Intimal Thickening in Normocholesterolemic Rhesus Monkeys Fed Low Supplements of Dietary Cholesterol
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Intimal Thickening in Normocholesterolemic Rhesus Monkeys
Fed Low Supplements of Dietary Cholesterol
By Mark L. Armstrong, Marjorie B. Megan, and Emory D. Warner
ABSTRACT
Rhesus monkeys were fed a high-fat diet containing either 0, 43, or 129 £tg/kcal of
cholesterol for 18 months. In the monkeys on the cholesterol-supplemented diets,
changes in plasma cholesterol remained within the range found in monkeys fed the cho-
lesterol-free diet. Monkeys on the cholesterol-supplemented diets were compared with
monkeys given no dietary cholesterol with range-matched plasma cholesterol; intimal
thickness of the aorta and branch arteries, distribution of lipoprotein cholesterol, and
tissue content of cholesterol in aorta and liver were considered. The monkeys on the cho-
lesterol-supplemented diets showed intimal thickening with more sudanophilia and in-
creased aortic cholesterol, a decrease in plasma high-density lipoprotein cholesterol, and
an increase in low-density lipoprotein cholesterol, and the monkeys fed the higher
amount of dietary cholesterol showed an increase in hepatic cholesterol. No null point for
the effect of dietary cholesterol on arterial intima was found even at an intake level far
below that conventionally used for the induction of experimental atherosclerosis in the
nonhuman primate. The intimal changes found in response to very low cholesterol intake
imply that subtle qualitative alterations in lipoproteins are of critical importance to our
understanding of lesion induction.
KEY WORDS plasma high-density lipoprotein cholesterol high-fat diet
plasma low-density lipoprotein cholesterol arterial cholesterol
distribution of lipoprotein cholesterol experimental atherosclerosis
• Rhesus monkeys fed high- or moderately high- the limits found in monkeys fed cholesterol-free
c h o l e s t e r o l diets d e v e l o p marked h y p e r - diets. In the present experiment we observed in-
cholesterolemia and atherosclerosis (1-8). The rise timal thickening associated with shifts in plasma
in total plasma cholesterol essentially involves an lipoprotein cholesterol toward low-density pre-
increase in low-density lipoprotein cholesterol (1, ponderance in monkeys on cholesterol-supple-
2, 5, 8, 9). When dietary intake of cholesterol and mented diets with plasma cholesterol levels within
fat is restricted to amounts comparable to those the control range.
recommended as a prudent diet for man (5), a slight
Methods
elevation in plasma cholesterol, areas of intimal
sudanophilia, and a few atheromatous lesions have Thirty adult male rhesus monkeys (6.7 ±0.19 kg)
were fed a semipurified diet (Table 1) in which approxi-
still been observed in the monkey (10). mately 40% of the total calories was derived from a mix-
As part of a dose-ranging study of the effects of ture of oils designed not to elevate (11) or lower (12)
cholesterol added at low to intermediate levels to plasma cholesterol. All monkeys were given 600 kcal/
the diet of rhesus monkeys, we investigated day to maintain positive caloric balances (13). After a
base-line period of 1 month, 50% of the monkeys were
whether morphologic changes occur in the arterial divided into three groups according to their plasma cho-
wall when plasma cholesterol is maintained within lesterol levels and given dietary cholesterol. Group 1 re-
ceived 43 /wg/kcal day"1, group 2 received 129 /xg/kcal
day"1, and group 3 received 387 j/g/kcal day"1. The re-
From the Arteriosclerosis Research Center and the Car- maining monkeys had no cholesterol added to their diet.
diovascular Division, Department of Internal Medicine, Univer- The experimental monkeys were by definition
sity of Iowa College of Medicine, Iowa City, Iowa 52242. monkeys on a cholesterol-supplemented diet with
This study was supported by U. S. Public Health Service plasma cholesterol levels below the upper limit (220
Grants HL-14230 and HL-14388 from the National Heart and mg/dliter) found in monkeys fed the cholesterol-free
Lung Institute. diet. The control group included five monkeys selected
Please address reprint requests to Mark L. Armstrong, M.D., from those fed the cholesterol-free diet; their plasma
Department of Internal Medicine, University Hospitals, Iowa cholesterol levels at the end of the study approximated
City, Iowa 52242.
This investigation was presented in part at the 45th Annual
the terminal mean and range values of the experimental
Meeting of the American Heart Association, November 1972. monkeys, 143 mg/dliter and 117 to 212 mg/dliter,
Received August 17, 1973. Accepted for publication January respectively. After 18 months the control and experi-
23, 1974. mental groups were autopsied after administration of
Circulation Research, VoL XXXIV, April 1974 447448 ARMSTRONG, MEGAN, WARNER
TABLE 1
Basic Diet
Fractional distribution
Source of food energy Within source Per total calories
Protein 0.20
Carbohydrate 0.41
Starch 0.75
Sucrose 0.25
Fat 0.39
Saturated 0.30
Monounsaturated 0.43
Polyunsaturated 0.27
Dietary ingredients included soya protein, cornstarch, cane sugar, safflower oil, peanut oil, and cocoa butter, with vitamins, salt mix,
and fiber as previously described (9).
phencyclidine anesthesia and exsanguination from the recovery were made with isotopic cholesterol and
abdominal aorta. cholesteryl oleate added to the combined extracts of
each sample.
MORPHOLOGIC STUDIES
The aortas were examined for visible lesions. Results
Histologic study was centered on the aorta at the arch RELATIONSHIP OF NORMOCHOLESTEROLEMIA TO DIETARY
and at the midpoints of the upper and lower halves of CHOLESTEROL CONTENT
the descending thoracic and the abdominal portions, on
the main coronary arteries at 0.4 cm (left) and 0.3 cm The responses of total plasma cholesterol to the
(right) from their ostia, on the subclavian artery midway three levels of dietary cholesterol are shown in Ta-
between the vertebral artery and the common trunk ble 2. All monkeys fed the two lower levels of cho-
(right) or the aorta (left), and on the common carotid lesterol, i.e., groups 1 and 2, maintained plasma
and femoral arteries at their midpoints. Paraffin sections cholesterol values within the control limit (220 mg/
were used for cross-sectional measurements of the in-
timal and medial areas by the projection technique pre- dliter) and were considered experimental monkeys.
viously described (14). The monkeys fed the highest level of cholesterol
(group 3) had plasma cholesterol values far above
PLASMA, AORTIC, AND HEPATIC LIPIDS
that of the control monkeys; they clearly failed to
Plasma cholesterol (15) was determined at 2-week or meet the criterion set for the experimental
monthly intervals in monkeys fasted overnight. The cho- monkeys and, therefore, were not included in this
lesterol content of high-density lipoproteins and low-
density lipoproteins was measured at the end of the study. Plasma cholesterol in the experimental
base-line diet period and at the end of the study by hep- monkeys rose above base-line values with terminal
arin-manganese precipitation (16, 17) after preparative increases averaging 12% and 45% for the two
ultracentrifugation of plasma at its own density and groups. The increases were evident within the first
removal of very low-density lipoprotein (18). The com-
pleteness of separation of high-density lipoproteins and month of cholesterol supplementation. In group 1
low-density lipoproteins was confirmed by paper elec- the increases at 2 weeks and 1 month averaged
trophoresis followed by elution of the appropriate areas 10% and 16%, respectively; and in group 2 the
with diethyl ether and qualitative analysis for cholester- increases were 34% and 46%, respectively. Only
ol by gas chromatography. random changes occurred in the control group.
Intima-inner media samples consisting of the inner Plasma triglycerides and phospholipids obtained
third of the aortic wall were removed under magnifica-
tion from normal-appearing portions of the upper ab- throughout the study as part of a more complete
dominal aorta. Hepatic tissue was taken from the right evaluation of plasma changes showed no sustained
lobe. All tissue was taken to constant dry weight at 41°C trends in experimental or control groups.
and extracted with a chloroform-methanol solution (2:1)
(19); cholesterol and cholesteryl ester were isolated by PLASMA CHANGES IN LIPOPROTEIN CHOLESTEROL
Florisil plates in a heptane-diethyl ether-acetic acid sol- Table 3 shows the high-density lipoprotein and
vent system (70:30:0.5). These lipid classes were the low-density lipoprotein cholesterol distribution
visualized by rhodamine 6G and eluted with three ex-
tractions of ether. Following hydrolysis of the ester band for control and experimental monkeys. Terminal
by the method of Abell et al. (15), cholesterol and high-density lipoprotein cholesterol values were
cholesteryl esters were determined by gas chromatogra- lower without exception in all experimental
phy as the trimethylsilyl derivative (20). Corrections for monkeys compared with base-line values, and theINTIMAL CHANGES WITH LOW CHOLESTEROL DIET 449
TABLE 2
Plasma Cholesterol Changes at Three Levels of Dietary Cholesterol
Plasma cholesterol
Dietary cholesterol Base line Final
Group (/xg/kcal) (mg/d liter) (mg/d liter)
1 43 115 ±2.5 130 ± 4.6
2 129 117 ± 2.6 168 ± 4.2
3 387 115 ± 2.0 392 ± 18.3
Plasma cholesterol values are means ± SE. Each group consisted offivemonkeys.
means of both experimental groups were lower keys had more intimal thickening expressed as
than the level found in the control monkeys. Ter- cross-sectional area than did the control group (Ta-
minal low-density lipoprotein cholesterol was high- ble 4). Whether the intimal thickening in experi-
er than the base-line value in all experimental mental monkeys was within a broader range of nor-
monkeys. The terminal low-density lipoprotein mal variation than that in the control group was in-
cholesterol level in group 1 averaged slightly less vestigated. The morphologic findings in the control
than that in the control group, but the ratio of low- group were suitably representative of numerous
density lipoprotein cholesterol to high-density control monkeys that we have studied previously.
lipoprotein cholesterol was slightly higher in group The average intimal thickening was generally
1. In group 2 the terminal low-density lipoprotein greater in group 2 than it was in group 1. Measure-
cholesterol level and the ratio of low-density ments of the transverse medial areas are not
lipoprotein cholesterol to high-density lipoprotein shown; they were quite similar among the groups
cholesterol were distinctly higher than they were and these indicate the comparability of arterial size
in the control group. at the sites preselected for measurement. The
ARTERIAL MORPHOLOGIC COMPARISONS histologic features found in experimental monkeys
Gross Aortic Lesions. —Macroscopic evidence of are shown in Figures 1-7. The involved areas con-
aortic intimal changes was minimal. The intimal tained significant fibrous and lipid elements. Foam
surface area that was estimated to differ from nor- cells were seen to a variable and sometimes promi-
mal appearance averaged 5% in the control groups, nent degree. The much smaller lesions in the con-
7.5% in group 1, and 11.8% in group 2. Clearly ele- trol group (not illustrated) were fibrotic thicken-
vated lesions were positively identified only in the ings of the intima that contained little lipid; foam
experimental monkeys; these lesions were small cells were sparse.
and consisted of both fatty and fibrous streaks. Tissue Cholesterol.—The cholesterol content of
Histologic Findings.—The experimental mon- normal-appearing aortic intima-inner media sam-
TABLE 3
Plasma Lipoprotein Cholesterol Changes with Dietary Cholesterol
Plasma cholesterol
(mg/dliter)
Dietary cholesterol
Group ( M g/kcal) HDL LDL Total LDL/HDL
Base line
Control 0 59 ±3.3 90 ±3.9 149 ± 5 1.52
1 0 56 ±3.7 59 ±3.8 115 ± 8 1.05
2 0 55 ±2.9 62 ±3.5 117 ± 6 1.12
Terminal
Control 0 58 ±4.1 88 ±4.1 146 ± 7 1.51
1 43 48 ± 3.9 82 ±8.1 130 ± 12 1.70
2 129 44 ± 3.4 125 ±9.6 168 ± 13 2.84
All values are means ± SE. Each group consisted offivemonkeys. HDL = high-density lipoprotein cholesterol and LDL — low-density
lipoprotein cholesterol.
Circulation Research, Vol. XXXIV, April 1974450 ARMSTRONG, MEGAN, WARNER
TABLE 4
Arterial Intimal Area (mm2)
Aorta
Group Thoracic Abdominal Carotid Subclavian Femoral Coronary
Control 0.014 ± 0.028 0.088 ± 0.044 0.000* 0.015 ± 0.014 0.000* 0.000*
1 0.356 ±0.113t 0.179 ± 0.033* 0.075 ±0.032 0.037 ± 0.021 0.056 ±0.035 0.006 ±0.003
2 0.502 ± 0.092f 0.255 ±0.061* 0.053 ±0.032 0.078 ± 0.027t 0.098 ±0.038 0.016 ±0.002
Values are means ± SE. Each group consisted of five monkeys.
'Average intimal area from endothelium to internal elastic membrane immeasurable at the magnification used (14).
tPINTIMAL CHANGES WITH LOW CHOLESTEROL DIET 4S1
FIGURE 3 FIGURE 4
Right main coronary artery from a monkey in group 2. The Common carotid artery from a monkey in group 1. Circum-
thickened intima shows marked cellularity beneath the flat en- ferential narrow fibrous bands surround a central intimal area
dothelium. Hemotoxylin and eosin stain; original magnification of myxoid appearance. Hemotoxylin and eosin stain; original
was 400x. magnification was 250x.
tended to be higher in the two groups fed choles- studies we have found that this redistribution pat-
terol. tern in lipoprotein cholesterol is characteristic of
The terminal plasma lipoprotein cholesterol monkeys on a cholesterol-supplemented diet.
values in the experimental monkeys differed from Lasser (21) has recently reported somewhat similar
base-line levels; a decrease in high-density lipopro- findings.
tein and an increase in low-density lipoprotein cho- The hepatic content of free cholesterol and
lesterol occurred after cholesterol feeding. Both cholesteryl esters was very similar in group 1 and
experimental groups had lower high-density the control group, but group 2, which received
lipoprotein cholesterol than did the control group, more dietary cholesterol, had more hepatic choles-
and group 2 had higher low-density lipoprotein terol, particularly cholesteryl ester. This difference
cholesterol. Such changes in plasma lipoprotein between group 2 and group 1 or the control group
cholesterol distribution suggest that the normo- is regarded as a reflection of the difference in
cholesterolemia of monkeys fed low levels of die- plasma low-density lipoprotein cholesterol level,
tary cholesterol is dissimilar from that found in since hepatic cholesterol content rises as plasma
monkeys on a cholesterol-free diet. In ongoing low-density lipoprotein cholesterol increases in
FIGURE 6
FIGURE 5 Femoral artery from a monkey in group 2. Variable
Subclavian artery from a monkey in group 1. Sudanophilia sudanophilia (black area) is seen in this lesion. Branch vessels in
(black area) is intense in the area of major intimal thickening. controls had no comparable lipid staining. Oil-red-O stain; ori-
Oil-red-0 stain; original magnification was 40x. ginal magnification was lOOx.
Circulation Rucarch, VoL XXXIV. April 1974452 ARMSTRONG, MEGAN, WARNER
the atherogenic potential of plasma; it estimates
the relative availability of the two major lipopro-
tein classes in postabsorptive plasma for uptake,
lipid exchange at the intimal surface, or both. How-
ever, the slightly higher terminal ratio in group 1
does not convincingly explain the obvious dif-
ference in intimal thickening between group 1 and
the control group. It remains to be established that
the ratio of low-density lipoprotein cholesterol to
high-density lipoprotein cholesterol is a better in-
dicator of the atherogenicity of plasma than is the
low-density lipoprotein cholesterol content in ex-
perimental atherosclerosis in primates. Moreover,
if we accept the view that a minor ratio difference
FIGURE 7
Femoral artery from a monkey in group 1. Intimal fibrous
during the 18-month experimental period favored
thickening and disruption of the internal elastic membrane sudanophilic intimal thickening, then we must also
differentiate this artery from control vessels. Verhoeff-Van accept the probability that, during a longer preex-
Gieson stain; orginal magnification was 250x. perimental period in adult monkeys, monkeys in
the control group had higher ratios than did those
in group 1 and that intimal thickening did not oc-
monkeys on a cholesterol diet (9, 22). cur.
The observed intimal thickening in the two ex- The data from group 1 suggest questions about
perimental groups cannot necessarily be attributed the effect of lipid food tides and about possible
to the same causes. In group 2, the clear increases qualitative lipoprotein changes in normocho-
in low-density lipoprotein cholesterol and in lesterolemic plasma that are consequent to choles-
hepatic cholesterol are changes involving the me- terol feeding. The fact that lipid absorbed from the
tabolism of cholesterol (22) and lipoprotein (9) that gut enters the arterial wall is well established (24,
cause or permit intimal thickening with associated 25). However, a role for postprandial hyperlipemia
increases in intimal lipid (23). The increase in in- per se as a contributing factor to intimal thickening
timal thickening in group 1, however, is more pre- has not been shown and was not evaluated in this
cariously ascribed to any metabolic change re- study. The results from group 1 parallel the find-
ported in this investigation. Plasma low-density ings of a number of in vivo and in vitro studies of ar-
lipoprotein cholesterol, hepatic free cholesterol, terial smooth muscle. Thomas et al. (26, 27) ob-
and hepatic cholesteryl ester levels were sta- tained in vivo evidence of a proliferative response
tistically similar to those levels in the control group in aortic smooth muscle cells after 72 hours of cho-
at the end of the experiment. Because group 1 had lesterol feeding and before hypercholesterolemia
lower terminal high-density lipoprotein cholester- was established. Fisher-Dzoga et al. (28) found that
ol, the ratio of terminal low-density lipoprotein cultured smooth muscle cells incorporated lipid
cholesterol to high-density lipoprotein cholesterol from postprandial serum without proliferation;
was higher than the ratio in the control group (Ta- they also observed, as have others (29), a prolifera-
ble 3). This ratio may possibly be a useful index of tive effect caused by low-density lipoproteins and a
TABLE 5
Cholesterol Content fmg/g dry weight) of Aortic Intima-lnner Media Samples
Group Free Ester* Total
Control 5.62 ± 0.82 0.76 ±0.24 6.36 ± 0.61
1 6.48 ± 0.96 2.53 ± 1.03f 9.01 ± l.Olf
2 7.78 ± 1.44 6.67 ± 1.35* 14.45 ± 2.28*
All values are means ± SE. Each group consisted of five monkeys.
'Cholesteryl moiety.
fP< 0.05 compared with control.
*P< 0.01 compared with control.
Circulation Retamti, Vol. XXXIV, April 1974INTIMAL CHANGES WITH LOW CHOLESTEROL DIET 453
TABLE 6
Cholesterol Content of Liver (mg/g dry weight)
Group Free Ester' Total
Control 5.13 ± 0.342 2.27 ±0.157 7.40 ± 0.410
1 4.97 ± 0.653 2.12 ± 0.200 7.09 ± 0.785
2 7.75 ± 0.260f 4.61 ± 0.531* 12.38 ± 0.681*
All values are means ± SE. Each group consisted offivemonkeys.
'Cholesteryl moiety.
t ? < 0.05 compared with control.
*P< 0.01 compared with control.
marked proliferation and incorporation of lipid, atherosclerosis in rhesus monkeys: I. Gross and light
microscopy features and lipid values in serum and aorta.
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