Increased atherosclerosis in young versus old hypercholesterolemic rabbits by a
mechanism independent of intimal cell proliferation.
María J. Cortés, Antonio Díez-Juan, Ignacio Pérez-Roger and Vicente Andrés
Unit of Vascular Biology, Instituto de Biomedicina de Valencia, Spanish Council for
Scientific Research, Valencia, Spain.
KEY WORDS: Atherosclerosis, aging, hypercholesterolemia
ABBREVIATIONS:
BrdU: 5-bromodeoxyuridine; OC: Old-Cholesterol-rich diet; ON: Old-Normal chow; YC:
Young-Cholesterol-rich diet; YN: Young-Normal chow; VSMC(s): vascular smooth
muscle cell(s).
Corresponding author:
Vicente Andrés, PhD
Instituto de Biomedicina de Valencia
Spanish Council for Scientific Research
C/Jaime Roig 11, 46010 Valencia (Spain)
Tel: +34-96-3391752
FAX: +34-96-3690800
E-mail: vandres@ibv.csic.es
ABSTRACT
We sought to determine the relative importance of aging and hypercholesterolemia on
atherosclerosis. Although plasma cholesterol increased similarly in young and old fat-fed rabbits,
aortic atherosclerotic lesions were more prominent in young animals. In both age groups, medial
cell proliferation was low and increased as a function of intimal lesion size. Importantly, intimal
cell replication was similar in young and old rabbits. These results show increased atherogenicity
in young rabbits challenged with an atherogenic diet compared with old animals by a mechanism
independent of intimal proliferation. They also suggest that reducing dietary fat content is
especially important in young individuals.
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1. INTRODUCTION
Atherosclerosis is the leading cause of mortality and morbidity in developed countries.
Animal and human studies indicate that atherosclerosis is initiated by alterations in the normal
function of the endothelium [1]. Endothelial dysfunction triggers a complex inflammatory process
that leads to the formation of fatty streaks, the earliest recognizable lesion of atherosclerosis, and
ultimately to fibrous and fibrocalcified plaques.
Aging and hypercholesterolemia are major cardiovascular risk factors [1-3]. Increasing
both dietary cholesterol content and the duration of exposure to cholesterol-rich diets result in
augmented experimental and human atherosclerosis. While aging is associated with structural and
functional alterations in the cardiovascular system [2,3], it is unclear whether age-dependent
intrinsic alterations in the arterial wall or increased exposure to risk factors contribute to the
increased prevalence of atherosclerosis in the elderly. In this study we evaluated the relative
contribution of aging and hypercholesterolemia to atherogenesis in New Zealand white rabbits. As
abnormal cell proliferation has been implicated on atherogenesis [1], we also investigated whether
age-dependent differences in atherosclerosis could be related to differences in arterial cell
proliferation.
2. MATERIALS AND METHODS
2.1 Experimental design. Young (4-5-months-old) and old (4-5-years-old) male New Zealand
rabbits fed a normal diet or a cholesterol-rich diet for 2 months were included in the following
groups: YN (Young-Normal chow, n=5), YC (Young-Cholesterol-rich diet, n=10), ON (OldNormal chow, n=5), and OC (Old-Cholesterol-rich diet, n=10). The cholesterol-rich diet contained
3
10 g cholesterol (Sigma) and 60 ml peanut oil per kilogram of rabbit chow (1% cholesterol).
Animal handling was in accordance with institutional guidelines. Blood was withdrawn before the
onset of the atherogenic diet and 2 days before sacrifice to monitor plasma cholesterol and
triglyceride levels. All animals received 4 intraperitoneal injections of 5-bromodeoxyuridine
(BrdU) (20 mg/Kg each) at 12-hour intervals starting 48 h before sacrifice. Rabbits were sacrificed
with an overdose of pentobarbital (iv injection). A cut was made in the cava vein and the systemic
circulation was thoroughly perfused with saline through the heart. The aortic arch, common carotid
and femoral artery were fixed in 100 % methanol for morphometric and immunohistological
studies.
2.2 Immunohistochemistry, immunofluorescence and quantification of atheroma. Methanolfixed arteries were paraffin-embedded and cut in 5 m cross-sections. Intimal and medial areas
were measured on elastic-trichrome stained specimens or RAM11-immunostained sections.
Quantification was done with the SigmaScan Pro 5.0 software (SPSS Science) using a
hemacytometer for calibration as described previously [4]. For each rabbit, 2-4 sections were
analyzed and the results were averaged. Immunohistochemistry using mouse monoclonal antiBrdU (1:50, DAKO) or anti-RAM11 (1:500, DAKO) antibodies was done with a
biotin/streptavidin-peroxidase detection system (Signet Laboratories) and 0.05 % (wt/vol) 3,3’diaminobenzidine tetrahydrochloride dihydrate substrate (Vector Laboratories). Antibodies for
immunofluorescence were: biotinylated anti-BrdU (1:60, Zymed Laboratories), FITC-conjugated
anti-BrdU (1:20, Roche), anti-RAM11 (1:1000, DAKO) and biotinylated anti-mouse (Signet
Laboratories). Biotinylated antibodies were detected with streptavidin-Texas red (1:500, Molecular
Probes). Hoechst 33258 (Roche) was used to counterstain the nuclei. Proliferation was quantified
4
as the number of BrdU-immunoreactive cells per mm2 of media or intima. For each rabbit, 2-3
sections were analyzed and the results were averaged.
2.3 Statistical analysis. Results are reported as mean  SEM. Differences in body weight and
triglyceride levels among the 4 groups were evaluated using ANOVA and Fisher’s post hoc test.
All other comparisons between YC and OC were performed using 2-tail, unpaired Student’s t test.
Statistically significant differences were considered at p<0.05. The relationship between intimal
thickening and medial and intimal proliferation was evaluated by regression analysis.
3. RESULTS AND DISCUSSION
All rabbits appeared healthy throughout the experimental protocol and were included in all
analysis. By the end of the study, body weight in ON and OC rabbits was similar (4.30 ± 0.16 and
4.29 ± 0.14 Kg, respectively) and greater than respective young animals (YN=3.34 ± 0.08, p<0.003
vs. ON; YC=3.45 ± 0.18 Kg; p<0.0004 vs. OC). No significant differences were observed in
plasma triglycerides (YN=88.4 ± 9.7, YC=107.7 ± 15.5, ON=87.2 ± 24.9, OC=120.4 ± 29.9
mg/dL). Total plasma cholesterol in normolipemic animals was below the detection level (<45
mg/dL), and was exceedingly increased in fat-fed rabbits without significant differences between
YC and OC (2488 ± 315 and 1747 ± 212 mg/dL, respectively). With the exception of one YC
rabbit that developed intimal lesions in all arteries examined (aortic arch, common carotid and
femoral arteries), intimal thickening was restricted to the aortic arch of fat-fed rabbits. Therefore,
subsequent analysis was performed on aortic arch specimen.
Consistent with previous studies analyzing early lesions in hypercholesterolemic rabbits
[5,6], intimal lesions consisted predominantly of RAM11-immunoreactive macrophages in both
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YC and OC animals (Fig. 1). Generally, lesions in YC rabbits were thicker and more localized than
in OC rabbits. Intimal SM-actin immunoreactive vascular smooth muscle cells (VSMCs) were
not detected (not shown). Whereas medial cross-sectional areas were similar in the aortic arch of
YC and OC rabbits (4.58 ± 0.43 and 5.69 ± 0.35 mm2, respectively, p>0.05), intimal thickening
was significantly higher in YC rabbits (Fig. 2).
Using genetically-modified mice, we have recently shown that excessive arterial cell
proliferation plays a critical role during atherogenesis [4]. Because arterial cell proliferation is
abundant at the onset of atherosclerosis in hypercholesterolemic rabbits [7-10], we investigated
whether age-dependent differences in cell replication might contribute to augmented
atherosclerosis in juvenile rabbits. As expected, arterial BrdU incorporation in normolipemic
rabbits was scattered (not shown). In contrast, the aortic arch of YC and OC rabbits disclosed
abundant BrdU-immunoreactive cells within the intima and media (Fig. 3A, 3B, and data not
shown). Double immunofluorescence experiments demonstrated that proliferating intimal cells
were predominantly RAM11-immunoreactive macrophages (Fig. 3C). The major proliferating
component within the media appeared to be VSMCs, as suggested by abundant medial SM-actin
immunoreactivity and scattered RAM11-positive medial cells (Fig. 1, 3C and data not shown).
In both age groups, the number of proliferating cells within intimal lesions greatly
exceeded that in the media (Fig. 3D). Medial proliferation occurred mainly near intimal lesions
and was approximately four times higher in YC (Fig. 3D), which developed greater lesions than
OC animals (Fig. 2). These findings are consistent with the response-to-injury hypothesis [1],
according to which medial proliferation depends upon mitogenic signals coming from intimal
cells. Indeed, a direct correlation between medial cell proliferation and lesion size was manifest in
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both age groups (Fig. 4). In marked contrast, intimal cell replication was similar in young and old
rabbits (Fig. 3D), and did not correlate with the size of the lesion (Fig. 4).
In summary, the major finding of the present study is that juvenile rabbits receiving a shortterm high-dose hypercholesterolemic diet developed more atherosclerosis within the aortic arch
than did old counterparts. These differences occurred in spite of similar proliferative activity
within the intimal lesion in both age groups. Our findings appear to disagree with previous studies
reporting that old rabbits receiving short- (2 months) and long-term (18 months) exposure to a lowdose hypercholesterolemic diet developed more atherosclerosis than young counterparts [11,12].
This discrepancy may be due to differences in the dose of dietary cholesterol (0.25% vs. 1%
cholesterol), which resulted in striking differences in total plasma cholesterol. Indeed, young and
old rabbits receiving for 2 months the 0.25% cholesterol diet averaged plasma cholesterol levels of
148-178 mg/dL [12], whereas animals challenged for the same period with 1% cholesterol diet
averaged 1747-2488 mg/dL (this study). Thus, under mildly proatherogenic circumstances, agedependent intrinsic alterations in the vessel wall appear to increase the susceptibility to
atherosclerosis in aged rabbits. In contrast, when challenged with a highly atherogenic diet, young
rabbits appear more susceptible to atherogenesis than old animals. When taken together with
previous studies suggesting that early atherosclerosis in young persons might be associated with
cholesterol concentration [13,14], our findings underscore the importance of lowering excessive
dietary fat content in young individuals.
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ACKNOWLEDGEMENTS
We are grateful to Rosa Arroyo for technical assistance and María J. Andrés for the
preparation of figures. This work was supported by grants from the Spanish Dirección General de
Educación Superior e Investigación Científica (DGESIC, PM97-0136) and from the American
Heart Association, Massachusetts Affiliate (9860022T) to V. Andrés. A. Díez-Juan received salary
support from grant 1FD97-1035-C02-02 (DGESIC and FEDER).
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Rosenfeld, M.E. and Ross, R. (1990) Arteriosclerosis 10, 680-687.
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Spagnoli, L.G., Orlandi, A., Mauriello, A., Santeusanio, G., De Angelis, C., Lucreziotti, R.
and Ramacci, M.T. (1991) Atherosclerosis 89, 11-24.
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[13]
Stary, H.C. (2000) Am. J. Clin. Nutr. 72, 1297S-1306S.
[14]
McGill, H.C., Jr., McMahan, C.A., Herderick, E.E., Malcom, G.T., Tracy, R.E. and Strong,
J.P. (2000) Am. J. Clin. Nutr. 72, 1307S-1315S.
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Figure 1: Immunohistochemical analysis of aortic arch cross-sections of fat-fed YC and OC
animals
using
anti-RAM11
antibody
and
hematoxylin
counterstain.
Representative
microphotographs are shown.
Figure 2: Intima-to-media ratio in the aortic arch of YC and OC animals (n = 10 each group).
Figure 3: Cell proliferation within the aortic arch examined by immunostaining using mouse
monoclonal anti-BrdU antibody. (A) BrdU incorporation detected with streptavidin-peroxidase
(brown nuclear signal). Specimens were counterstained with hematoxylin. The right panel shows
and adjacent section stained with elastic-trichrome. Representative photomicrographs from one YC
rabbit is shown (Arrowheads: internal elastic lamina). (B) Same as in A, but BrdU-containing
immunocomplexes were detected using streptavidin-Texas Red (left). Hoechst 33258 nuclear
staining of the same microscopic field is shown on the right. Elastic fibers disclosed blue
autofluorescence. (C) Double immunofluorescence using anti-RAM11 antibody (revealed with
streptavidin-Texas Red) and anti-BrdU antibody conjugated to FITC (green signal). Nuclei are
counterstained with Hoechst 33258 (blue signal). Top: double exposure showing RAM11 and
Hoechst 33258 staining. Bottom: BrdU signal in the same microscopic field. Elastic fibers
disclosed green autofluorescence. Note the presence of proliferating macrophages in the intima
(co-localization of RAM11 and BrdU). (D) Number of BrdU-immunoreactive cells per surface of
media and intima in the aortic arch of YC and OC rabbits.
10
Figure 4: Number of BrdU-immunoreactive cells within the media and the intimal lesion of young
and old hipercholesterolemic rabbits as a function of intimal lesion size in the aortic arch. Each
point represents one animal.
11
FIGURE 1
Young-C
Old-C
12
FIGURE 2
0.12
Intima
p < 0.007
0.08
Media
0.04
Young-C
13
Old-C
FIGURE 3
C
A
Young
Old
-RAM11/Hoechst 33258
BrdU+ cells / mm2
-BrdU
D
Elastic Stain
MEDIA
20
15
10
5
BrdU+ cells / mm2
B
-BrdU
Hoechst 33258
-BrdU
14
p < 0.05
INTIMA
1200
800
400
FIGURE 4
Old
R2 = 0.7212
p < 0.002
0.3
Intima / Media
Intima / Media
Young
0.2
0.1
10
20
BrdU+ cells /
30
40
mm2
R2 = 0.6025
p < 0.009
0.08
0.06
0.04
0.02
50
2
(MEDIA)
4
BrdU+ cells /
R2 = 0.2501
0.2
0.1
500
1000
1500
8
mm2
10
12
(MEDIA)
Old
Intima / Media
Intima / Media
Young
0.3
6
2000
0.08
R2 = 0.3864
0.06
0.04
0.02
1000
BrdU+ cells / mm2 (INTIMA)
2000
3000
BrdU+ cells / mm2 (INTIMA)
15