© 1995 American Heart Association, Inc.
Articles |
Selective Increase in Cholesterol at Atherosclerosis-Susceptible Aortic Sites After Short-term Cholesterol Feeding
From the Department of Pathology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC.
Correspondence to Dawn C. Schwenke, PhD, Department of Pathology, Bowman Gray School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1072. E-mail dschwenke@cpm.bgsm.wfu.edu.
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Abstract |
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Abstract In rabbits, the aortic arch and branch sites of the descending thoracic and abdominal aortas are susceptible to atherosclerosis. This study investigated the hypothesis that the reported focal increase in LDL concentration and mean residence time at susceptible aortic sites after feeding cholesterol for 4 to 8 days precede atherosclerotic change as indicated by increased aortic cholesterol concentration. Cholesterol concentrations for all aortic sites of normal rabbits were similar (
2.8 µmol/g). No change in aortic
cholesterol concentration could be detected after feeding
cholesterol for 8 days. However, after feeding
cholesterol for 12 and 16 days, cholesterol
concentrations for abdominal branch sites were increased compared with
abdominal branch sites of normal rabbits (4.47±0.50, n=8, and
4.85±0.33, n=11, µmol/g, respectively, versus 2.87±0.27, n=12,
µmol/g; P<.025 and P<.005, respectively). In
contrast, the cholesterol concentration of
atherosclerosis-resistant nonbranch
abdominal aorta was unchanged after feeding cholesterol for
16 days and was much less than that of the branch sites (2.72±0.12
versus 4.85±0.33 µmol/g, n=11; P<.001).
Cholesterol concentrations for other susceptible sites were
also increased after feeding cholesterol for 12 and 16
days. Cholesterol concentrations for susceptible sites were
linearly related to a combined measure of duration and extent of
hypercholesterolemia (P<.001 to
P<.0001), whereas no such relationship could be detected
for resistant sites. Most (59% to 93%) of the
cholesterol accumulating in susceptible aortic sites after
feeding cholesterol for 12 and 16 days was nonesterified,
suggesting that the increased cholesterol concentration did
not reflect development of foam cells or the insudation of plasma
lipoproteins. This study suggests that the reported focal increases in
LDL concentration and mean residence time at susceptible aortic sites
during cholesterol feeding precede
atherosclerosis.
Key Words: atherosclerosis • cholesterol • aorta • susceptibility • rabbit
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Introduction |
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Atherosclerotic lesions develop in nonrandom, characteristic arterial sites in human individuals1 2 and experimental animals3 including rabbits.4 5 6 7 8 9 In hypercholesterolemic rabbits, the aortic sites susceptible to atherosclerosis include the aortic arch and branch sites of the thoracic and abdominal aortas, whereas the uniform (nonbranch) areas of the thoracic and abdominal aorta are relatively resistant to atherosclerosis. Several detailed studies in rabbits have mapped the distribution of atherosclerotic lesions at sequential early times after feeding cholesterol,4 10 after feeding cholesterol for several months,4 6 7 8 and during the regression of atherosclerosis.5
Cholesterol and cholesterol ester are characteristic components of atherosclerotic lesions of human beings.11 12 13 Studies in experimental animals,14 15 16 17 including rabbits,15 16 17 have reported the NEC and EC content of arteries after the development of macroscopically evident atherosclerotic lesions. Other studies have characterized the cholesterol composition of normal (or relatively normal) artery and atherosclerotic lesions within the same arteries.18 19 Limited characterization of the cholesterol compositions of selected arterial sites susceptible and resistant to atherosclerosis has been reported for artery of normal pigeons and those fed cholesterol for increasing intervals up to 8 weeks20 and for normal pigs and those fed cholesterol for 6 weeks.21 Some data are also available for the cholesterol concentration for the aortic arch compared with the less susceptible thoracic and abdominal aortas of normal rabbits.17 22 23 However, as far as we know, cholesterol concentration and composition have not been reported for atherosclerosis-susceptible branch sites of the thoracic or abdominal aortas of normal rabbits nor for these same sites during very early stages of cholesterol feeding (ie, before the development of atherosclerotic lesions).
The aortic rate of LDL degradation and estimated aortic concentrations of undegraded LDL are focally elevated in the atherosclerosis-susceptible aortic sites of rabbits fed a cholesterol-free diet.24 25 These differences are exaggerated during 16 days of feeding a cholesterol-containing diet.25 Retention of LDL in the artery is similar among all aortic sites of normal rabbits.26 However, retention of LDL is increased in the atherosclerosis-susceptible aortic sites after feeding cholesterol for 4 days.26 Differences between corresponding susceptible sites of normal and cholesterol-fed rabbits are evident as early as 8 days after beginning cholesterol feeding for both aortic concentrations of undegraded LDL and aortic rates of LDL degradation.25 26 The goal of the studies reported here was to determine how such observed changes in aortic rates of LDL degradation, estimated aortic concentrations of undegraded LDL, and aortic retention of LDL were related to temporal changes in ACCs, an index of the development of atherosclerosis. As shown below, cholesterol concentrations for atherosclerosis-susceptible aortic sites were not altered after feeding cholesterol for 8 days but had increased after feeding cholesterol for 12 days. These data suggest that changes in aortic concentrations of undegraded LDL and aortic rates of LDL degradation precede by about 4 days atherosclerotic change as indicated by increased ACC.
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Methods |
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Rabbits
To facilitate comparison with earlier work,24 25 26 the rabbits used in the present studies were of the same breed, sex, and weight and were housed under similar conditions to the rabbits used in the previous studies. Thirty-eight of the total of 48 rabbits used in the present studies were obtained from Franklin's Rabbitry and Supply; 10 were obtained from Robinson Services, Inc. All rabbits were acclimated to the animal facility for about 1 week before entering the study, during which time they were fed a standard cholesterol-free rabbit chow (Prolab; Agway). Rabbits weighed
2.5 kg before entering the study and 2.60±0.03 kg (n=47) at
the end of the study. Twelve of these rabbits continued to receive the
cholesterol-free rabbit chow and served as controls.
The remaining 36 rabbits were studied after they had consumed the same
diet supplemented with cholesterol and 2.5% corn oil for 2
to 4 (n=5), 8 (n=11), 12 (n=9), or 16 (n=11) days. The
cholesterol concentration in the diet was initially 0.5%;
most rabbits fed cholesterol were given this same diet
throughout the study. However, for two of the rabbits (one 8- and one
12-day rabbit), dietary cholesterol was reduced to 0.2%
midway in the study so that their plasma cholesterol
concentrations did not exceed the average of those observed in rabbits
in earlier studies.25 26 All rabbits were fed 100 g of
their respective diets each day. For most of the rabbits fed
cholesterol, 1-mL blood samples were collected from the
peripheral vein of either ear after an overnight fast
during the period when the rabbits consumed the
cholesterol-free rabbit chow and at 4-day intervals
during cholesterol feeding. One-milliliter blood
samples were collected after an overnight fast from all rabbits
immediately before death. All blood samples were collected into 0.01
vol of 0.4 mol/L disodium EDTA. Most rabbits were killed by an overdose
of pentobarbital sodium (100 mg/kg body wt IV), but a few were
exsanguinated after being deeply anesthetized with
ketamine hydrochloride (60 mg/kg body wt IM) and xylazine (6
mg/kg body wt IM). Typically, similar numbers of untreated rabbits and
those fed cholesterol for 8, 12, and 16 days were studied
at a given time. Because rabbits from different suppliers may respond
differently to dietary cholesterol, data for the 38 rabbits
obtained from Franklin's Rabbitry were analyzed separately,
and these results were compared with corresponding analyses for
pooled data for rabbits from both suppliers. Because corresponding
results were qualitatively and quantitatively similar, we report pooled
results for rabbits obtained from the two suppliers. These studies were
approved by the Animal Care and Use Committee of the Bowman Gray School
of Medicine of Wake Forest University.
Aortic Sampling
After euthanasia, the heart and aorta extending to the iliac
bifurcation were removed en block. The aorta was separated from the
heart at the aortic valve. The abdominal aorta was separated from the
aortic arch and descending thoracic aorta 1 to 2 mm above the celiac
orifice. Adventitial tissue was removed from both sections of the
aorta. Aortic segments were opened longitudinally, pinned flat, and
photographed. The aortic arch was separated from the descending
thoracic aorta 1 to 2 mm distal to the ductus arteriosus. Next,
triangular samples of aorta were removed from branch orifice areas of
the (atherosclerosis-susceptible) abdominal aorta,
and trapezoidal samples of aorta (including pairs of orifices) were
obtained from intercostal orifice areas in the
(atherosclerosis-susceptible) descending thoracic
aorta (Fig 1
). The aortic samples were
photographed again. Because atherosclerotic change should be manifested
in the intimal layer of the artery,12 27 the thickest
aortic sample (aortic arch) was separated into an inner layer (intima
plus inner media) and an outer layer (outer media). During these
procedures, aortic samples were maintained at 4°C whenever possible.
After subdividing the aortic samples as described above, all aortic
samples were weighed and frozen at -20°C until
analysis. The intima plus inner media of the aortic arch
accounted for 39.9±2.5% (n=12, mean± SEM), 45.9±3.2% (n=5),
36.7±1.9% (n=11), 41.8±2.2% (n=9), and 39.6±1.7% (n=11) of the
wet weight of the full thickness of the aortic arch for the normal
rabbits and those studied after feeding cholesterol for 2
to 4, 8, 12, and 16 days, respectively.
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Plasma Cholesterol Concentrations and
ACCs
Cholesterol concentrations in plasma were determined
with an enzymatic method.28 Lipids were extracted from all
aortic samples with chloroform/methanol (2:1, vol/vol), and the
resulting extracts were washed with water.29
[3H]Cholesterol that was >98% pure by
thin-layer chromatography on silica gel 60 (EM
Science) with development in hexane/diethyl ether/acetic acid (50:50:1,
vol/vol/vol) was present as an internal standard to correct for
procedural losses. TC (NEC plus EC) concentration was determined in
duplicate30 after saponification31 of an
aliquot of the lipid extract. For most aortic samples, NEC and EC were
separated from another aliquot of the lipid extract by thin-layer
chromatography as described above. NEC was
determined30 and corrected for recovery from
thin-layer chromatography. EC concentrations of
these aortic samples were calculated as differences between measured TC
and NEC concentrations. For some samples the measured concentration of
NEC was slightly greater than that of TC, resulting in a negative value
for EC. Such negative values were retained when calculating averages to
avoid overestimation of very low values of EC (for which measurement
errors might result in some positive and some negative values).
CEI
The aortic exposure to elevated plasma cholesterol
(ie, the CEI) was calculated by determining the area under the curve of
the increment in plasma cholesterol concentration versus
time of cholesterol feeding. The increment in plasma
cholesterol concentration at the individual times of
cholesterol feeding was determined by subtracting the
plasma cholesterol concentration before
cholesterol feeding from the plasma cholesterol
concentration at the various times of cholesterol feeding.
CEIs for normal rabbits are then zero. In some cases it was not
possible to collect plasma samples before cholesterol
feeding or at one or several times after beginning
cholesterol feeding. In these cases, values of plasma
cholesterol concentrations before beginning
cholesterol feeding were estimated as the average of all
measured plasma cholesterol concentrations before
cholesterol feeding (1.15±0.07 mmol/L, n=25). Missing
values for plasma cholesterol concentrations after feeding
cholesterol for 4, 8, or 12 days were estimated from the
plasma cholesterol concentration just before euthanasia and
the relationships determined between measured plasma
cholesterol concentrations after feeding
cholesterol for different intervals.
Statistical Methods
Possible differences between data for adjacent
atherosclerosis-susceptible and
atherosclerosis-resistant aortic sites were
compared by paired t tests.32 ANOVA with a
multiple-measures design33 was used to assess the
overall effect of atherosclerosis susceptibility on
ACC. ANOVAs were performed with BMDP program 2V (BMDP
Statistical Software, Inc). To determine whether
cholesterol feeding increased ACCs over those of normal
rabbits, data for aortic sites of rabbits fed cholesterol
for different times were compared with those for the corresponding
aortic sites of normal rabbits either by independent-samples
t tests or Wilcoxon's two-sample rank test
(when group variances were dissimilar).32 Probability
values were adjusted by using the Bonferroni criteria to account for
the multiple comparisons with the same control.34 The
influence of duration of cholesterol feeding on ACCs was
investigated by linear regression.32 The combined
influences of extent and duration of
hypercholesterolemia on ACC were evaluated by
regression of ACC on the CEI.32
Sequential measurements of plasma cholesterol concentrations in rabbits fed cholesterol for the different times were compared with the value before cholesterol feeding for the same rabbits by using Wilcoxon's two-sample rank test32 and the Bonferroni criteria.34 Plasma cholesterol concentrations at death for rabbits fed cholesterol were compared with those for the normal rabbits in a similar manner. Data are presented as mean±SEM. A probability value of less than .05 was considered significant.
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Results |
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Plasma Cholesterol Concentrations
Plasma cholesterol concentrations increased approximately linearly during cholesterol feeding to values 15-, 20-, and 24-fold above the normal value, respectively, for rabbits killed after feeding cholesterol for 8, 12, and 16 days (Fig 2
). To relate data for the ACCs
presented here to previous data for aortic rates of LDL
degradation, estimated aortic concentrations of undegraded LDL, and
aortic retention of LDL,25 26 it is important that the
rabbits used for the present study were very similar to those of
the previous studies. For this reason, the present study was
conducted with young adult rabbits of the same breed and sex and
similar weight and that were housed in conditions similar to those used
in the previous studies. In addition, it was important to establish
that aortic exposure to hypercholesterolemia
was similar for both groups of rabbits. As shown in the
Table, plasma cholesterol concentrations
at death for the normal rabbits described here were 25%
(P<.025) lower than those described in the previous study.
However, plasma cholesterol concentrations at death for
rabbits studied after feeding cholesterol for 8 and 16 days
(the two periods of cholesterol feeding in common between
the two studies) differed less than 5% between the two studies
(Table). In addition, the time dependence of the increase in plasma
cholesterol concentrations observed in this study (Fig 2) was similar regardless of whether rabbits were
maintained on the cholesterol-containing diet for 8,
12, or 16 days. These data suggest that valid comparisons can be made
between temporal changes in aortic rates of LDL degradation, estimated
aortic concentrations of undegraded LDL, and aortic retention of
LDL25 26 and the temporal changes in ACCs reported
below.
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Comparison of Cholesterol Concentrations of
Atherosclerosis-Susceptible and Adjacent
Atherosclerosis-Resistant Aortic
Sites
TC concentrations were first compared between adjacent aortic
sites susceptible and resistant to
atherosclerosis. For normal rabbits, TC concentrations
for atherosclerosis-susceptible and
atherosclerosis-resistant aortic sites were
similar (2.75 µmol/g fresh wt; Fig 3). After
feeding cholesterol for 8 days, the TC concentration was
3.03±0.25 µmol/g in branch sites of abdominal aorta compared with
2.55±0.10 µmol/g for adjacent
atherosclerosis-resistant abdominal aorta,
a 19% increase (P<.05). ANOVA with multiple measures on
aortic sites revealed an overall 11% (P<.05) higher TC
concentration for susceptible aortic sites after feeding
cholesterol for 8 days. During the next 4 to 8 days of
cholesterol feeding, the TC concentrations continued to
increase selectively in atherosclerosis-susceptible
aortic sites. After feeding cholesterol for 12 days, TC
concentrations for aortic arch intima plus inner media and branch sites
of the descending thoracic and abdominal aortas were 18%
(P<.001), 9.5% (P<.05), and 56%
(P<.005) higher, respectively, than those for the adjacent
atherosclerosis-resistant sites. After
feeding cholesterol for 16 days, the differences in TC
concentrations between these pairs of
atherosclerosis-susceptible and
atherosclerosis-resistant sites increased
to 36% (P<.01), 20% (P<.01), and 80%
(P<.001), respectively.
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) aortic sites during 16 days of cholesterol
feeding. For rabbits never fed cholesterol, n=12 (except
n=11 for thoracic branch sites); rabbits fed cholesterol
for 8 days, n=11; rabbits fed cholesterol for 12 days, n=9
(except n=8 for nonbranch thoracic aorta and abdominal branch sites);
and rabbits fed cholesterol for 16 days, n=11 (except n=10
for arch outer media). *P<.05, **P<.01,
***P<.001 atherosclerosis-susceptible
vs adjacent resistant sites by paired t test.
+P<.05, ++ P<.01 corresponding aortic sites of
normal rabbits vs those fed cholesterol by
independent-samples t test with the Bonferroni
correction for multiple comparisons.
Changes in ACC During Cholesterol Feeding
To determine when aortic TC concentrations were altered by
cholesterol feeding, aortic data for rabbits fed
cholesterol were compared with data for the corresponding
aortic sites of normal rabbits. No difference in TC concentrations
could be detected between corresponding aortic sites of normal rabbits
and those fed cholesterol for 8 days. However, after
feeding cholesterol for 12 days, cholesterol
concentrations for atherosclerosis-susceptible
aortic arch intima plus inner media and abdominal aorta branch sites
were 3.48±0.22 and 4.47±0.50 µmol/g, respectively, compared with
2.87±0.10 and 2.87±0.27 µmol/g for the corresponding susceptible
aortic sites of normal rabbits, increases of 21% (P<.05)
and 56% (P<.025), respectively. After feeding
cholesterol for 16 days, cholesterol
concentrations for the aortic arch intima plus inner media and
abdominal aorta branch sites were, respectively, 36%
(P<.005) and 69% (P<.003) greater than for the
corresponding sites of normal rabbits. A 30% increase
(P<.01) in TC concentration could also be detected
for thoracic aorta branch sites. TC concentrations showed significant
linear increases during cholesterol feeding for
atherosclerosis-susceptible aortic arch intima plus
inner media and branch sites of thoracic and abdominal aortas
(P<.0001, P<.002, and P<.0001,
respectively, by regression analysis; Fig 3
).
In contrast, duration of cholesterol feeding did not
influence TC concentrations for
atherosclerosis-resistant aortic sites.
NEC Concentrations for Atherosclerosis-Susceptible
and Adjacent Atherosclerosis-Resistant
Aortic Sites
To determine the nature of any changes in cholesterol
concentrations of aortic sites, aortic TC was separated into NEC and
EC. Fig 4 (left) shows NEC concentrations for aortic
sites susceptible and resistant to
atherosclerosis. For normal rabbits and those fed
cholesterol 8 days, 93% to 100% of the
cholesterol present in all aortic sites was
nonesterified. NEC concentrations did not differ between adjacent
susceptible and resistant sites of normal rabbits or those fed
cholesterol 8 days. However, after feeding
cholesterol for 8 days, NEC concentrations were 16% higher
for all atherosclerosis-susceptible sites combined
compared with all atherosclerosis-resistant
sites combined (P<.05 by multiple-measures ANOVA).
After feeding cholesterol for 12 days, NEC concentrations
were increased in two of three susceptible aortic sites compared with
the adjacent resistant sites (aortic arch intima plus inner
media, 19%; P<.01; thoracic aorta branch sites, 29%;
P<.01). The abdominal branch sites showed a trend in the
same direction. After feeding cholesterol for 16 days, NEC
concentrations were increased by 22% and 52% for branch sites of
thoracic and abdominal aorta, respectively, compared with the
corresponding adjacent
atherosclerosis-resistant sites (both
P<.005).
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EC Concentrations for Atherosclerosis-Susceptible
and Adjacent Atherosclerosis-Resistant
Aortic Sites
EC concentrations (Fig 4, right) were very low in
all aortic sites of normal rabbits and did not differ between
susceptible and resistant sites. Aortic EC concentrations
remained very low after feeding cholesterol for 8 days.
However, at this time the EC concentration was slightly but
significantly greater for
atherosclerosis-susceptible aortic arch intima plus
inner media compared with
atherosclerosis-resistant aortic arch outer
media (P<.02). After feeding cholesterol for 12
days, the EC concentration was higher for abdominal aorta branch sites
compared with the adjacent resistant site (0.70±0.14 versus
0.01±0.14 µmol/g, P<.01). After this time of
cholesterol feeding, EC concentrations did not differ
between susceptible and resistant sites of the aortic arch. EC
could not be detected in thoracic branch sites (-0.39±0.26
µmol/g) and was lower in those sites than in the adjacent
resistant site (0.13±0.06 µmol/g, P<.05). After
feeding cholesterol for 16 days, the EC concentration of
atherosclerosis-susceptible aortic arch intima plus
inner media was higher than the outer media (0.53±0.19 versus
-0.09±0.14 µmol/g, P<.02). The numerically larger
difference between EC concentrations for susceptible and
resistant sites of abdominal aorta after feeding
cholesterol for 16 days was of borderline significance
(P<.07).
Changes in EC and NEC Concentrations of Aortic Sites During
Cholesterol Feeding
Compared with corresponding aortic sites of normal rabbits, an
increase in NEC concentration was first detected after feeding
cholesterol for 12 days, but only in thoracic aorta branch
sites (3.51±0.28 versus 2.57±0.21 µmol/g, P<.05). After
feeding cholesterol for 16 days, NEC concentrations were
increased in all atherosclerosis-susceptible aortic
sites: 24% (P<.006) in aortic arch intima plus inner media
and 35% (P<.02) and 55% (P<.005),
respectively, for thoracic and abdominal aorta branch sites.
Compared with normal rabbits, an increase in aortic EC concentration was first detected after feeding cholesterol for 12 days. The absolute increase in EC concentration for susceptible sites was much smaller than the increase in NEC concentration. However, after feeding cholesterol for 12 days, the EC concentration of abdominal branch sites was increased 304-fold (0.70±0.14 versus 0.002±0.078 µmol/g, P<.005). After feeding cholesterol for 16 days, the EC concentration was increased 11-fold in aortic arch intima plus inner media (0.53±0.19 versus 0.046±0.026 µmol/g, P<.02). The 348-fold increase in EC concentration in abdominal branch sites after feeding cholesterol for 16 days failed to reach significance (P<.15) due to a large degree of variability in the responses of individual rabbits to cholesterol feeding. Despite these relatively large increases in EC concentrations for susceptible sites during cholesterol feeding, NEC still accounted for 84% to 99% of the TC concentration for susceptible aortic sites after feeding cholesterol for 16 days.
NEC showed significant linear increases as determined by using regression analysis during cholesterol feeding for aortic arch intima plus inner media (P<.002) and branch sites of abdominal (P<.002) and thoracic (P<.004) aortas. EC also showed significant linear increases during cholesterol feeding for aortic arch intima plus inner media (P<.025) and abdominal branch sites (P<.003). There was a small but significant linear increase in NEC during cholesterol feeding for nonbranch thoracic aorta (P<.05 by regression analysis). Cholesterol feeding did not influence the NEC concentrations of the other atherosclerosis-resistant aortic sites or the EC concentration of any atherosclerosis-resistant site.
Relation Between Aortic Exposure to
Hypercholesterolemia and
Cholesterol Concentrations of
Atherosclerosis-Susceptible and
Atherosclerosis-Resistant Aortic
Sites
While cholesterol concentrations of
atherosclerosis-susceptible aortic sites after
feeding cholesterol for 12 or 16 days were higher than
those of corresponding sites of normal rabbits, considerable
variability was observed for ACCs among rabbits fed
cholesterol for any given time. Additionally, individual
rabbits showed variation in degree of
hypercholesterolemia while consuming the same
cholesterol-containing diet. To investigate the
hypothesis that aortic cholesterol accumulation in rabbits
fed cholesterol was determined not only by duration but
also by degree of hypercholesterolemia, aortic
TC concentrations were related to the aortic exposure to
hypercholesterolemia (ie, the CEI). As shown in
Fig 5, TC concentrations for
atherosclerosis-susceptible aortic arch intima plus
inner media (Fig 5A) and branch sites of descending
thoracic (Fig 5C) and abdominal (Fig 5E) aortas were significantly related to the CEI
(P<.0001, P<.001, and P<.0001,
respectively). In contrast, aortic TC concentration and CEI were not
related for atherosclerosis-resistant
aortic arch outer media (Fig 5B) and nonbranch thoracic
(Fig 5D) and abdominal (Fig 5F) aortas.
Additional analyses were performed to investigate the relation
between aortic NEC and EC concentrations and the CEI. For susceptible
sites of aortic arch and abdominal aorta, both NEC and EC were linearly
related to the CEI (NEC, P<.01 and P<.04, and
EC, P<.005 and P<.001 for susceptible sites of
aortic arch and abdominal aorta, respectively; data not shown). No such
relation could be demonstrated for thoracic aorta branch sites or any
atherosclerosis-resistant aortic site.
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); rabbits fed cholesterol for 8
days, n=11 ( |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In rabbits, the aortic arch and branch sites within the thoracic and abdominal aortas are susceptible to atherosclerosis in the presence of hypercholesterolemia, whereas uniform (nonbranch) thoracic and abdominal aortas are relatively resistant to atherosclerosis.4 5 6 7 8 9 10 One aim of this study was to characterize the time dependence of aortic accumulation of TC, EC, and NEC (indices of the development of atherosclerosis) after short periods of cholesterol feeding. A second aim was to compare the interval of cholesterol feeding at which an increase in ACC could first be detected in atherosclerosis-susceptible aortic sites with the interval that induced changes in aortic rates of LDL degradation, estimated aortic concentrations of undegraded LDL, and aortic retention of LDL.25 26 For this purpose, we studied rabbits of the same breed, sex, and weight as in the earlier studies,25 26 and we carefully matched plasma cholesterol concentrations during cholesterol feeding to the earlier studies (Table
). We investigated the same atherosclerosis-susceptible aortic sites as we had studied previously, ie, aortic arch and branch sites of the thoracic and abdominal aortas.24 25 26 To increase the probability of identifying small changes in cholesterol concentration for the small thoracic intercostal orifice sites, in the present study we changed the shape of the aortic sample taken from these sites from a triangle to a trapezoid, which included the orifice area but less nonbranch artery than did the triangles. In addition, because atherosclerotic change should be manifested in the intimal layer of the artery,12 27 we divided the aortic arch (the thickest aortic sample) into the intima plus inner media and the outer media.
For normal rabbits, the cholesterol concentrations for atherosclerosis-susceptible and atherosclerosis-resistant aortic sites were similar. To determine when ACCs were altered by cholesterol feeding, cholesterol concentrations of corresponding aortic sites of normal rabbits and those fed cholesterol were compared. Differences in ACCs between rabbits fed cholesterol and those not fed cholesterol were first evident after feeding cholesterol for 12 days, whereas differences between atherosclerosis-susceptible and atherosclerosis-resistant aortic sites were first evident after feeding cholesterol for 8 days. Differences in ACCs between corresponding sites of normal and cholesterol-fed rabbits and between adjacent susceptible and resistant aortic sites were further exaggerated after feeding cholesterol for 16 days. As far as we know, no comparable data have been reported. These results further emphasize the very focal development of atherosclerosis in characteristic aortic sites of rabbits.
In comparison, rates of aortic LDL degradation and aortic concentrations of undegraded LDL are increased in atherosclerosis-susceptible abdominal branch sites and aortic arch of rabbits never fed cholesterol,24 and retention of LDL within the artery is increased in atherosclerosis-susceptible aortic sites after feeding cholesterol for only 4 days.26 After feeding rabbits cholesterol for 3 days, Day and Proudlock35 report that incorporation of radiolabeled oleate into cholesterol ester is increased more than twofold in the atherosclerosis-susceptible aortic arch intima, whereas no change in cholesterol esterification was observed in the aortic arch media or in the intima or media of the relatively atherosclerosis-resistant thoracic and abdominal aortas. This metabolic change was observed in the absence of a change in arterial cholesterol concentration.35
Consistent changes in interaction of LDL with the aorta predicted to be atherogenic (increased absolute rates and decreased fractional rates of aortic LDL degradation, increased aortic concentrations of undegraded LDL, and prolongation of aortic retention of LDL) occur preferentially in susceptible aortic sites during 4 to 16 days of cholesterol feeding.25 26 Fractional rates of aortic LDL degradation are first significantly decreased compared with normal rabbits for abdominal branch sites after feeding cholesterol for 8 days.26 This suggests that atherogenic changes in interaction of LDL with the aorta occur about 4 days before atherosclerotic change in the aorta as indicated by increases in ACC. These atherogenic changes in interaction of LDL with the aorta may provide a mechanism to explain a portion of the increased susceptibility of characteristic aortic sites to atherosclerosis.
During the 16-day period of cholesterol feeding, most (59% to 93%) of the increase in cholesterol concentration in atherosclerosis-susceptible aortic sites was NEC, although the relative increase in the EC concentration was greater. Even after feeding cholesterol for 16 days, NEC accounted for 84% to 99% of the cholesterol in atherosclerosis-susceptible aortic sites, in agreement with results for pooled aorta of rabbits fed cholesterol for 2 weeks.36 This predominance of NEC in susceptible aortic sites of rabbits fed cholesterol for 16 days would not be consistent with the development of intimal foam cells, which typically accumulate EC.37 38 In addition, data from our earlier study25 suggest that less than 1.4% of the intimal surface of atherosclerosis-susceptible aortic sites contains foam cells after feeding cholesterol for 16 days. Thus it seems unlikely that intimal accumulation of foam cells accounts for a significant portion of the increase in cholesterol concentration observed for atherosclerosis-susceptible aortic sites after feeding cholesterol for 16 days.
It is relevant to compare the cholesterol accumulation in
atherosclerosis-susceptible aortic sites with that
which might be present as part of lipoproteins retained within the
artery. After feeding cholesterol for 16 days, the
concentration of undegraded LDL retained in abdominal branch sites was
estimated to be 3.12% of the plasma LDL concentration.25
After feeding cholesterol for 16 days, plasma
cholesterol concentrations of rabbits in this study
averaged 29.5 mmol/L (Table). If LDL accounts for 15.3% of the plasma
cholesterol concentration, as it did for rabbits with
similar plasma cholesterol concentrations,25
the plasma LDL cholesterol concentrations of rabbits
studied here after feeding cholesterol for 16 days would be
4.42 mmol/L. At this plasma LDL concentration, retention of undegraded
LDL by abdominal branch sites at 3.12% of the plasma concentration
would contribute 0.141 µmol cholesterol/g artery. This
amount is 7.2% of the observed increase in TC for these sites after
feeding cholesterol for 16 days (Fig 3).
Assuming that 71% of LDL cholesterol is
esterified,39 the amount of undegraded LDL present in
abdominal branch sites would amount to 0.0409 µmol NEC/g artery and
0.100 µmol EC/g artery. These values are 2.7% and 12.5% of the
observed increases in NEC and EC concentrations, respectively, after
this same interval of cholesterol feeding. Similar
calculations indicated that the increase in undegraded LDL present
in abdominal branch sites after feeding cholesterol for 8
days25 would be expected to increase aortic
cholesterol only by 2.5%, consistent with the
present inability to detect a significant increase in
cholesterol for this aortic site after feeding
cholesterol for 8 days.
Lipoproteins other than LDL may be retained within the artery, and calculations similar to those above can be done for all plasma lipoproteins. For this purpose, we assumed that all lipoproteins in plasma are retained in the artery in proportion to their concentrations in plasma, and we used the weighted average of the percent of cholesterol (77%) that is esterified in the various lipoprotein fractions in plasma.39 Such calculations suggest that retention of all plasma lipoproteins in the artery in proportion to their concentrations in plasma would contribute 0.920 µmol TC/g abdominal branch sites for rabbits fed cholesterol for 16 days, 46.7% of the observed increase in TC for this aortic site. Of this amount, 0.212 and 0.708 µmol would be NEC and EC, respectively, accounting for 14.2% and 88.5% of the NEC and EC present in this aortic site after feeding cholesterol for 16 days. Qualitatively similar results were obtained when similar calculations were done for aortic arch (intima plus inner media and outer media in order to compare with the earlier data25 ) and thoracic aorta branch sites. Thus, retention of plasma lipoproteins within the artery may account for a significant fraction of the increase in EC concentration found for atherosclerosis-susceptible aortic sites after feeding rabbits cholesterol for 16 days. However, most NEC in these sites must be present in other forms.
Lipid vesicles rich in NEC and containing little or no EC have been demonstrated not only in atherosclerotic lesions of human beings40 41 and rabbits36 40 42 43 44 45 but also in normal rabbit aorta.36 Another group observed extracellular accumulation of membranous material (NEC and phospholipids) and smaller amounts of neutral lipid droplets (EC) in arteries of rabbits fed cholesterol for 1 to 2 weeks.46 While some investigators have stated that lipid vesicles within the aorta increase after feeding cholesterol for 10 days to 2 weeks,43 47 the only quantitative study36 showed little if any increase in NEC-rich vesicles after feeding rabbits a 1% cholesterol diet for 2 weeks, and only a small increase after feeding the same diet for 4 weeks. However, to obtain sufficient material from which to isolate the vesicles, Chao et al36 pooled entire aortas from a number of rabbits, potentially diluting what could be substantial changes in the small atherosclerosis-susceptible sites. Thus, the focal increase in NEC observed here in atherosclerosis-susceptible aortic sites during cholesterol feeding might reflect an increase in such NEC-rich vesicles in these sites at these early times of cholesterol feeding. Alternatively or additionally, the increase in NEC in atherosclerosis-susceptible aortic sites during cholesterol feeding could reflect the expansion of cellular NEC pools, including cellular membrane cholesterol. Indeed, cells with some characteristics of smooth muscle cells but enriched with NEC have been isolated from aortas of rabbits fed cholesterol for 4 weeks.37 Increases in cellular membrane NEC of a magnitude similar to the increase in NEC observed in abdominal branch sites after feeding cholesterol for 16 days alter cellular calcium homeostasis and cellular membrane fluidity.48 Decreased fluidity of endothelial cell membranes resulting from cholesterol enrichment promotes monocyte binding that could facilitate emigration of monocytes into the intima.49 Such changes in membrane fluidity have a number of other deleterious effects that might contribute to atherogenesis, including alteration of cellular permeability, receptor function, enzyme activity, lipoprotein degradation, cholesterol efflux, and cellular proliferation.50 51
We observed significant linear increases in cholesterol
concentrations for atherosclerosis-susceptible
aortic sites during cholesterol feeding (Fig 3). By squaring correlation coefficients for
relationships between duration of cholesterol feeding and
ACCs, it was possible to determine the portion of aortic
cholesterol accumulation that could be explained by
duration of cholesterol feeding. This analysis
suggested that duration of cholesterol feeding could
explain 30% more of the variation in TC concentrations in
atherosclerosis-susceptible aortic sites during
cholesterol feeding. The same method was used to consider
the combined influences of duration and extent of
hypercholesterolemia on aortic
cholesterol accumulation (Fig 5). This
analysis suggested that these combined influences could explain
an average of 39% of the variation in cholesterol
concentrations for atherosclerosis-susceptible
aortic sites during 16 days of cholesterol feeding. For
this latter analysis data from the normal rabbits were
included, as they had been for investigating the effect of duration of
cholesterol feeding alone on ACC. Comparing these results
indicated that the combined influences of extent and duration of
cholesterol feeding could explain an additional 30% of the
variation in ACCs than could duration of cholesterol
feeding alone. Interestingly, intercepts from the regression of ACCs on
the CEI, which were determined by using only data for rabbits fed
cholesterol, predicted ACCs of normal rabbits very closely
(Fig 5). Predicted TC concentrations for aortic arch
intima plus inner media and branch sites of thoracic and abdominal
aortas were 2.71, 2.65, and 2.83 µmol/g, respectively, compared with
mean measured values of 2.87, 2.59, and 2.87 µmol/g,
respectively.
Other investigators have shown that arterial cholesterol concentrations are related to the extent of hypercholesterolemia for a given duration of cholesterol feeding, either assessed as the CEI52 as described here or as mean plasma cholesterol concentration during cholesterol feeding.53 54 55 However, we are the first to demonstrate that this relation can be observed after very short periods of hypercholesterolemia, when it occurs selectively in atherosclerosis-susceptible aortic sites.
In summary, we found that cholesterol concentrations for atherosclerosis-susceptible and atherosclerosis-resistant aortic sites of normal rabbits did not differ. After feeding cholesterol for 8 days, small differences in cholesterol concentrations were found between atherosclerosis-susceptible and atherosclerosis-resistant aortic sites. However, increases in ACCs compared with normal rabbits were first evident after feeding cholesterol for 12 days, and only in susceptible sites. These results for cholesterol concentrations for atherosclerosis-susceptible and atherosclerosis-resistant aortic sites underscore the very selective development of atherosclerosis in characteristic aortic sites. Increases in ACCs could not be detected until about 4 days after alterations in aortic rates of LDL degradation and estimated aortic concentrations of undegraded LDL.25 26 The increases in cholesterol concentrations for atherosclerosis-susceptible aortic sites were related both to the extent and duration of hypercholesterolemia, whereas little or no relationship was observed for atherosclerosis-resistant aortic sites. Retention of plasma lipoproteins may account for much of the EC accumulation in atherosclerosis-susceptible aortic sites after feeding cholesterol for 16 days. However, further study will be needed to explain the accumulation of NEC at these sites.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grant HL45027. The author gratefully acknowledges the skillful technical assistance of Deanna Wiederhold and John Mason. The author is an Established Investigator of the American Heart Association.
Received April 4, 1995; accepted August 16, 1995.
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References |
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