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Effect of removal of atherogenic diet on protein synthesis and cholesterol retention in rabbit aorta and lung
Atherosclerosis, 54 (1985) l-10 Elsevier Scientific Publishers Ireland.
Ltd.
ATH 03545
Effect of Removal of Atherogenic Diet on Protein Synthesis and Cholesterol Retention in Rabbit Aorta and Lung James P. Gilligan
* and Ronald 0. Langner
School of Pharmacy, University of Connecticut, Storrs, CT 06268 (U.S.A.) (Received 4 April, 1984) (Revised, received 22 June, 1984) (Accepted 25 June, 1984)
Summary
The purpose of these studies was to determine the effect of cholesterol feeding in rabbits on the synthesis of collagen and non-collagen proteins in both aortic and lung tissues. Rabbits were fed a 2% cholesterol diet for 30 or 60 days, followed by 30 days of a low cholesterol diet (i.e. 30-30 or 60-30). After 30 days of cholesterol feeding non-collagen protein synthetic rates were significantly elevated in aortic tissues, but not in the lung. After 60 days of cholesterol feeding, both collagen and non-collagen synthetic rates were elevated in the aorta but not in the lung. Both tissues demonstrated significant increases in cholesterol content. When cholesterol was removed from the diet, cholesterol continued to accumulate in the aorta but decreased in the lung. The 60-30 group demonstrated both the largest increase in aortic cholesterol, and the largest increase in the per cent of collagen being synthesized in the aorta. These data therefore demonstrate that cholesterol feeding stimulates both collagen and non-collagen protein synthesis and suggests that there may be some differences in the lag phase before significant changes are apparent in both parameters. Following removal of cholesterol from the diet the per cent collagen synthesized in the aorta increased further, due to an apparent reduction in non-collagen protein synthesis rather than a further acceleration of collagen synthesis. These changes may be important in explaining how intermittent-cholesterol feeding produces a more fibrous aortic lesion. Key words: Aorta - Atherosclerosis - Cholesterol - Collagen - Lung - Rabbits
* Present address: Unigene Laboratories, 110 Little Falls Road, These studies were supported in part by NIH grant HL-26122. 0021-9150/85/$03.30
0 1985 Elsevier Scientific
Publishers
Ireland,
Fairfield,
Ltd.
NJ 07006, U.S.A.
2
Introduction Early human atherosclerotic lesions which evolve from serum hyperlipidemia contain increased amounts of free and esterified cholesterol. As the lesions become more advanced, they accumulate increased amounts of collagen which is thought to be responsible for the irreversibility of complex atherosclerotic plaques [1,2]. The factors responsible for stimulating collagen synthesis are unknown. Studies by several authors [3-61 have shown that continuous cholesterol feeding in excess of 90 days will result in an increased aortic collagen synthetic rate. This increase in collagen synthesis was later demonstrated to be located at the primary site of cholesterol accumulation [7]. In a recent study Langner and Modrak [8] reported that hypercholesterolemia caused an increase in collagen synthesis in rabbit aorta and liver, but no change in lung. These data suggested that the connective tissue response to hyperlipemic insult is present in some tissues, but not all tissues. The increase in collagen synthesis is also accompanied by increased rates of non-collagen protein synthesis. When rabbits were fed cholesterol continuously for 120 days there was a proportional increase in both collagen and non-collagen synthesis [5]. Studies by Ehrhart and Holderbaum [6], using a high-cholesterol diet supplemented with peanut-oil, demonstrated that the increase in collagen synthesis was greater than the change in non-collagen protein synthesis, and concluded that this accounted for the more fibrous lesion normally seen with the peanut-oil supplemented diet. When rabbits are fed an intermittent high-cholesterol diet they also develop a more fibrous type lesion compared to rabbits continuously fed a high-cholesterol diet [9]. The mechanisms for this increased fibrous tissue deposition is unknown. It is possible however, that removal of cholesterol from the diet stimulates a greater increase in collagen synthesis relative to non-collagen protein synthesis similar to that observed with the peanut-oil diet. Another possible mechanism could be that the increases in both the synthesis of collagen and non-collagen protein occur independently of each other at different times. For example, if increases in collagen synthesis were to occur significantly before changes in non-collagen protein synthesis then one could expect a more fibrous type lesion. Therefore, one purpose of this study was to determine both the time of onset and the relative degree of change for both collagen and non-collagen synthetic rates following removal of cholesterol from the diet. A second objective of this study was to gain further information concerning the role of increased protein synthesis on cholesterol accumulation and retention. Previous studies [lo] have demonstrated a close correlation between increased collagen synthesis and increased rates of tissue cholesterol deposition in the aorta. The lung has been shown to have an increased cholesterol content in response to cholesterol feeding with no change in protein synthesis [8]. Therefore, this study was designed to compare cholesterol accumulation and efflux in tissues demonstrating increased protein synthetic activity (i.e. aorta) to a tissue with basal levels of protein synthesis (i.e. lung). The hypothesis to be tested is that in tissues demonstrating an
3
elevated retention
rate of collagen of accumulated
and non-collagen cholesterol.
protein
synthesis,
there is an increased
Methods White male New Zealand rabbits weighing between 2.0-2.5 kg were used in these studies. Rabbits were randomly divided into 5 groups: Group 1 (34 rabbits) were fed Purina Rabbit Chow and served as control; Group 2 (15 rabbits) were fed a 2% cholesterol-supplemented diet for 30 days (30); Group 3 (7 rabbits) were fed 2% cholesterol diet for 30 days followed by the control diet for 30 days (30-30); Group 4 (15 rabbits) were fed the 2% cholesterol-supplemented diet for 60 days (60); Group 5 (10 rabbits) were fed the 2% cholesterol diet for 60 days followed by the cholesterol-free diet for 30 days (60-30). Control and experimental animals were killed on the same day by cervical dislocation. The aorta and lungs were rapidly removed, weighed, and sections taken for histology. The aorta was cleaned of loose adhering tissue, opened longitudinally and macroscopic lesions graded, using the O-4 grading system described by Lorenzen [ll]. Grade 0 indicated the complete absence of lesions and grade 4 indicated severe lesion development with lesions extending into the abdominal aorta. Both tissues were then weighed and placed in oxygen-rich Krebs bicarbonate buffer, pH 7.4, for a 20 min preincubation period at 37 o C under a 95% 0,/5% CO, atmosphere. Following preincubation the tissues were transferred to incubation vessles, containing fresh oxygenated Krebs bicarbonate buffer and 0.2 mM L-proline with 3 PCi of [U-‘4C]proline in a final volume of 10 ml. Following the incubation period (90 min aorta; 60 min lung) the tissues were immediately rinsed with ice-cold trichloroacetic acid (TCA) to prevent further incorporation of [r4C]proline. Tissues were then homogenized using a glass co-axial homogenizer (Kontes) and appropriated dilutions were made for biochemical analysis. Collagen and non-collagen proteins were separated using the hot trichloroacetic acid (TCA) procedure described by Newman and Langner [12]. The tissues were repeatedly washed with ice-cold 5% TCA to remove non-incorporated [‘4C]proline. The washed pellets were then extracted for 1 h at 90 ‘C, and the supematant containing hydrolyzed collagen fragments was collected. The 90 ‘C TCA extraction was then repeated. The combined supematants were evaporated to dryness, resolubilized in distilled water, and counted in a liquid scintillation counter. These samples were used as an estimate of collagen synthetic activity. The pellet remaining after the removal of collagen, which contained non-collagen protein, was digested at 45 o C overnight, and was then counted in a liquid scintillation counter. Hydroxyproline content of aorta and lung homogenates was estimated by the method of Kivirikko et al. [13] and served as an index of collagen content. Non-collagen protein concentrations were determined on the tissue homogenates by the method of Lowry et al. [14] using bovine serum albumin as the standard. The cholesterol content of aortic and lung tissues was determined after delipidation utilizing a modified procedure of Folch et al. [15]. Following extraction free and esterified cholesterol were separated on thin layer plates of silica gel 60HR extra
4
pure (E.M.), 0.5 mm thick. The plates were developed in n-heptane/diethyl ether/acetic acid (75 : 25 : 2) and visualized in iodine. The iodine was allowed to dissipate and the free and esterified cholesterol spots scraped into conical tubes. Cholesterol levels were determined using the method described by Franey and Amador [16]. Serum cholesterol levels were determined weekly. Blood was collected from the medial ear artery and allowed to clot. Serum was collected and the cholesterol levels were determined on the ethanolic extract by the method of Franey and Amador [16]. Histological sections were taken from aorta at a level just below the subclavian artery and the midthoracic region. The sections were fixed in buffered formalin and stained with hematoxylin-eosin, Verhoeff or Van Gieson stains. The statistical significance of any observed differences was determined by use of analysis of variance or Student t-tests [17]. Results
Control animals were killed at each of the experimental time points. Since there was no apparent difference between control groups, the data was combined into a single control group in Tables l-5. Rabbits fed the 2% cholesterol-supplemented diet exhibited a characteristic rise in serum cholesterol values. By day 30 all experimental animals had serum cholesterol levels in excess of 600 mg/lOO ml. Continuation of the cholesterol diet for an additional 30 days resulted in serum cholesterol levels greater than 1300 mg/lOO ml (Table 1). When the animals were placed on the low-cholesterol control diet there was a rapid fall in serum cholesterol values, but they were still elevated above control values after 30 days. TABLE
1
EFFECTS OF 2% CHOLESTEROL FORMATION All data expressed
as mean
Control 30 B 30-30 60 60-30
ON TISSUE WET WEIGHTS
AND VISIBLE
LESION
f SE.
Serum cholesterol
(w/l~
FEEDING
n-4
60.0f 4.0 615.0k47.6 * 105.2 f 27.0 * 1352 k69.1 * 361.7+46 *
Lung
Thoracic
Wet Weight/ Body Weight
Wet Weight/ Body Weight
(g/kg)
(g/kg)
3.56k0.11 3.55 f 0.01 3.32 f 0.21 4.05 f 0.23 * 3.78 kO.22
0.226 + 0.008 0.28OkO.013 * 0.320*0.013 * 0.352 f 0.017 * 0.420 f 0.02 *
* Significantly different from controls (P -c 0.05). * 30 and 60 = Either 30 or 60 days of 2% cholesterol cholesterol diet followed by 30 days normal diet.
diet;
aorta
30-30
Macroscopic
lesions
O-l-2-3-4
or 60-60
27-7-O-O-O 3-7-3-2-O o-o-2-4-1 O-O-2-5-8 O-O-2-3-5
= 30 or 60 days of 2%
5
Rabbits fed cholesterol for 30 days developed aortic lesions (Table 1) which were characterized by several layers of intimal foam cells. The media in these early lesions appeared to be normal, with the only difference between grade 2 and 4 lesions being the increased surface area involved and the thickness of the lesions. The rabbits maintained on the 60-day continuous cholesterol diet and those on the 60-30 intermittent regimen had the most severe disease as evidenced by the large number of grade 3 and 4 lesions. Several histological sections taken from the 60-30 group of rabbits demonstrated some focal cellular necrosis, with increased evidence of lesion fibrosis. As also seen in Table 1 rabbits fed the cholesterol diet showed a significant increase in thoracic wet weight ratios. These increases persisted even when the animals were placed on a normal cholesterol diet. The wet weight/body weight ratio of the lungs was only elevated in the 60-day cholesterol group, suggesting increased cholesterol accumulation. The total and esterified cholesterol content of the thoracic aortas were significantly increased in all cholesterol-fed rabbits (Table 2). In the 30-day group, both total cholesterol and cholesterol-esters were significantly greater than control, however the levels were significantly lower than those observed in the 30-30 group of animals. In the rabbits fed cholesterol continuously for 60 days, both measures of aortic cholesterol content were significantly increased when compared to either the 30-30 or control rabbits. The 60-30 group had the highest levels of both cholesterol and esterified cholesterol, even though their serum cholesterol levels were much reduced. The cholesterol and esterified cholesterol content of the lungs was significantly elevated in both groups of continuously-fed cholesterol animals (Table 2). The lungs differed from the aortas in that removal of cholesterol from the diet resulted in the
TABLE
2
EFFECT OF 2% CHOLESTEROL CHOLESTEROL CONTENT All data reported
DIET
FOR
* ** *** a
PERIODS
OF
TIME
ON
TISSUE
as mean + SE.
Lung
Control 30 a 30-30 60 60-30
VARYING
Aorta
Total pg cholesterol/ mg protein
pg cholesterol-ester,’ mg protein
Total pg cholesterol/ mg protein
pg cholesterol-ester/ mg protein
25.8 + 1.6 43.2 + 3.7 ** 23.9*1.6 63.3k4.3 *** 29.6 f 2.9
2.9 + 0.4 18.9k3.4 ** 6.5k1.4 * 33.4k3.1 *** 5.3*1.0*
7.1* 0.5 11.7+ 0.8 * 19.6+ 3.3 * 75.9* 15.5 ** 95.7+ 10.8 **
0.8kO.l 4.0* 1.1 * 10.5 f 2.9 51.8+4.4 ** 60.1 f 6.8 **
Significantly different from controls (P < 0.05). Significantly (P -Z 0.05) different from 30-30 animals. Significantly (P c 0.05) different from 60-30 animals. 30 and 60 = Either 30 or 60 days of 2% cholesterol diet; 30-30 cholesterol diet followed by 30 days normal diet.
or 60-30
= 30 or 60 days of 2%
6
return of total cholesterol values in the lung toward control levels. The cholesterol-ester content in the lungs of both the 30-30 and the 60-30 group also decreased when cholesterol was removed from the diet but remained slightly, but significantly elevated when compared to control. Since the color developed in the protein assay described by Lowry et al. [14] is dependent upon the presence of amino acids essentially lacking in collagen, this assay was used to estimate non-collagen protein content. The amount of non-collagen protein present in these tissues did not significantly change in any of the experimental groups (Table 3). The total amount of collagen in either the lung or aorta was estimated by measuring the amount of hydroxyproline present. The aortas from the 30, 30-30 or 60 groups did not contain any change in total collagen content. The aortas from the 60-30 group did however demonstrate a significant increase in collagen content. The lungs from the 60-30 animals did not demonstrate any change in collagen content from control levels. The rate of collagen and non-collagen protein synthesis in aorta and lung is presented in Tables 4 and 5. Following 30 days of cholesterol feeding there was a significant increase in thoracic aorta non-collagen protein synthesis, but there was no significant change in collagen synthetic activity. The relative per cent of collagen and non-collagen proteins being synthesized in the 30-day rabbit aortas was not different from control. Thoracic aortas taken from rabbits in the 30-30 intermittent cholesterol-fed group demonstrated a significant 2-fold increase in collagen synthesis (Table 4). The rate of non-collagen protein synthesis was also elevated, with the per cent of collagen being synthesized no different from controls. The rabbits receiving the 60- and 60-30-day treatment demonstrated further increases in collagen synthesis with an approximate 4-fold increase in collagen synthetic activity. Non-collagen protein synthesis in both the 60- and 60-30 groups was significantly elevated above
TABLE
3
EFFECT OF 2% CHOLESTEROL LAGEN PROTEIN CONTENT All data reported
DIET ON LUNG
AND
AORTA
COLLAGEN
AND
NON-COL-
as mean f SE. Aorta
Lung mg protein/ g tissue
pg hypro “/ mg protein
mg protein/ g tissue
ng hypro/ mg protein
Control 30 a
109.5 f 3.5 112.8k 3.4
30-30 60 60-30
99.0+4.3 101.7 f 3.0 115.6 f 3.5
13.4kO.6 11.9hO.6 12.0k0.6 12.4kO.8 13.2* 1.5
103.7 * 5.7 106.2 f 4.4 116.9+7.1 106.0 k 4.1 114.9 + 3.2
52.6 f 4.0 53.5 _I 3.0 58.6k6.0 54.5 f 3.0 50.2 f 4.2
’ 30 and 60 = Either 30 or 60 days of 2% cholesterol cholesterol diet followed by 30 days of control diet. b pg hydroxyproline.
diet;
30-30
or 60-30
= 30 or 60 days of 2%
TABLE
4
EFFECT OF 2% CHOLESTEROL FEEDING ON “C-PROLINE COLLAGEN AND NON-COLLAGEN PROTEIN All data reported
Control 30 a 30-30 60 60-30
INCORPORATION
INTO AORTIC
as mean + SE. Collagen DPM/ mg protein
Non-collagen mg protein
81.1 f 13.7 141.0+ 19.0 195.0*30.3 * 361.2+39.0 * 415.3 + 57.2 *
135.5 270.5 379.2 508.6 395.7
f + + + k
* Significantly different from controls (P < 0.05). * 30 and 60 = Either 30 or 60 days of 2% cholesterol cholesterol diet followed by 30 days of control diet. b % collagen synthesized = collagen DPM/(Non-collagen
13.4 31.8 108.0 71.6 58.4
DPM/
% Collagen
* * * *
11.6kO.4 10.9kO.9 11.2*1.2 15.8 +0.9 * 19.8k1.3 *
diet:
30-30
or 60-30
b
= 30 or 60 days of 2%
DPM X 5.1) + (collagen
DPM x 100)
control levels (Table 4). The per cent of collagen synthesized in the thoracic aortas from 60 and 60-30 animals was significantly increased above controls, suggesting a more fibrous type of tissue response in these animals. The per cent of collagen synthesized was calculated by dividing collagen DPM by non-collagen DPM, multipled by a factor of 5.1, plus collagen DPM X 100. The correction factor of 5.1 was used to correct for the fact that collagen contains approximately 5.1 times as much proline as does non-collagen protein. Lung collagen and non-collagen protein synthesis remained unaltered in all experimental groups (Table 5). Discussion Previous studies from our laboratory have shown that in rabbits fed a 2% cholesterol diet for 70 days, collagen synthesis was increased only in the inner layers
TABLE
5
EFFECT OF 2% CHOLESTEROL FEEDING ON [14C]PROLINE COLLAGEN AND NON-COLLAGEN PROTEIN
Control 30 b 30-30 60 60-30
Collagen DPM/ mg protein
Non-collagen mg protein
474.5 515.5 318.0 440.0 438.7
1360.0 f 47.2 1458.0 f 96.3 96.3 1278.0* 56.8 1307.0* 1271.2* 141.6
f 39.7 *66.1 f 34.5 f 28.2 f 64.5
DPM/
INCORPORATION
% Collagen
INTO LUNG
a
6.OkO.4 6.5 f 0.7 4.5 +_0.4 5.9*0.3 6.2kO.4
a % collagen synthesized = collagen DPM/(non-collagen DPM x 5.1) + (collagen DPM x 100). b 30 and 60 = Either 30 or 60 days of 2% cholesterol diet; 30-30 or 60-30 = 30 or 60 days of 2% cholesterol diet followed by 30 days of control diet. All data reported as mean f SE.
8
of the arterial wall, the primary site of cholesterol accumulation [7]. These results suggested that cholesterol deposition in the arterial wall was capable of initiating localized fibrotic response which may have an effect on further cholesterol accumulation. Ehrhart and Holderbaum reported that when rabbits are fed cholesterol for 140-180 days, both collagen and non-collagen synthetic rates are elevated [5]. Previous work from our laboratory [3] had reported that collagen synthesis was elevated after only 60 days of 2% cholesterol feeding, however in these studies we did not measure non-collagen protein synthetic rates. In the present studies we investigated changes in both collagen and non-collagen protein synthetic rates after varying periods of cholesterol feeding. The purpose of these studies was to determine whether both measures of protein synthesis increase at the same time. As seen in Table 1 following 30 days of cholesterol feeding there was a significant increase in non-collagen protein synthesis. Collagen synthetic rates were also elevated at this time, but were not statistically significant. By 60 days both collagen and non-collagen synthetic rates were significantly elevated, and this occurred whether the rabbits were fed cholesterol for 60 days or for 30 days followed by 30 days of normal diet. These data demonstrate that cholesterol feeding induces an increase in both collagen and non-collagen protein synthesis, but that the rate of response for non-collagen protein synthesis may be more rapid than that observed for collagen synthesis. Many investigators have shown that continuous feeding of a high-cholesterol diet to rabbits produces a lipid-laden foam cell lesion. In the studies reported by Ehrhart and Holderbaum [5] they found an equal increase in both collagen and non-collagen protein synthetic rates. In the present study the ratio of collagen to non-collagen protein synthesis was measured in 4 separate groups and is reported in Table 4. In the rabbits fed for only a short time (i.e. 30 and 30-30 groups) there appears to be an equal increase in both collagen and non-collagen protein synthesis. However in the 60 and 60-30 groups there is a significant change in the per cent collagen being synthesized. The 60-30 rabbits had the largest change primarily because of an apparent reduction in non-collagen protein synthesis when compared to the 60-day animals. The data in Table 4 differs substantially from that reported by Ehrhart and Holderbaum [5], who found no change in the per cent of proteins being synthesized as collagen. In their experiments they studied rabbits which had been on a highcholesterol diet for a much longer time and they reported collagen synthetic rates as a function of extractable proteins. In our studies rabbits were fed cholesterol for much shorter periods and we reported our data as a function of total proteins. These differences in procedure may account in part for the apparent differences. In addition, the per cent increase in collagen synthesis may, in fact, be linked to the severity of the lesions and not directly correlated to the chronologic exposure to cholesterol. Several investigators have reported that removal of cholesterol promotes a more fibrous type of aortic lesion in rabbits. It has been speculated that the removal of cholesterol from the diet produces a stimulation of collagen synthesis. Previous studies from our laboratory reported that removal of cholesterol did not, by itself, result in a stimulation of collagen synthesis [la]. Since collagen synthesis in the 60
9
and 60-30 groups was essentially the same (Table 4) it again suggests that removal of cholesterol from the diet does not stimulate collagen synthesis. However, based upon the changes in non-collagen protein synthetic rates, it is possible that the reason cholesterol removal produces a more fibrous lesion is that non-collagen protein synthesis tends to become reduced when cholesterol is withdrawn resulting in a relative increase in the per cent of collagen being synthesized. Additional research will be required to investigate this hypothesis. Another objective of these studies was to study further the relationship between elevated rates of protein synthesis and tissue cholesterol accumulation and retention. Studies by Modrak and Langner [lo] demonstrated a close correlation between increased rates of aortic collagen synthesis and aortic rates of cholesterol accumulation. In a separate series of experiments [8] we also demonstrated that in cholesterolfed rabbits the lungs will accumulate increased levels of cholesterol and cholesterol-esters, but will not respond with increased rates of collagen synthesis. In the present studies when rabbits were placed on low-cholesterol diets after 30 or 60 days of high-cholesterol feeding, there was a rapid fall in serum cholesterol levels (Table 1). The cholesterol content of the thoracic aorta in both regression groups continued to increase even though serum levels were falling. The rate of increase in aortic cholesterol was greater in the animals fed cholesterol for 60 days followed by 30 days of normal diet (i.e. 60-30 group) than in the 30-30 group. As seen in Table 2, over the same 30-day low-cholesterol period, the rabbits fed cholesterol for 60 days accumulated more than 3 times as much cholesterol as did the rabbits fed for just 30 days. The reason for this difference is not known Since the final serum levels in the 60-30 group was higher than that observed in the 30-30 group, this may account for some of the difference. Another possible reason may be that in the aortas of the 60-30 animals, there is an increased rate of protein synthesis with a higher percentage of collagen being synthesized (Table 4). This difference in protein synthesis may alter aortic permeability and allow more cholesterol to enter the tissue or it may alter cholesterol efflux, and prevent cholesterol from leaving the tissue. Either or both mechanisms would result in an increase in tissue cholesterol deposition. The lungs taken from these animals presented a much different pattern of response from what was observed in the aorta. After 30 or 60 days of cholesterol feeding the lungs had significantly elevated levels of tissue cholesterol (Table 2). When the animals were placed on the regression diet, instead of continuing to accumulate cholesterol as seen in the aorta, the lungs rapidly released their accumulated cholesterol and returned to control levels after 30 days. As seen in Table 5, the lungs exhibited no alteration in either collagen or non-collagen protein synthetic rates. Since the aorta, which has increased rates of total protein synthesis, continues to accumulate cholesterol in the presence of the same falling cholesterol levels, the data suggest that the continued accumulation of cholesterol occurs because of some alteration of tissue metabolism. This is contrary to the earlier suggestions of Friedman and Byers [19], who suggested that during the first 3 months of falling serum cholesterol levels, additional plaque growth occurs because of the continuing hypercholesterolemia and not because of some peculiar metabolism of the plaque itself.
10
In summary, the feeding of cholesterol will induce an increase in both collagen and non-collagen protein synthesis. When cholesterol feeding is continued there is a relative increase in the percentage of aortic collagen being synthesized. The significance of this change in collagen synthesis is not known. Our data, however, do show that in a tissue which has an elevated protein synthetic rate, reduction of serum cholesterol does not promote a regression of tissue cholesterol content. These observations are not inconsistent with the hypothesis that changing rates of aortic protein synthesis play an important role in promoting the development of aortic lesions. The data further suggest that drugs which could return this altered metabolic activity toward control levels may be helpful in promoting regression of established atherosclerosis. References 1 Constantinides, P., Overview of studies on regression of atherosclerosis, Artery, 9 (1981) 30. 2 Armstrong, M.L., Regression of atherosclerosis, Atheroscl. Rev., 1 (1976) 137. 3 Langner, R.O. and Modrak, J.B., Collagen metabolism during the early stages of cholesterol-induced atherogenesis in rabbits, Blood Vessels, 13 (1976) 257. 4 Fischer, G.M., Cherian, K. and Swain, M.L., Increased synthesis of aortic collagen and elastin in experimental atherosclerosis, Atherosclerosis, 39 (1981) 463. 5 Ehrhart, L.A., and Holderbaum, D., Stimulation of aortic protein synthesis in experimental rabbit atherosclerosis, Atherosclerosis, 27 (1977) 477. 6 Ehrhart, L.A. and Holderbaum, D., Aortic collagen, elastin and non-fibrous protein synthesis in rabbits fed cholesterol and peanut oil, Atherosclerosis, 37 (1980) 423. 7 Langner, R.O., Gilligan, J.P. and Ehrhart, L.A., The effect of cholesterol feeding on protein synthesis in different regions of the rabbit arterial wall, Exp. Molec. Path., 31 (1979) 308. 8 Langner, R.O. and Modrak, J.B., Alteration of collagen synthesis in different tissues of the atherosclerotic rabbit, Artery, 9 (1981) 253. 9 Constantinides, P., Booth, J. and Carlson, G., Production of advanced cholesterol atherosclerosis in the rabbit, Arch. Path., 70 (1960) 712. 10 Modrak, J.B. and Langner, R.O., Possible relationship of cholesterol accumulation and collagen synthesis in rabbit aortic tissues, Atherosclerosis, 37 (1980) 211. 11 Lorenzen, I., Alterations in acid mucopolysaccharides and collagen of rabbit aorta related to age of epinephrine thyroxine induced atherosclerotic lesions, Acta Endocrin., 39 (1962) 615. 12 Newman, R.A. and Langner, R.O., Comparison of TCA and collagenase in the isolation of tissue collagen, Anal. B&hem., 66 (1966) 175. 13 Kivirikko, K.I., Laitinen, 0. and Prockop, D.J., Modification of a specific assay for hydroxyproline in urine, Anal. Biochem., 19 (1967) 249. 14 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 15 Folch, J., Lees, M. and Sloane Stanley, G.J., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 16 Franey, R.J. and Amador, E., Serum cholesterol measurement based on ethanol extraction and ferric chloride-sulfuric acid, Clin. Chim. Acta, 21 (1968) 255. 17 Snedecor, G.W. and Cochran, W.G., Statistical Methods, 7th edition, Iowa State University Press, Ames, IA, 1980, p. 96. 18 Langner, R.O. and Modrak, J.B., Aortic collagen synthesis in the rabbit following removal of atherogenic diet, Exp. Mol. Path., 26 (1977) 310. 19 Friedman, M. and Byers, SO., Observations concerning the evolution of atherosclerosis in the rabbit after cessation of cholesterol feeding, Amer. J. Path., 43 (1963) 349.
Ltd.
ATH 03545
Effect of Removal of Atherogenic Diet on Protein Synthesis and Cholesterol Retention in Rabbit Aorta and Lung James P. Gilligan
* and Ronald 0. Langner
School of Pharmacy, University of Connecticut, Storrs, CT 06268 (U.S.A.) (Received 4 April, 1984) (Revised, received 22 June, 1984) (Accepted 25 June, 1984)
Summary
The purpose of these studies was to determine the effect of cholesterol feeding in rabbits on the synthesis of collagen and non-collagen proteins in both aortic and lung tissues. Rabbits were fed a 2% cholesterol diet for 30 or 60 days, followed by 30 days of a low cholesterol diet (i.e. 30-30 or 60-30). After 30 days of cholesterol feeding non-collagen protein synthetic rates were significantly elevated in aortic tissues, but not in the lung. After 60 days of cholesterol feeding, both collagen and non-collagen synthetic rates were elevated in the aorta but not in the lung. Both tissues demonstrated significant increases in cholesterol content. When cholesterol was removed from the diet, cholesterol continued to accumulate in the aorta but decreased in the lung. The 60-30 group demonstrated both the largest increase in aortic cholesterol, and the largest increase in the per cent of collagen being synthesized in the aorta. These data therefore demonstrate that cholesterol feeding stimulates both collagen and non-collagen protein synthesis and suggests that there may be some differences in the lag phase before significant changes are apparent in both parameters. Following removal of cholesterol from the diet the per cent collagen synthesized in the aorta increased further, due to an apparent reduction in non-collagen protein synthesis rather than a further acceleration of collagen synthesis. These changes may be important in explaining how intermittent-cholesterol feeding produces a more fibrous aortic lesion. Key words: Aorta - Atherosclerosis - Cholesterol - Collagen - Lung - Rabbits
* Present address: Unigene Laboratories, 110 Little Falls Road, These studies were supported in part by NIH grant HL-26122. 0021-9150/85/$03.30
0 1985 Elsevier Scientific
Publishers
Ireland,
Fairfield,
Ltd.
NJ 07006, U.S.A.
2
Introduction Early human atherosclerotic lesions which evolve from serum hyperlipidemia contain increased amounts of free and esterified cholesterol. As the lesions become more advanced, they accumulate increased amounts of collagen which is thought to be responsible for the irreversibility of complex atherosclerotic plaques [1,2]. The factors responsible for stimulating collagen synthesis are unknown. Studies by several authors [3-61 have shown that continuous cholesterol feeding in excess of 90 days will result in an increased aortic collagen synthetic rate. This increase in collagen synthesis was later demonstrated to be located at the primary site of cholesterol accumulation [7]. In a recent study Langner and Modrak [8] reported that hypercholesterolemia caused an increase in collagen synthesis in rabbit aorta and liver, but no change in lung. These data suggested that the connective tissue response to hyperlipemic insult is present in some tissues, but not all tissues. The increase in collagen synthesis is also accompanied by increased rates of non-collagen protein synthesis. When rabbits were fed cholesterol continuously for 120 days there was a proportional increase in both collagen and non-collagen synthesis [5]. Studies by Ehrhart and Holderbaum [6], using a high-cholesterol diet supplemented with peanut-oil, demonstrated that the increase in collagen synthesis was greater than the change in non-collagen protein synthesis, and concluded that this accounted for the more fibrous lesion normally seen with the peanut-oil supplemented diet. When rabbits are fed an intermittent high-cholesterol diet they also develop a more fibrous type lesion compared to rabbits continuously fed a high-cholesterol diet [9]. The mechanisms for this increased fibrous tissue deposition is unknown. It is possible however, that removal of cholesterol from the diet stimulates a greater increase in collagen synthesis relative to non-collagen protein synthesis similar to that observed with the peanut-oil diet. Another possible mechanism could be that the increases in both the synthesis of collagen and non-collagen protein occur independently of each other at different times. For example, if increases in collagen synthesis were to occur significantly before changes in non-collagen protein synthesis then one could expect a more fibrous type lesion. Therefore, one purpose of this study was to determine both the time of onset and the relative degree of change for both collagen and non-collagen synthetic rates following removal of cholesterol from the diet. A second objective of this study was to gain further information concerning the role of increased protein synthesis on cholesterol accumulation and retention. Previous studies [lo] have demonstrated a close correlation between increased collagen synthesis and increased rates of tissue cholesterol deposition in the aorta. The lung has been shown to have an increased cholesterol content in response to cholesterol feeding with no change in protein synthesis [8]. Therefore, this study was designed to compare cholesterol accumulation and efflux in tissues demonstrating increased protein synthetic activity (i.e. aorta) to a tissue with basal levels of protein synthesis (i.e. lung). The hypothesis to be tested is that in tissues demonstrating an
3
elevated retention
rate of collagen of accumulated
and non-collagen cholesterol.
protein
synthesis,
there is an increased
Methods White male New Zealand rabbits weighing between 2.0-2.5 kg were used in these studies. Rabbits were randomly divided into 5 groups: Group 1 (34 rabbits) were fed Purina Rabbit Chow and served as control; Group 2 (15 rabbits) were fed a 2% cholesterol-supplemented diet for 30 days (30); Group 3 (7 rabbits) were fed 2% cholesterol diet for 30 days followed by the control diet for 30 days (30-30); Group 4 (15 rabbits) were fed the 2% cholesterol-supplemented diet for 60 days (60); Group 5 (10 rabbits) were fed the 2% cholesterol diet for 60 days followed by the cholesterol-free diet for 30 days (60-30). Control and experimental animals were killed on the same day by cervical dislocation. The aorta and lungs were rapidly removed, weighed, and sections taken for histology. The aorta was cleaned of loose adhering tissue, opened longitudinally and macroscopic lesions graded, using the O-4 grading system described by Lorenzen [ll]. Grade 0 indicated the complete absence of lesions and grade 4 indicated severe lesion development with lesions extending into the abdominal aorta. Both tissues were then weighed and placed in oxygen-rich Krebs bicarbonate buffer, pH 7.4, for a 20 min preincubation period at 37 o C under a 95% 0,/5% CO, atmosphere. Following preincubation the tissues were transferred to incubation vessles, containing fresh oxygenated Krebs bicarbonate buffer and 0.2 mM L-proline with 3 PCi of [U-‘4C]proline in a final volume of 10 ml. Following the incubation period (90 min aorta; 60 min lung) the tissues were immediately rinsed with ice-cold trichloroacetic acid (TCA) to prevent further incorporation of [r4C]proline. Tissues were then homogenized using a glass co-axial homogenizer (Kontes) and appropriated dilutions were made for biochemical analysis. Collagen and non-collagen proteins were separated using the hot trichloroacetic acid (TCA) procedure described by Newman and Langner [12]. The tissues were repeatedly washed with ice-cold 5% TCA to remove non-incorporated [‘4C]proline. The washed pellets were then extracted for 1 h at 90 ‘C, and the supematant containing hydrolyzed collagen fragments was collected. The 90 ‘C TCA extraction was then repeated. The combined supematants were evaporated to dryness, resolubilized in distilled water, and counted in a liquid scintillation counter. These samples were used as an estimate of collagen synthetic activity. The pellet remaining after the removal of collagen, which contained non-collagen protein, was digested at 45 o C overnight, and was then counted in a liquid scintillation counter. Hydroxyproline content of aorta and lung homogenates was estimated by the method of Kivirikko et al. [13] and served as an index of collagen content. Non-collagen protein concentrations were determined on the tissue homogenates by the method of Lowry et al. [14] using bovine serum albumin as the standard. The cholesterol content of aortic and lung tissues was determined after delipidation utilizing a modified procedure of Folch et al. [15]. Following extraction free and esterified cholesterol were separated on thin layer plates of silica gel 60HR extra
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pure (E.M.), 0.5 mm thick. The plates were developed in n-heptane/diethyl ether/acetic acid (75 : 25 : 2) and visualized in iodine. The iodine was allowed to dissipate and the free and esterified cholesterol spots scraped into conical tubes. Cholesterol levels were determined using the method described by Franey and Amador [16]. Serum cholesterol levels were determined weekly. Blood was collected from the medial ear artery and allowed to clot. Serum was collected and the cholesterol levels were determined on the ethanolic extract by the method of Franey and Amador [16]. Histological sections were taken from aorta at a level just below the subclavian artery and the midthoracic region. The sections were fixed in buffered formalin and stained with hematoxylin-eosin, Verhoeff or Van Gieson stains. The statistical significance of any observed differences was determined by use of analysis of variance or Student t-tests [17]. Results
Control animals were killed at each of the experimental time points. Since there was no apparent difference between control groups, the data was combined into a single control group in Tables l-5. Rabbits fed the 2% cholesterol-supplemented diet exhibited a characteristic rise in serum cholesterol values. By day 30 all experimental animals had serum cholesterol levels in excess of 600 mg/lOO ml. Continuation of the cholesterol diet for an additional 30 days resulted in serum cholesterol levels greater than 1300 mg/lOO ml (Table 1). When the animals were placed on the low-cholesterol control diet there was a rapid fall in serum cholesterol values, but they were still elevated above control values after 30 days. TABLE
1
EFFECTS OF 2% CHOLESTEROL FORMATION All data expressed
as mean
Control 30 B 30-30 60 60-30
ON TISSUE WET WEIGHTS
AND VISIBLE
LESION
f SE.
Serum cholesterol
(w/l~
FEEDING
n-4
60.0f 4.0 615.0k47.6 * 105.2 f 27.0 * 1352 k69.1 * 361.7+46 *
Lung
Thoracic
Wet Weight/ Body Weight
Wet Weight/ Body Weight
(g/kg)
(g/kg)
3.56k0.11 3.55 f 0.01 3.32 f 0.21 4.05 f 0.23 * 3.78 kO.22
0.226 + 0.008 0.28OkO.013 * 0.320*0.013 * 0.352 f 0.017 * 0.420 f 0.02 *
* Significantly different from controls (P -c 0.05). * 30 and 60 = Either 30 or 60 days of 2% cholesterol cholesterol diet followed by 30 days normal diet.
diet;
aorta
30-30
Macroscopic
lesions
O-l-2-3-4
or 60-60
27-7-O-O-O 3-7-3-2-O o-o-2-4-1 O-O-2-5-8 O-O-2-3-5
= 30 or 60 days of 2%
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Rabbits fed cholesterol for 30 days developed aortic lesions (Table 1) which were characterized by several layers of intimal foam cells. The media in these early lesions appeared to be normal, with the only difference between grade 2 and 4 lesions being the increased surface area involved and the thickness of the lesions. The rabbits maintained on the 60-day continuous cholesterol diet and those on the 60-30 intermittent regimen had the most severe disease as evidenced by the large number of grade 3 and 4 lesions. Several histological sections taken from the 60-30 group of rabbits demonstrated some focal cellular necrosis, with increased evidence of lesion fibrosis. As also seen in Table 1 rabbits fed the cholesterol diet showed a significant increase in thoracic wet weight ratios. These increases persisted even when the animals were placed on a normal cholesterol diet. The wet weight/body weight ratio of the lungs was only elevated in the 60-day cholesterol group, suggesting increased cholesterol accumulation. The total and esterified cholesterol content of the thoracic aortas were significantly increased in all cholesterol-fed rabbits (Table 2). In the 30-day group, both total cholesterol and cholesterol-esters were significantly greater than control, however the levels were significantly lower than those observed in the 30-30 group of animals. In the rabbits fed cholesterol continuously for 60 days, both measures of aortic cholesterol content were significantly increased when compared to either the 30-30 or control rabbits. The 60-30 group had the highest levels of both cholesterol and esterified cholesterol, even though their serum cholesterol levels were much reduced. The cholesterol and esterified cholesterol content of the lungs was significantly elevated in both groups of continuously-fed cholesterol animals (Table 2). The lungs differed from the aortas in that removal of cholesterol from the diet resulted in the
TABLE
2
EFFECT OF 2% CHOLESTEROL CHOLESTEROL CONTENT All data reported
DIET
FOR
* ** *** a
PERIODS
OF
TIME
ON
TISSUE
as mean + SE.
Lung
Control 30 a 30-30 60 60-30
VARYING
Aorta
Total pg cholesterol/ mg protein
pg cholesterol-ester,’ mg protein
Total pg cholesterol/ mg protein
pg cholesterol-ester/ mg protein
25.8 + 1.6 43.2 + 3.7 ** 23.9*1.6 63.3k4.3 *** 29.6 f 2.9
2.9 + 0.4 18.9k3.4 ** 6.5k1.4 * 33.4k3.1 *** 5.3*1.0*
7.1* 0.5 11.7+ 0.8 * 19.6+ 3.3 * 75.9* 15.5 ** 95.7+ 10.8 **
0.8kO.l 4.0* 1.1 * 10.5 f 2.9 51.8+4.4 ** 60.1 f 6.8 **
Significantly different from controls (P < 0.05). Significantly (P -Z 0.05) different from 30-30 animals. Significantly (P c 0.05) different from 60-30 animals. 30 and 60 = Either 30 or 60 days of 2% cholesterol diet; 30-30 cholesterol diet followed by 30 days normal diet.
or 60-30
= 30 or 60 days of 2%
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return of total cholesterol values in the lung toward control levels. The cholesterol-ester content in the lungs of both the 30-30 and the 60-30 group also decreased when cholesterol was removed from the diet but remained slightly, but significantly elevated when compared to control. Since the color developed in the protein assay described by Lowry et al. [14] is dependent upon the presence of amino acids essentially lacking in collagen, this assay was used to estimate non-collagen protein content. The amount of non-collagen protein present in these tissues did not significantly change in any of the experimental groups (Table 3). The total amount of collagen in either the lung or aorta was estimated by measuring the amount of hydroxyproline present. The aortas from the 30, 30-30 or 60 groups did not contain any change in total collagen content. The aortas from the 60-30 group did however demonstrate a significant increase in collagen content. The lungs from the 60-30 animals did not demonstrate any change in collagen content from control levels. The rate of collagen and non-collagen protein synthesis in aorta and lung is presented in Tables 4 and 5. Following 30 days of cholesterol feeding there was a significant increase in thoracic aorta non-collagen protein synthesis, but there was no significant change in collagen synthetic activity. The relative per cent of collagen and non-collagen proteins being synthesized in the 30-day rabbit aortas was not different from control. Thoracic aortas taken from rabbits in the 30-30 intermittent cholesterol-fed group demonstrated a significant 2-fold increase in collagen synthesis (Table 4). The rate of non-collagen protein synthesis was also elevated, with the per cent of collagen being synthesized no different from controls. The rabbits receiving the 60- and 60-30-day treatment demonstrated further increases in collagen synthesis with an approximate 4-fold increase in collagen synthetic activity. Non-collagen protein synthesis in both the 60- and 60-30 groups was significantly elevated above
TABLE
3
EFFECT OF 2% CHOLESTEROL LAGEN PROTEIN CONTENT All data reported
DIET ON LUNG
AND
AORTA
COLLAGEN
AND
NON-COL-
as mean f SE. Aorta
Lung mg protein/ g tissue
pg hypro “/ mg protein
mg protein/ g tissue
ng hypro/ mg protein
Control 30 a
109.5 f 3.5 112.8k 3.4
30-30 60 60-30
99.0+4.3 101.7 f 3.0 115.6 f 3.5
13.4kO.6 11.9hO.6 12.0k0.6 12.4kO.8 13.2* 1.5
103.7 * 5.7 106.2 f 4.4 116.9+7.1 106.0 k 4.1 114.9 + 3.2
52.6 f 4.0 53.5 _I 3.0 58.6k6.0 54.5 f 3.0 50.2 f 4.2
’ 30 and 60 = Either 30 or 60 days of 2% cholesterol cholesterol diet followed by 30 days of control diet. b pg hydroxyproline.
diet;
30-30
or 60-30
= 30 or 60 days of 2%
TABLE
4
EFFECT OF 2% CHOLESTEROL FEEDING ON “C-PROLINE COLLAGEN AND NON-COLLAGEN PROTEIN All data reported
Control 30 a 30-30 60 60-30
INCORPORATION
INTO AORTIC
as mean + SE. Collagen DPM/ mg protein
Non-collagen mg protein
81.1 f 13.7 141.0+ 19.0 195.0*30.3 * 361.2+39.0 * 415.3 + 57.2 *
135.5 270.5 379.2 508.6 395.7
f + + + k
* Significantly different from controls (P < 0.05). * 30 and 60 = Either 30 or 60 days of 2% cholesterol cholesterol diet followed by 30 days of control diet. b % collagen synthesized = collagen DPM/(Non-collagen
13.4 31.8 108.0 71.6 58.4
DPM/
% Collagen
* * * *
11.6kO.4 10.9kO.9 11.2*1.2 15.8 +0.9 * 19.8k1.3 *
diet:
30-30
or 60-30
b
= 30 or 60 days of 2%
DPM X 5.1) + (collagen
DPM x 100)
control levels (Table 4). The per cent of collagen synthesized in the thoracic aortas from 60 and 60-30 animals was significantly increased above controls, suggesting a more fibrous type of tissue response in these animals. The per cent of collagen synthesized was calculated by dividing collagen DPM by non-collagen DPM, multipled by a factor of 5.1, plus collagen DPM X 100. The correction factor of 5.1 was used to correct for the fact that collagen contains approximately 5.1 times as much proline as does non-collagen protein. Lung collagen and non-collagen protein synthesis remained unaltered in all experimental groups (Table 5). Discussion Previous studies from our laboratory have shown that in rabbits fed a 2% cholesterol diet for 70 days, collagen synthesis was increased only in the inner layers
TABLE
5
EFFECT OF 2% CHOLESTEROL FEEDING ON [14C]PROLINE COLLAGEN AND NON-COLLAGEN PROTEIN
Control 30 b 30-30 60 60-30
Collagen DPM/ mg protein
Non-collagen mg protein
474.5 515.5 318.0 440.0 438.7
1360.0 f 47.2 1458.0 f 96.3 96.3 1278.0* 56.8 1307.0* 1271.2* 141.6
f 39.7 *66.1 f 34.5 f 28.2 f 64.5
DPM/
INCORPORATION
% Collagen
INTO LUNG
a
6.OkO.4 6.5 f 0.7 4.5 +_0.4 5.9*0.3 6.2kO.4
a % collagen synthesized = collagen DPM/(non-collagen DPM x 5.1) + (collagen DPM x 100). b 30 and 60 = Either 30 or 60 days of 2% cholesterol diet; 30-30 or 60-30 = 30 or 60 days of 2% cholesterol diet followed by 30 days of control diet. All data reported as mean f SE.
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of the arterial wall, the primary site of cholesterol accumulation [7]. These results suggested that cholesterol deposition in the arterial wall was capable of initiating localized fibrotic response which may have an effect on further cholesterol accumulation. Ehrhart and Holderbaum reported that when rabbits are fed cholesterol for 140-180 days, both collagen and non-collagen synthetic rates are elevated [5]. Previous work from our laboratory [3] had reported that collagen synthesis was elevated after only 60 days of 2% cholesterol feeding, however in these studies we did not measure non-collagen protein synthetic rates. In the present studies we investigated changes in both collagen and non-collagen protein synthetic rates after varying periods of cholesterol feeding. The purpose of these studies was to determine whether both measures of protein synthesis increase at the same time. As seen in Table 1 following 30 days of cholesterol feeding there was a significant increase in non-collagen protein synthesis. Collagen synthetic rates were also elevated at this time, but were not statistically significant. By 60 days both collagen and non-collagen synthetic rates were significantly elevated, and this occurred whether the rabbits were fed cholesterol for 60 days or for 30 days followed by 30 days of normal diet. These data demonstrate that cholesterol feeding induces an increase in both collagen and non-collagen protein synthesis, but that the rate of response for non-collagen protein synthesis may be more rapid than that observed for collagen synthesis. Many investigators have shown that continuous feeding of a high-cholesterol diet to rabbits produces a lipid-laden foam cell lesion. In the studies reported by Ehrhart and Holderbaum [5] they found an equal increase in both collagen and non-collagen protein synthetic rates. In the present study the ratio of collagen to non-collagen protein synthesis was measured in 4 separate groups and is reported in Table 4. In the rabbits fed for only a short time (i.e. 30 and 30-30 groups) there appears to be an equal increase in both collagen and non-collagen protein synthesis. However in the 60 and 60-30 groups there is a significant change in the per cent collagen being synthesized. The 60-30 rabbits had the largest change primarily because of an apparent reduction in non-collagen protein synthesis when compared to the 60-day animals. The data in Table 4 differs substantially from that reported by Ehrhart and Holderbaum [5], who found no change in the per cent of proteins being synthesized as collagen. In their experiments they studied rabbits which had been on a highcholesterol diet for a much longer time and they reported collagen synthetic rates as a function of extractable proteins. In our studies rabbits were fed cholesterol for much shorter periods and we reported our data as a function of total proteins. These differences in procedure may account in part for the apparent differences. In addition, the per cent increase in collagen synthesis may, in fact, be linked to the severity of the lesions and not directly correlated to the chronologic exposure to cholesterol. Several investigators have reported that removal of cholesterol promotes a more fibrous type of aortic lesion in rabbits. It has been speculated that the removal of cholesterol from the diet produces a stimulation of collagen synthesis. Previous studies from our laboratory reported that removal of cholesterol did not, by itself, result in a stimulation of collagen synthesis [la]. Since collagen synthesis in the 60
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and 60-30 groups was essentially the same (Table 4) it again suggests that removal of cholesterol from the diet does not stimulate collagen synthesis. However, based upon the changes in non-collagen protein synthetic rates, it is possible that the reason cholesterol removal produces a more fibrous lesion is that non-collagen protein synthesis tends to become reduced when cholesterol is withdrawn resulting in a relative increase in the per cent of collagen being synthesized. Additional research will be required to investigate this hypothesis. Another objective of these studies was to study further the relationship between elevated rates of protein synthesis and tissue cholesterol accumulation and retention. Studies by Modrak and Langner [lo] demonstrated a close correlation between increased rates of aortic collagen synthesis and aortic rates of cholesterol accumulation. In a separate series of experiments [8] we also demonstrated that in cholesterolfed rabbits the lungs will accumulate increased levels of cholesterol and cholesterol-esters, but will not respond with increased rates of collagen synthesis. In the present studies when rabbits were placed on low-cholesterol diets after 30 or 60 days of high-cholesterol feeding, there was a rapid fall in serum cholesterol levels (Table 1). The cholesterol content of the thoracic aorta in both regression groups continued to increase even though serum levels were falling. The rate of increase in aortic cholesterol was greater in the animals fed cholesterol for 60 days followed by 30 days of normal diet (i.e. 60-30 group) than in the 30-30 group. As seen in Table 2, over the same 30-day low-cholesterol period, the rabbits fed cholesterol for 60 days accumulated more than 3 times as much cholesterol as did the rabbits fed for just 30 days. The reason for this difference is not known Since the final serum levels in the 60-30 group was higher than that observed in the 30-30 group, this may account for some of the difference. Another possible reason may be that in the aortas of the 60-30 animals, there is an increased rate of protein synthesis with a higher percentage of collagen being synthesized (Table 4). This difference in protein synthesis may alter aortic permeability and allow more cholesterol to enter the tissue or it may alter cholesterol efflux, and prevent cholesterol from leaving the tissue. Either or both mechanisms would result in an increase in tissue cholesterol deposition. The lungs taken from these animals presented a much different pattern of response from what was observed in the aorta. After 30 or 60 days of cholesterol feeding the lungs had significantly elevated levels of tissue cholesterol (Table 2). When the animals were placed on the regression diet, instead of continuing to accumulate cholesterol as seen in the aorta, the lungs rapidly released their accumulated cholesterol and returned to control levels after 30 days. As seen in Table 5, the lungs exhibited no alteration in either collagen or non-collagen protein synthetic rates. Since the aorta, which has increased rates of total protein synthesis, continues to accumulate cholesterol in the presence of the same falling cholesterol levels, the data suggest that the continued accumulation of cholesterol occurs because of some alteration of tissue metabolism. This is contrary to the earlier suggestions of Friedman and Byers [19], who suggested that during the first 3 months of falling serum cholesterol levels, additional plaque growth occurs because of the continuing hypercholesterolemia and not because of some peculiar metabolism of the plaque itself.
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In summary, the feeding of cholesterol will induce an increase in both collagen and non-collagen protein synthesis. When cholesterol feeding is continued there is a relative increase in the percentage of aortic collagen being synthesized. The significance of this change in collagen synthesis is not known. Our data, however, do show that in a tissue which has an elevated protein synthetic rate, reduction of serum cholesterol does not promote a regression of tissue cholesterol content. These observations are not inconsistent with the hypothesis that changing rates of aortic protein synthesis play an important role in promoting the development of aortic lesions. The data further suggest that drugs which could return this altered metabolic activity toward control levels may be helpful in promoting regression of established atherosclerosis. References 1 Constantinides, P., Overview of studies on regression of atherosclerosis, Artery, 9 (1981) 30. 2 Armstrong, M.L., Regression of atherosclerosis, Atheroscl. Rev., 1 (1976) 137. 3 Langner, R.O. and Modrak, J.B., Collagen metabolism during the early stages of cholesterol-induced atherogenesis in rabbits, Blood Vessels, 13 (1976) 257. 4 Fischer, G.M., Cherian, K. and Swain, M.L., Increased synthesis of aortic collagen and elastin in experimental atherosclerosis, Atherosclerosis, 39 (1981) 463. 5 Ehrhart, L.A., and Holderbaum, D., Stimulation of aortic protein synthesis in experimental rabbit atherosclerosis, Atherosclerosis, 27 (1977) 477. 6 Ehrhart, L.A. and Holderbaum, D., Aortic collagen, elastin and non-fibrous protein synthesis in rabbits fed cholesterol and peanut oil, Atherosclerosis, 37 (1980) 423. 7 Langner, R.O., Gilligan, J.P. and Ehrhart, L.A., The effect of cholesterol feeding on protein synthesis in different regions of the rabbit arterial wall, Exp. Molec. Path., 31 (1979) 308. 8 Langner, R.O. and Modrak, J.B., Alteration of collagen synthesis in different tissues of the atherosclerotic rabbit, Artery, 9 (1981) 253. 9 Constantinides, P., Booth, J. and Carlson, G., Production of advanced cholesterol atherosclerosis in the rabbit, Arch. Path., 70 (1960) 712. 10 Modrak, J.B. and Langner, R.O., Possible relationship of cholesterol accumulation and collagen synthesis in rabbit aortic tissues, Atherosclerosis, 37 (1980) 211. 11 Lorenzen, I., Alterations in acid mucopolysaccharides and collagen of rabbit aorta related to age of epinephrine thyroxine induced atherosclerotic lesions, Acta Endocrin., 39 (1962) 615. 12 Newman, R.A. and Langner, R.O., Comparison of TCA and collagenase in the isolation of tissue collagen, Anal. B&hem., 66 (1966) 175. 13 Kivirikko, K.I., Laitinen, 0. and Prockop, D.J., Modification of a specific assay for hydroxyproline in urine, Anal. Biochem., 19 (1967) 249. 14 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 15 Folch, J., Lees, M. and Sloane Stanley, G.J., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 16 Franey, R.J. and Amador, E., Serum cholesterol measurement based on ethanol extraction and ferric chloride-sulfuric acid, Clin. Chim. Acta, 21 (1968) 255. 17 Snedecor, G.W. and Cochran, W.G., Statistical Methods, 7th edition, Iowa State University Press, Ames, IA, 1980, p. 96. 18 Langner, R.O. and Modrak, J.B., Aortic collagen synthesis in the rabbit following removal of atherogenic diet, Exp. Mol. Path., 26 (1977) 310. 19 Friedman, M. and Byers, SO., Observations concerning the evolution of atherosclerosis in the rabbit after cessation of cholesterol feeding, Amer. J. Path., 43 (1963) 349.