxidant properties of probucol. It is possible that probucol has
several mechanisms of action that contribute to its protective effect
against the early phases of atherogenesis in the nonhuman primate.
In this study, probucol treatment was started after the monkeys had been on the cholesterol diet for 3.5 months. By this time, fibrofatty lesions were present in the lower abdominal aorta and iliac arteries,22 23 while only early fatty streaks were present in the thoracic and upper abdominal aorta. Because of the reproducibility of lesion distribution observed in monkeys with the same lipid levels, it was possible to study the effects of probucol on lesion development and lesion progression. The decrease in lesion size was statistically significant only in the thoracic aorta, but there was a striking trend toward smaller intimal lesion size associated with increased resistance of plasma LDL to ex vivo oxidative modification.11 Similarly, in cholesterol-fed rabbits, probucol treatment preserved endothelial function and was correlated with LDL resistance to modification.53 In this study, the decrease in lesion size was accompanied by decreases in primarily macrophage but also smooth muscle cell content in the thoracic and abdominal aorta. These observations provide further evidence that probucol may prevent or decrease the inflammatory response and thus early lesion formation and progression but may not alter the cellular characteristics of the more advanced lesions of atherosclerosis.54 Thus, the fibroproliferative response that occurs as a late component after the early inflammatory response (fatty streak formation) may be less affected by reduction in subsequent inflammation. However, in this study the animals were analyzed 11 months after initiation of the diet and 7.5 months after initiation of treatment. Thus, the prolonged effects of probucol on lesion progression were not assessed in this single–time point analysis and need to be addressed in longer-term studies with analyses at multiple time points.
The decrease in intimal lesion area in the probucol-treated animals appears to be due in part to a decrease in the cellular content of the lesions, with a smaller percentage of the cells entering into cell cycle traverse. The decrease in expression of PCNA mRNA levels was consistent with the decrease in PCNA-positive cells determined by quantitative immunohistochemistry. However, the greater decrease in PCNA immunostaining than in mRNA level suggests that probucol altered PCNA protein expression posttranscriptionally. Probucol may affect cell proliferation in part by decreasing mRNA levels of PDGF-B and CSF-1, two potent growth factors, as shown by Northern analysis of probucol-treated animals. The decreased expression of PDGF B-chain, PDGF ß-receptor, and CSF-1 but not c-fms indicates that, at least in the case of PDGF B-chain and PDGF ß-receptor, probucol treatment altered expression of both a growth factor and its receptor. Whether these results reflect a direct effect of probucol on specific growth-regulatory molecules is not known. The changes in mRNA levels may be due in part to the relative decrease in macrophage content of the lesions or to the lower ratio of intimal to medial thickness (data not shown) of aortic tissue in the probucol-treated animals. Detectable decreases in mRNA levels were not observed for a number of other growth-regulatory molecules, including PDGF A-chain, HB-EGF, and TGF-ß. The possibility that these molecules may be regulated posttranscriptionally is being investigated.
The apparent prolonged inhibition of cell proliferation in this primate model is associated with vascular sites characterized by early and moderate inflammatory responses at the time of drug initiation. This inhibition is notable in such a model of chronic vascular injury, because even in models of acute injury of the normal artery, sustained inhibition of intimal lesion formation has been difficult to achieve. Why has there been no effect on cell proliferation in the iliac arteries that we know had more advanced lesions, characterized by the presence of endothelial denudation and smooth muscle cell infiltration? Is it that advanced lesions, with their associated fibrosis, may require a longer time to remodel? Might an effect have been observed if the probucol treatment had been continued for a longer time, or is probucol ineffective beyond a certain stage? Can either of these possibilities explain the lack of effect of probucol on femoral atherosclerosis in humans?55 Are there unique characteristics of lipid peroxidation, such as nonenzymatic peroxidation as opposed to 15-lipoxygenase in earlier lesions?56 In regions in which apparent prolonged inhibition of cell cycle traverse has been achieved, how has probucol altered growth factor and cytokine mRNA levels? Has it blocked early initiators of their expression? Addressing these questions will be critical to the use of specific antioxidants, such as probucol, in the possible prevention of clinical events associated with atherosclerosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by NIH grants HL-18645 (to R.R., E.W.R., and A.C.), HL-47151 (to R.R. and E.W.R.), HL-07312 (to M.Y.C.), and RR-00166 (to the Regional Primate Center) and an unrestricted grant for cardiovascular research from Bristol-Myers Squibb Company (to R.R.). We would like to thank Dr Rodney Schmidt for advice with the image analysis, Karen Engel and Roderick Browne for technical assistance, and Kris Carroll for preparation of figures and photographic assistance.
Received April 26, 1995; accepted July 28, 1995.
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