Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1928-1937

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schwenke, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schwenke, D. C.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1928-1937.)
© 1995 American Heart Association, Inc.

Articles

Selective Increase in Cholesterol at Atherosclerosis-Susceptible Aortic Sites After Short-term Cholesterol Feeding

Dawn C. Schwenke

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.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 ({approx}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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {approx}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 1Down). 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.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 1. Illustration of the en face view of open regions of aorta showing the location, shape, and approximate size of samples of branch orifice areas obtained from the thoracic and abdominal aortas. Each small trapezoidal sample of the branch sites of the descending thoracic aorta (left) included the orifices of two intercostal arteries. Triangular samples of branch sites of the abdominal aorta (right) included, from top to bottom, the orifices of the celiac, superior mesenteric, left renal, and right renal arteries. The aortic arch was separated from the descending thoracic aorta 1 to 2 mm distal to the ductus scar.

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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 2Down). 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 TableDown, 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 (TableDown). In addition, the time dependence of the increase in plasma cholesterol concentrations observed in this study (Fig 2Down) 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.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. Line graph showing time dependence of plasma cholesterol concentration (Conc.) after feeding the cholesterol-containing diet. Symbols overlap because values before starting cholesterol feeding (0 days) were very similar. Terminal values are mean±SEM of 12, 11, 9, and 11 normal rabbits and those fed cholesterol for 8 ({bullet}···{bullet}), 12 ({blacktriangleup}—{blacktriangleup}), and 16 ({blacksquare}---{blacksquare}) days, respectively. Earlier values for cholesterol-fed rabbits are means from 3 to 11 rabbits. Terminal plasma cholesterol concentrations for rabbits fed cholesterol for 8, 12, and 16 days differed significantly from terminal values for normal rabbits (P<.0003, P<.0006, and P<.0003, respectively, by Wilcoxon's two-sample rank test with Bonferroni correction for multiple comparisons).


View this table:
[in this window]
[in a new window]
  		Table 1. Total Plasma Cholesterol Concentrations at Death for Rabbits Fed Cholesterol up to 16 Days

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 ({approx}2.75 µmol/g fresh wt; Fig 3Down). 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.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. Line graphs showing time dependence of TC concentrations for atherosclerosis-susceptible ({bullet}) and atherosclerosis-resistant ({circ}) 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 3Up). 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 4Down (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).



View larger version (34K):
[in this window]
[in a new window]
  		Figure 4. Line graphs showing time dependence of NEC and EC concentrations for atherosclerosis-susceptible ({bullet}) and atherosclerosis-resistant ({circ}) aortic sites during 16 days of cholesterol feeding. For rabbits never fed cholesterol (indicated here by 0 days), n=11 (except n=10 for thoracic branch sites); rabbits fed cholesterol for 8 days, n=10; 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=9 (except n=8 for arch outer media). *P<.05, **P<.02, ***P<.005 atherosclerosis-susceptible vs adjacent resistant sites by paired t test. +P<.05, ++P<.02, +++P<.006 corresponding aortic sites of normal rabbits vs those fed cholesterol by independent-samples t test with the Bonferroni correction for multiple comparisons.

EC Concentrations for Atherosclerosis-Susceptible and Adjacent Atherosclerosis-Resistant Aortic Sites
EC concentrations (Fig 4Up, 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 5Down, TC concentrations for atherosclerosis-susceptible aortic arch intima plus inner media (Fig 5ADown) and branch sites of descending thoracic (Fig 5CDown) and abdominal (Fig 5EDown) 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 5BDown) and nonbranch thoracic (Fig 5DDown) and abdominal (Fig 5FDown) 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.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 5. Plots showing relationships between TC concentrations for atherosclerosis-susceptible and atherosclerosis-resistant aortic sites and the CEI. For rabbits never fed cholesterol and those fed cholesterol for up to 4 days, n=17 (except n=16 for thoracic branch sites) ({diamondsuit}); rabbits fed cholesterol for 8 days, n=11 ({bullet}); rabbits fed cholesterol for 12 days, n=9 (except n=8 for nonbranch thoracic aorta and abdominal branch sites) ({blacktriangleup}); rabbits fed cholesterol 16 days, n=11 (except n=10 for arch outer media) ({blacksquare}). Regression lines were determined from data from cholesterol-fed rabbits only (n=36). r values for regressions including data for normal rabbits are .66, .57, and .64 for atherosclerosis-susceptible aortic arch intima plus inner media and thoracic and abdominal aorta branch sites, respectively, and .14, .13, and .08 for atherosclerosis-resistant aortic arch outer media and nonbranch thoracic and abdominal aorta, respectively.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 (TableUp).

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 (TableUp). 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 3Up). 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 3Up). 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 5Up). 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 5Up). 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.


*    Selected Abbreviations and Acronyms
 
ACC = aortic cholesterol concentration
CEI = cholesterol exposure index
EC = esterified cholesterol
NEC = nonesterified cholesterol
TC = total cholesterol


*    Acknowledgments
 
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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Velican C, Velican D. Differences in the pattern of atherosclerotic involvement between non-branched regions and adjacent branching points of human coronary arteries. Atherosclerosis. 1985;54:333-342. [Medline] [Order article via Infotrieve]
  2. Wissler RW, Robertson AL, Cornhill JF, Mcgill HC, Mcmahan CA, Strong JP. Natural history of aortic and coronary atherosclerotic lesions in youth: findings from the PDAY study. Arterioscler Thromb. 1993;13:1291-1298. [Abstract/Free Full Text]
  3. Cornhill JF, Barrett WA, Herderick EE, Mahley RW, Fry DL. Topographic study of sudanophilic lesions in cholesterol-fed minipigs by image analysis. Arteriosclerosis. 1985;5:415-426. [Abstract/Free Full Text]
  4. Roach MR, Fletcher J, Cornhill JF. The effect of the duration of cholesterol feeding on the development of sudanophilic lesions in the rabbit aorta. Atherosclerosis. 1976;25:1-11. [Medline] [Order article via Infotrieve]
  5. Roach MR, Fletcher J. Alterations in distribution of sudanophilic lesions in rabbits after cessation of a cholesterol-rich diet. Atherosclerosis. 1979;32:1-10. [Medline] [Order article via Infotrieve]
  6. Roach MR, Cornhill JF, Fletcher J. A quantitative study of the development of sudanophilic lesions in the aorta of rabbits fed a low-cholesterol diet for up to six months. Atherosclerosis. 1978;29:259-264. [Medline] [Order article via Infotrieve]
  7. Kratky RG, Ivey J, Rogers KA, Daley S, Roach MR. The distribution of fibro-fatty atherosclerotic lesions in the aortae of casein- and cholesterol-fed rabbits. Atherosclerosis. 1993;99:121-131. [Medline] [Order article via Infotrieve]
  8. Cornhill JF, Roach MR. A quantitative study of the localization of atherosclerotic lesions in the rabbit aorta. Atherosclerosis. 1976;23:489-501. [Medline] [Order article via Infotrieve]
  9. Daley SJ, Herderick EE, Cornhill JF, Rogers KA. Cholesterol-fed and casein-fed rabbit models of atherosclerosis, I: differing lesion area and volume despite equal plasma cholesterol levels. Arterioscler Thromb. 1994;14:95-104. [Abstract/Free Full Text]
  10. Zeindler CM, Kratky RG, Roach MR. Quantitative measurements of early atherosclerotic lesions on rabbit aortae from vascular casts. Atherosclerosis. 1989;76:245-255. [Medline] [Order article via Infotrieve]
  11. Smith EB, Slater RS. The microdissection of large atherosclerotic plaques to give morphologically and topographically defined fractions for analysis, Part I: the lipids in the isolated fractions. Atherosclerosis. 1972;15:37-56. [Medline] [Order article via Infotrieve]
  12. Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W Jr, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW. A definition of initial, fatty streak, and intermediate lesions of atherosclerosis: report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1994;89:2462-2478. [Abstract/Free Full Text]
  13. Mukhin DN, Orekhov AN, Andreeva ER, Schindeler EM, Smirnov VN. Lipids in cells of atherosclerotic and uninvolved human aorta, III: lipid distribution in intimal sublayers. Exp Mol Pathol. 1991;54:22-30. [Medline] [Order article via Infotrieve]
  14. Portman OW, Alexander M, Maruffo CA. Nutritional control of arterial lipid composition in squirrel monkeys: major ester classes and types of phospholipids. J Nutr. 1967;91:35-46.
  15. Rosenfeld ME, Chait A, Bierman EL, King W, Goodwin P, Walden CE, Ross R. Lipid composition of aorta of Watanabe heritable hyperlipemic and comparably hypercholesterolemic fat-fed rabbits: plasma lipid composition determines aortic lipid composition of hypercholesterolemic rabbits. Arteriosclerosis. 1988;8:338-347. [Abstract/Free Full Text]
  16. Stein Y, Stein O, Delplanque B, Fesmire JD, Lee DM, Alaupovic P. Lack of effect of probucol on atheroma formation in cholesterol-fed rabbits kept at comparable plasma cholesterol levels. Atherosclerosis. 1989;75:145-155. [Medline] [Order article via Infotrieve]
  17. Fischer GM, Swain ML, Cherian K. Increased vascular collagen and elastin synthesis in experimental atherosclerosis in the rabbit: variation in synthesis among major vessels. Atherosclerosis. 1980;35:11-20. [Medline] [Order article via Infotrieve]
  18. Hough JL, Zilversmit DB. Effect of 17 beta estradiol on aortic cholesterol content and metabolism in cholesterol-fed rabbits. Arteriosclerosis. 1986;6:57-63. [Abstract/Free Full Text]
  19. Schwenke DC, St. Clair RW. Influx, efflux, and accumulation of LDL in normal arterial areas and atherosclerotic lesions of White Carneau pigeons with naturally occurring and cholesterol-aggravated aortic atherosclerosis. Arterioscler Thromb. 1993;13:1368-1381. [Abstract/Free Full Text]
  20. Schwenke DC, St. Clair RW. Accumulation of 125I-tyramine cellobiose–labeled low density lipoprotein is greater in the atherosclerosis-susceptible region of White Carneau pigeon aorta and further enhanced once atherosclerotic lesions develop. Arterioscler Thromb. 1992;12:446-460. [Abstract/Free Full Text]
  21. Bell FP. Modification in the transmural concentration of cholesterol and the transmural distribution of 131I-albumin in the aortas of pigs fed cholesterol: a preliminary report. Artery. 1983;12:24-32. [Medline] [Order article via Infotrieve]
  22. Nielsen LB, Nordestgaard BG, Stender S, Kjeldsen K. Aortic permeability to LDL as a predictor of aortic cholesterol accumulation in cholesterol-fed rabbits. Arterioscler Thromb. 1992;12:1402-1409. [Abstract/Free Full Text]
  23. Swindell AC, Krupp MN, Twomey TM, Reynolds JA, Chichester CO. Effects of doxazosin on atherosclerosis in cholesterol-fed rabbits. Atherosclerosis. 1993;99:195-206. [Medline] [Order article via Infotrieve]
  24. Schwenke DC, Carew TE. Quantification in vivo of increased LDL content and rate of LDL degradation in normal rabbit aorta occurring at sites susceptible to early atherosclerotic lesions. Circ Res. 1988;62:699-710. [Abstract/Free Full Text]
  25. Schwenke DC, Carew TE. Initiation of atherosclerotic lesions in cholesterol-fed rabbits. I: focal increases in arterial LDL concentration precede development of fatty streak lesions. Arteriosclerosis. 1989;9:895-907. [Abstract/Free Full Text]
  26. Schwenke DC, Carew TE. Initiation of atherosclerotic lesions in cholesterol-fed rabbits, II: selective retention of LDL vs. selective increases in LDL permeability in susceptible sites of arteries. Arteriosclerosis. 1989;9:908-918. [Abstract/Free Full Text]
  27. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]
  28. Lipid Research Clinics Program. Manual of Laboratory Operations: Lipid and Lipoprotein Analysis. Washington, DC: US Government Printing Office; 1982. US Department of Health, Education, and Welfare publication NIH 75-629.
  29. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226:497-509. [Free Full Text]
  30. Rudel LL, Morris MD. Determination of cholesterol using o-phthalaldehyde. J Lipid Res. 1973;14:364-366. [Abstract]
  31. Abell LL, Levy BB, Brodie BB, Kendall FE. A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J Biol Chem. 1952;195:357-366. [Free Full Text]
  32. Snedecor GW, Cochran WG. Statistical Methods. 6th ed. Ames, Iowa: Iowa State University Press; 1979:3-593.
  33. Winer BJ. Statistical Principles in Experimental Design. 2nd ed. New York, NY: McGraw-Hill; 1971.
  34. Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res. 1980;47:1-9. [Abstract/Free Full Text]
  35. Day AJ, Proudlock JW. Changes in aortic cholesterol-esterifying activity in rabbits fed cholesterol for 3 days. Atherosclerosis. 1974;19:253-258. [Medline] [Order article via Infotrieve]
  36. Chao F-F, Blanchette-Mackie EJ, Dickens BF, Gamble W, Kruth HS. Development of unesterified cholesterol-rich lipid particles in atherosclerotic lesions of WHHL and cholesterol-fed NZW rabbits. J Lipid Res. 1994;35:71-83. [Abstract]
  37. Haley NJ, Shio H, Fowler S. Characterization of lipid-laden aortic cells from cholesterol-fed rabbits, I: resolution of aortic cell populations by metrizamide density gradient centrifugation. Lab Invest. 1977;37:287-296. [Medline] [Order article via Infotrieve]
  38. Rosenfeld ME, Khoo JC, Miller E, Parthasarathy S, Palinski W, Witztum JL. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts. J Clin Invest. 1991;87:90-99.
  39. Kelley JL, Suenram CA, Rozek MM, Schaffer SA, Schwartz CJ. Influence of the acyl-CoA:cholesterol O-acyltransferase inhibitor, CL277082, on cholesteryl ester accumulation in rabbit macrophage-rich granulomas and hepatic tissue. Biochim Biophys Acta. 1988;960:83-90. [Medline] [Order article via Infotrieve]
  40. Chao F-F, Amende LM, Blanchette-Mackie EJ, Skarlatos SI, Gamble W, Resau JH, Mergner WT, Kruth HS. Unesterified cholesterol-rich lipid particles in atherosclerotic lesions of human and rabbit aortas. Am J Pathol. 1988;131:73-83. [Abstract]
  41. Kruth HS. Localization of unesterified cholesterol in human atherosclerotic lesions: demonstration of filipin-positive, oil-red-O-negative particles. Am J Pathol. 1984;114:201-208. [Abstract]
  42. Mora R, Simionescu M, Simionescu N. Purification and partial characterization of extracellular liposomes isolated from the hyperlipidemic rabbit aorta. J Lipid Res. 1990;31:1793-1807. [Abstract]
  43. Mora R, Lupu F, Simionescu N. Cytochemical localization of B-lipoproteins and their components in successive stages of hyperlipidemic atherogenesis of rabbit aorta. Atherosclerosis. 1989;79:183-195. [Medline] [Order article via Infotrieve]
  44. Kruth HS. Filipin-positive, oil red O-negative particles in atherosclerotic lesions induced by cholesterol feeding. Lab Invest. 1983;50:87-93. [Medline] [Order article via Infotrieve]
  45. Simionescu N, Vasile E, Lupu F, Popescu G. Prelesional events in atherogenesis: accumulation of extracellular cholesterol-rich liposomes in the arterial intima and cardiac valves of the hyperlipidemic rabbit. Am J Pathol. 1986;123:109-125. [Abstract]
  46. Guyton JR, Klemp KF. Early extracellular and cellular lipid deposits in aorta of cholesterol-fed rabbits. Am J Pathol. 1992;141:925-936. [Abstract]
  47. Frank JS, Fogelman AM. Ultrastructure of the intima in WHHL and cholesterol-fed rabbit aortas prepared by ultra-rapid freezing and freeze-etching. J Lipid Res. 1989;30:967-978. [Abstract]
  48. Gleason MM, Medow MS, Tulenko TN. Excess membrane cholesterol alters calcium movements, cytosolic calcium levels, and membrane fluidity in arterial smooth muscle cells. Circ Res. 1991;69:216-227. [Abstract/Free Full Text]
  49. Pritchard KA Jr, Schwarz SM, Medow MS, Stemerman MB. Effect of low-density lipoprotein on endothelial cell membrane fluidity and mononuclear cell attachment. Am J Physiol. 1991;260:C43-C49. [Abstract/Free Full Text]
  50. Cooper RA. Abnormalities of cell-membrane fluidity in the pathogenesis of disease. N Engl J Med. 1977;297:371-377. [Medline] [Order article via Infotrieve]
  51. Jackson RL, Gotto AM Jr. Hypothesis concerning membrane structure, cholesterol, and atherosclerosis. Atheroscler Rev. 1976;1:1-21.
  52. Walton KW, Dunkerley DJ, Johnson AG, Khan MK, Morris C, Watts RB. Investigation by immunofluorescence of arterial lesions in rabbits on two different lipid supplements and treated with pyridinol carbamate. Atherosclerosis. 1976;23:117-139. [Medline] [Order article via Infotrieve]
  53. Ho KJ, Pang LC, Taylor CB. Mode of cholesterol accumulation in various tissues of rabbits with prolonged exposure to various serum cholesterol levels. Atherosclerosis. 1974;19:561-566. [Medline] [Order article via Infotrieve]
  54. Ross AC, Minick CR, Zilversmit DB. Equal atherosclerosis in rabbits fed cholesterol-free, low-fat diet or cholesterol-supplemented diet. Atherosclerosis. 1978;29:301-315. [Medline] [Order article via Infotrieve]
  55. Brattsand R. The effect of niceritrol (pentaerythritoltetranicotinate) and clofibrate upon hyperlipemia and atherosclerosis induced in rabbits by cholesterol-free semisynthetic diets. Atherosclerosis. 1974;20:453-467.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 J. Exp. Med.Home page
J. Jongstra-Bilen, M. Haidari, S.-N. Zhu, M. Chen, D. Guha, and M. I. Cybulsky
Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis
J. Exp. Med., September 4, 2006; 203(9): 2073 - 2083.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
J. W. Ruberti, C. A. Curcio, C. L. Millican, B. P. M. Menco, J.-D. Huang, and M. Johnson
Quick-Freeze/Deep-Etch Visualization of Age-Related Lipid Accumulation in Bruch's Membrane
Invest. Ophthalmol. Vis. Sci., April 1, 2003; 44(4): 1753 - 1759.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
J. H. von der Thusen, T. J.C. van Berkel, and E. A.L. Biessen
Induction of Rapid Atherogenesis by Perivascular Carotid Collar Placement in Apolipoprotein E-Deficient and Low-Density Lipoprotein Receptor-Deficient Mice
Circulation, February 27, 2001; 103(8): 1164 - 1170.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. C. Schwenke
Metabolic evidence for sequestration of low-density lipoprotein in abdominal aorta of normal rabbits
Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1128 - H1140.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L. Hajra, A. I. Evans, M. Chen, S. J. Hyduk, T. Collins, and M. I. Cybulsky
The NF-kappa B signal transduction pathway in aortic endothelial cells is primed for activation in regions predisposed to atherosclerotic lesion formation
PNAS, August 1, 2000; 97(16): 9052 - 9057.