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Nutritional control of coronary artery atherosclerosis in the squirrel monkey
Journal of Atherosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
N U T R I T I O N A L CONTROL OF CORONARY A R T E R Y A T H E R O S C L E R O S I S IN T H E S Q U I R R E L M O N K E Y
C. A. MARUFFO AND O. W. PORTMAN Departments of Pathology and Primate Nutrition, Oregon Regional Primate Research Center, Beaverton, and Departments of Pathology and Biochemistry, University of Oregon Medical School, Portland, Oreg. ( U.S.d.)
(Revised, received August 23rd, 1967)
SUMMARY
A study was made of the coronary arteries from squirrel monkeys which were fed one of two diets, one which induced aortic atherosclerosis and one which did not, for 3, 6, and 8 months. A third series of monkeys was fed the atherogenic diet for 3 months followed b y 3 or 5 months on the control diet. Atherosclerotic changes consisting of extensive intra- and extracellular lipid accumulation in the intima and media without significant cell proliferation occurred in the coronary arteries of squirrel monkeys after various intervals on a diet containing butter plus additional cholesterol. Correlations of coronary artery lesions with the terminal plasma cholesterol and aorta and esterified cholesterol concentrations were statistically significant. In animals transferred from the atherogenic diet to the basal diet, a lesion characteristic of the early plaque was seen: lipid-poor and rich in PAS-positive material. Muscle cells of the media at the base of the plaque showed ultrastructural alterations. Cytoplasmic changes in the endothelial cells were relatively subtle: a few heterogeneous lipid droplets, an increase in pinocytotic vacuoles, a prominent endoplasmic reticulum and large lysosomes. Fragmentation of the elastic lamellae and calcium deposition in the inner media were often observed.
INTRODUCTION
Numerous experiments have been performed in monkeys in order to gain an insight into the pathogenesis of atherosclerosis. Although some of the experimentallyinduced lesions cannot be considered identical to their naturally-occurring counterparts in humans, studies of experimental atherosclerosis have advanced our knowledge and understanding of the development of the condition in man. J. Atheroscler. Res., 1968, 8:237-247
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Several different procedures have been used to obtain arterial lesions in primates: endocrine manipulationsa, 2, pyridoxine deficiency3-6, magnesium deficiency7, and high cholesterol and/or high fat intakes s-15. We have recently described the sequence of early nutritionally induced changes in the composition of the major lipid classes and subclasses of phospholipids from aortas of squirrel monkeys and the potential for regression of lipid-rich lesions with dietary alterations a6. This communication describes a histological study of coronary atherosclerosis induced in those squirrel monkeys as well as the ultrastructural detail of the arterial lesions. The effect of diet on the induction and progression, as well as on the regression, of coronary artery lesions is described. MATERIALS AND METHODS
Eighty-seven sexually mature and apparently healthy female squirrel monkeys were used. A detailed report of the experimental procedures has already been presentedl6; a brief summary of those procedures follows. These animals were obtained from two sources. One importer obtained Brazilian squirrel monkeys (Saimiri madeirae juruanus) from a collecting station in Leticia, Colombia; the other obtained squirrel monkeys (Saimiri boliviensis nigric@s) from Iquitos, Peru. In all of the experiments the animals from each origin were evenly distributed between groups. Although squirrel monkeys from both sources were similar in many of the measurements made, the Brazilian squirrels appeared to be somewhat more susceptible to dietary increases in arterial lipids and also healthier than the squirrel monkeys from Peru. They were quarantined at the Oregon Regional Primate Research Center for 60 days, treated with antihelminthics, and X-rayed. Hematological studies and tuberculin testing were repeated at varying intervals.
Experimental diets used The two semipurified diets used most extensively produced the fewest and the most aortic sudanophilic lesions during earlier studies of squirrel monkeysaT, 18. The first of the diets provided 8% (16% of calories) corn oil and contained no added cholesterol; the second contained 25.2% butter (45% of calories) and contained cholesterol at 0.1 g/kcal. Casein was supplied at 20% of the diet and a salt mixture at 4% (ref. 19). The following quantities of vitamins were supplied per kg of diet: vitamin A acetate 12,500 units, a-tocopherol 0.1 g, crystalline vitamin Da 100 #g (4000 units), menadione 0.04 g, ascorbic acid 0.5 g, inositol 1.0 g, choline chloride 5.0 g, niacin 0.049 g, riboflavin 0.01 g, thiamine 0.01 g, pyridoxine 0.01 g, calcium pantothenate 0.03 g, biotin 0.2 rag, folie acid 1.0 rag, and vitamin B12 0.02 rag. The diets were made to 100~ by the addition of sucrose.
Preparation of arterial tissue The animals were sacrificed at three different intervals. Usually 5 animals from
J. Alheroscler. Res., 1968, 8:237 247
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239
each group were available at each sacrifice period. The dietary treatments and durations are indicated in Table 1. At 3 months a group of control animals (corn oil) and a group of high butter were sacrificed. At 6 months three groups of animals were used: one control (corn oil), one high butter, and a third group composed of animals which had been on the high butter diet for the first 3 months and had transferred to the corn oil (control) diet for the last 3 months before the sacrifice. At 8 months,the same experimental pattern was repeated: a control group (corn oil), a high butter group, and a group on the high butter diet during the first 3 months and on the control diet (corn oil) during the last 5 months before termination of the experiment. TABLE
1
EXPERIMENTAL DIET SERIES
Group
Diet
Control, 3 months corn oil 3 months high butter Control, 6 months corn oil 6 months high butter 3 months high butter + 3 months corn oil Control, 8 months corn oil 8 months high butter 3 months high butter + 5 months corn oil
The monkeys were sacrificed after Sernylan (phencyclidine (1-(1-phenylcyclohexyl) piperidine hydrochloride)) was administered by opening the thoracic and ab dominal cavities with a mento-pubic incision and withdrawing blood from the abdominal aorta at its bifurcation. A complete autopsy was performed on each animal. The heart was fixed in 10 % buffered formalin and cut into 5 transverse blocks parallel to the A-V groove. From each block frozen sections (10 20 # thick) were stained with Sudan IV and hematoxylin in order to grade microscopically the coronary atherosclerotic lesions, which were classified according to the extent of sundanophilia and cell proliferation (plaques). For each monkey, all graded arteries (0.5-3.0 in ascending order of severity of involvement) were added and results expressed as mean coronary score per 100 coronary arterial cross-sections 2~ The remaining portions of the blocks were embedded in paraffin, and several sections were stained with the following techniques21: hematoxylin and eosin, Gomori's aldehyde fuchsin for elastic fibers, periodic acid-Schiff (PAS) preceded by treatment of hydrated sections for 10 rain with saliva, and Von Kossa for calcium. For light microscopy of thin sections and for electron microscopy (6 animals), tissue from the anterior aspect of the heart (below the A-V groove) was cut in crosssection under cold phosphate-buffered osmium tetroxide into 1-2 mm cubes which were fixed at approximately 4~ for 60-90 min. In addition, several 1 mm slices of myocardium were fixed in 2 % glutaraldehyde 22 for 90 rain, washed overnight in phosphate buffer (sucrose 4.5 %), cut into 1 mm cubes and postfixed in osmiumtetroxJ. Atheroscler. Res., 1968, 8:237-247
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c . A . MARUFFO, O. W. PORTMAN
ide for 60-90 min at approximately 4~ The blocks were then dehydrated through graded alcohols and embedded in araldite; sections were cut with glass and diamond knives on an L K B Ultrotome. From each block several 0.5-1/z sections were mounted on glass slides, stained with 1% toluidine blue 23 and examined with a light microscope. Ultrathin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with RCA-3F and Philips EM200 electron microscopes. RESULTS
General observations All the animals were regarded as young adults of comparable age, as estimated by long bone development, eruption of the third molar, and wear of the canine teeth. The body weights were similar in all groups of animals 16 and remained essentially static throughout the experiment. No skeletal alterations were observed. Only 3 cases of peritoneal filariasis (achanthocheilonema) were found. A few animals of the high butter group showed mild f a t t y metamorphosis of the liver. Coronary atherosclerosis. Effect of diet and time on extent of lesions The results of the grading of coronary atherosclerotic lesions and the statistical tests of significance are presented in Table 2. After 3 months the monkeys fed the butter-containing diet had significantly higher coronary artery disease scores than monkeys of the control group. After 6 and 8 months on these dietary regimens the extent of coronary artery lesions was much more severe in the monkeys fed the butter diet than was the case at 3 months. In groups 5 and 8, which were fed the lesion-
TABLE 2 THE EFFECT
OF DIET AND DURATION
OF FEEDING
ON THE SEVERITY
OF CORONARY
ATHEROSCLEROSIS
See t e x t for m e a n i n g of the c o r o n a r y score which is equivalent to the p r o d u c t of the p r o p o r t i o n of involved c o r o n a r y a r t e r y sections and the m e a n e x t e n t of involvement. Meanings of g r o u p designations are indicated in Table 1. Group
No. of arterial crosssections observed
Mean coronary score a
~ S.E. mean
1 2
462 530
0 12
0 2.2
4 5 6 7 8
438 536 513 484 395
52 10 2 61 23
12.8 1.2 1.6 3.0 4.2
Significance b
i
I
] ]
[
a The m e a n c o r o n a r y score for 6 squirrel m o n k e y s m a i n t a i n e d on a diet of P u r i n a M o n k e y Chow for 2 m o n t h s and sacrificed before initiation of t h e feeding e x p e r i m e n t was 2 ~ 1.3. b G r o u p m e a n s which are connected b y a line are statistically different ( S t u d e n t ' s t test) at the P = 0.025 level or less. j . Atheroscler. Res., 1968, 8 : 2 3 7 - 2 4 7
NUTRITIONALCONTROL OF CORONARYARTERY ATHEROSCLEROSISIN MONKEY
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inducing diet for 3 months followed by 3 and 5 months on the control diet, the coronary artery scores appeared to stabilize or increase only slightly after the time of diet cross-over. Correlation of coronary atherosclerosis with aortic atherosclerosis and the serum cholesterol level A previous study 17 showed that aortic sudanophilia correlated extremely well with the total and esterified cholesterol concentration of the inner layers of the aorta. The correlation coefficients (r) of the coronary artery score with the aortic cholesterol ester and aortic total cholesterol concentrations for 45 pairs of values were 0.399 and 0.547 respectively. The correlation coefficient for the coronary artery score with the terminal plasma cholesterol level was 0.428. The probabilities of these correlations occurring by chance is less than 0.01. Light microscopic observations. Control animals Groups 1, 3, and 6, which were on a corn oil diet during 3, 6, and 8 months, showed only a minimal amount of intimal sudanophilia; these lipids were mainly extracellular, located beneath the endothelium. There were no alterations of the internal elastic lamina, no increase in mucopolysaccharides or calcium deposition. Besides these small foci of subendothelial lipids, several plaques were observed in these groups of control animals. These lesions were lipid-poor and occupied approximately 10-20 % of the arterial lumen (Fig. 2); the internal elastic lamina at the base of the plaque was seldom disrupted; the proliferation consisted mainly of elongated cells (spindle cells) and a snlall increase of PAS-positive material; no calcium precipitation was demonstrated in these lesions. Experimental animals Groups 2, 4, and 7, which were on a high butter diet during 3, 6, and 8 months, showed a clear response to the atherogenic diet with numerous severe arterial lesions. The most prevalent type of atherosclerotic plaque found in these animals was different from the ones observed in the previously described control groups. I t consisted of a large accumulation of lipids in the intima and media of the arteries. The lipid accumulation, which involved only the inner media at 3 months, sometimes occupied almost the whole thickness of the media after 8 months on the high butter diet (Fig. 1). The lesions were observed all along the course of the coronary arteries, but the maximal narrowing always occurred near the base of the heart. No significant cell proliferation was present in these plaques and calcium deposition was minimal. In groups 5 and 8 which were fed the high butter diet for 3 months followed b y 3 and 5 months on the corn oil diet, the lesions remained with microscopic features similar to those of arteries from group 2. The animals of groups 5 and 8 which were transferred to the corn oil diet after 3 months had significantly lower coronary artery disease scores than the groups which were maintained on the butter diet throughout the experimental period. J. Atheroscler. Res., 1968, 8:237-247
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Fig. 1. Severe lipid-sudanophilic deposition (S) can be o b s e r v e d t h r o u g h o u t a cross-frozen s e c t i o n of a c o r o n a r y arteriole. M o n k e y g r o u p 7 (8 m o n t h s h i g h b u t t e r ) . Stained w i t h S u d a n IV a n d h e m a t oxylin. • 350. Fig. 2. L a r g e s p o n t a n e o u s atherosclerotic p l a q u e in a c o r o n a r y a r t e r y of a c o n t r o l a n i m a l . Mild s u d a n o p h i l i a (arrows) is o b s e r v e d in t h e m e d i a a t t h e base of t h e plaque. F r o z e n section, s t a i n e d with S u d a n IV a n d h e m a t o x y l i n . • 220.
j . Atheroscler. Res., 1968, 8 : 2 3 7 247
NUTRITIONAL CONTROL OF CORONARY ARTERY ATHEROSCLEROSIS IN MONKEY
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Fig. 3. Early atherosclerotic lesion in a coronary artery from ~ m o n k e y of group 8 (3 m o n t h s high b u t t e r + 5 m o n t h s corn oil). Subendothelial and medial increase of PAS-positive material (M) is observed; t h e tunica elastics has varying thickness t h r o u g h o u t t h e plaque (arrow). Aralditee m b e d d e d , PAS and 1 % toluidine blue stained. • 1600. Fig. 4. Electron micrograph of a portion of t h e lesion shown in Fig. 3. The absence of lipids is a p p a r e n t in this early plaque; t h e negative image of a calcium particle, lost in the process of sectioning (Ca), is observed close to a site of mild disruption (arrow) of t h e elastics (EL). M: mucopolysaccharides; L: lumen; C: collagen fibers. Araldite-embedded, uranyl a c e t a t e and lead citrate stain. Fixed in glutaraldehyde, postfixed in OsO4. • 7000.
J. Atheroscler. Res., 1968, 8 : 2 3 7 - 2 4 7
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c . A . MARUFFO, O. W. PORTMAN
Coronary arteries of the latter groups (5 and 8) showed, with light microscopy of araldite-embedded myocardium, another type of lesion which was infrequent in the corn oil controls or high butter groups (Fig. 3). This lesion consisted of a thickening of the intima which included large amounts of PAS-positive material in the subendothelial spaces and inner media; these lesions were Sudan negative although minute granules of calcium were detected beneath the internal elastic lamina.
Electron microscopic observations A comprehensive description of the ultrastructure of the normal coronary artery 24, arterioles, and capillaries25, 2~ has been reported in other species; no special features were observed in the present monkeys. In the high butter groups, the alterations observed in the coronary arteries were similar to those described in previous electron microscopic studies of other species u7-29. Large amounts of lipids of varying electron density were observed in endothelial cells, in irregular subendothelial cells (not identified as smooth muscle cells), and in smooth muscle cells of the media; the lipids appeared as homogeneous cytoplasmic inclusions limited by a single agranular membrane. The extraceUular lipids had a higher electron density than the previously described inclusions and evidence of phagocytosis was observed at the periphery of the plaques. These lipid inclusions were coarsely granular and also limited by a single membrane. The alterations of the media in experimental coronary atherosclerosis were severe and consisted mainly of fragmentation of the elastic lamina and cytoplasmic modifications of the smooth muscle cells within the media; they showed lipid deposition with abundant "myelin figures", dilatation of the cisterna of the endoplasmic reticulum and large numbers of free ribosomes. The lesions observed in tile groups of animals (groups 5 and 8) which were transferred from the atherogenic to the basal diet were of two different types. One was the lesion already described in the high butter groups but containing less lipid. The other was a lipid-free, early lesion that was not detected in frozen sections but was frequently observed in thick sections of araldite-embedded myocardium. Cytoplasmic changes in the endothelial cells were relatively subtle and consisted of very few heterogeneous lipid droplets without limiting membranes. There was also an increase in pinocytotic vacuoles and a prominent endoplasmic reticulum; large lysosomes were frequently seen. Tile subendothelial space was considerably enlarged and contained mucopolysaccharides as shown by its positivity to the PAS reaction at light microscopy (araldite-embedded) and by an electron density consistent with, but not definitive for, mucopolysaccharide (Fig. 4); some globular material was also present in this area. The internal elastic membrane was often fragmented and the media at the base of tile plaque showed calcium deposition and proliferation of collagen fibers. The smooth muscle cells did not contain vacuoles; however, they showed a prominent endoplasmic reticulum and free RNA particles.
j . Atherosder. Res., 1968, 8 : 2 3 7 - 2 4 7
N U T R I T I O N A L C O N T R O L OF C O R O N A R Y A R T E R Y A T H E R O S C L E R O S I S I N M O N K E Y
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DISCUSSION
There have been relatively few controlled studies of the relationship of diet to coronary atherosclerosis in primates. The present study demonstrates that the semipurified diet associated with atherosclerosis in squirrel monkeys is also results in increased coronary atherosclerosis. After initiation of atherosclerosis in the coronary artery b y feeding the butter-containing diet, further progression of the process was stopped or partially inhibited by transferring the animals to the corn oil-containing control diet. Actual regression in the extent of lesions was not demonstrated in the diet cross-over group within the duration of these experiments, although the coronary arteries from the cross-over group had certain unique histological characteristics. There are a few descriptions of experimentally-induced atherosclerosis in squirrel monkeys. MIDDLETON AND LOFLAND30 reported a potentiating effect of high protein-high cholesterol intakes on coronary and aortic atherosclerosis in this species. ANDRUS AND PORTMAN17,18 reported that squirrel monkeys resembled other nonhuman primates in their serum cholesterol responses to four different semipurified diets but were more susceptible to induced sudanophilia than the other species tested. MALINOW et al. 14 observed aortic and coronary atherosclerosis induced in a large series of squirrel monkeys by diets which included margarine, butter, and cholesterol. These reports have shown the susceptibility of this species to the induction of experimental aortic and coronary atherosclerosis; however, it is clear in our animals as well as in those studied b y MIDDLETON et al. 31 that naturally-occurring lesions have a quite different pattern from those produced by special conditions such as the feeding of diets which contain high levels of butter and/or cholesterol. In the present study we have observed in the control groups lesions which were well circumscribed and proliferative with a moderate amount of intracellular medial sudanophilia at the base of the lesion; on the other hand, the lesions of the experimental animals were diffuse and had abundant intra- and extracellular lipids. With the electron microscope we have not been able to demonstrate smooth muscle cells within discrete experimental atherosclerotic plaques, as has been reported in aortas of rabbits fed diets with cholesterol for 3 months 32 and in the aortas of dogs fed cholesterol for 4 months 2~, as well as in the aortas of squirrel monkeys I7. Tile types of cells observed in these lesions were characterized b y varying degree of electron density, a round contour, and b y the absence of myofilaments; similar cells have been described in human aorta f a t t y streaks 33 and in experimental aortic atherosclerosis in rabbits a4. I t is possible as PARKER AND ODLAND29 suggest that the cells in these lesions m a y have derived from smooth muscle cells. The alterations of smooth muscle cells in the media adjacent to lesions were marked and in m a n y respects conformed to the description of PARKER AND ODLAND. Nevertheless, ANDRUS AND PORTMAN1~ have also noted the relative absence of musculo-elastic lesions and mature atherosclerosis in the coronary arteries of squirrel monkeys. The early plaques observed in the animals of the "diet cross-over groups" were characterized b y abundant extracellular PAS-positive material and b y dense granular j . Atheroscler. Res., 1968, 8:237-247
246
c . A . MARUFFO, O. W. PORTMAN
c o m p o n e n t s in tile p l a q u e e x t r a c e l lu la r space as r e v e a l e d b y electron microscopy; b o t h o b s e r v a t i o n s v e r y likely indicate th e presence of m u co p o l y sacch ar i d es in increased q u a n t i t i e s or in m a t e r i a l m o r e r e a d il y d e m o n s t r a t e d histologically. D i s r u p t i o n of t h e elastic lamellae, calcium deposition, a n d collagen fiber proliferation were o t h e r comp o n e n t s of these early lesions. T h e s e v e r i t y of c o r o n a r y atherosclerosis in m o n k e y s which were r e t u r n e d to t h e c o n t r o l diets after h a v i n g been fed t h e high b u t t e r diet was essentially stabilized b y t h e diet cross-over. Monkeys which were fed t h e atherogenic diet for the full e x p e r i m e n t a l period (8 months) h a d progressively more severe c o r o n a r y atherosclerosis w i t h time. T h e r e was on t h e o th e r h a n d no evidence of a decrease in t h e e x t e n t of atherosclerosis d u r i n g 3 or 5 m o n t h s after cross-over to th e basal diet. I t seems desirable to i n v e s t i g a t e longer periods of feeding t h e lesion-inducing diet an d p a r t i c u l a r l y longer i n t e r v a l s on t he basal diet in these cross-over e x p e r i m e n t s . ACKNOWLEDGEMENTS This s t u d y was s u p p o r t e d b y a g r a n t - i n - a i d from t h e N a t i o n a l I n s t i t u t e s of H e a l t h ( H E 09744), Bethesda, Md. P u b l i c a t i o n No. 295 from the Oregon R e g i o n a l P r i m a t e R e s e a r c h Center, s u p p o r t e d in p a r t b y G r a n t No. F R 00163 f r o m t h e N a t i o n a l I n s t i t u t e s of H e a l t h .
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(Papio ursinus) with special reference to age and endocrine status, S. Aft. J. reed. Sci., 1960, 25 : 59. 3 RINEHART, J. F. AND L. D. GREENBERG, Arteriosclerotic lesions in pyridoxine deficient monkeys, Fed. Proc., 1948, 7: 278. 4 RINEHART, J. F. AND L. D. GREENBERG, Arteriosclerotic lesions in pyridoxine deficient monkeys, Amer. J. Path., 1949, 25: 481. 5 RINEHART, J. F. AND L. D. GREENBERG, Pathogenesis of experimental arteriosclerosis in pyridoxine deficiency, Arch. Path., 1951, 51: 12. 6 RINEHART, J. F. AND L. D. GREENBERG, Vitamin B deficiency in the rhesus monkey. With particular reference to the occurrence of atherosclerosis, dental caries, and hepatic cirrhosis, Amer. J. clin. Nutr., 1956, 4: 318. 7 VITALE,J. J., H. VELEZ,C. GUZMANANDP. CORREA,Magnesium deficiency in the cebus monkey, Circulat. Res., 1963, 12: 642. 8 MANN, G. V., S. B. ANDRUS,A. MCNALLYAND F. J. STARE, Experimental atherosclerosis in cebus monkeys, J. exp. Ned., 1953, 98: 195. 9 Cox, G. E., C. B. TAYLOR, L. G. COX AND M. A. COUNTS,Atherosclerosis in rhesus monkeys. Part 1 (Hypercholesterolemia induced by dietary fat and cholesterol), Arch. Path., 1958, 66: 32. 10 MANN, G. V. AND S. B. ANDRUS, Xanthomatosis and atherosclerosis produced by diet in an adult rhesus monkey, J. Lab. din. Ned., 1956, 48: 533. 11 STARE, F. J., S. B. ANDRUSAND O. W. PORTMAN, Primates in medical research with special reference to New World monkeys. In: D. E. PICKERING(Ed.), Research with Primates, Tektronix Foundation, Beaverton, Oreg., 1962, pp. 59-66. 12 TAYLOR, C. B., G. E. COX, P. MANALO-ESTRELLAAND J. SOUTHWORTH,Atherosclerosis in rhesus monkeys. Part 2 (Arterial lesions associated with hypercholesteremia induced by dietary fat and cholesterol), Arch. Path., 1962, 74: 16. J. Athe~oscler. Res., 1968, 8:237-247
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(Marked diet-induced hypercholesteremia with xanthomatosis and severe atherosclerosis), Arch. Path., 1963, 76: 239. 14 MALINOW, M. R., C. A. MARUFFO AND A. M. PERLEY, Experimental atherosclerosis in squirrel monkeys (Saimiri sciurea), J. Path. Bact., 1966, 92: 491. 15 WISSLER, R. W., L. E. FRAZIER, R. H. HUGHES AND R. A. RASMUSSEN, Atherogenesis in the cebus monkey. Part 1 (A comparison of three food fats under controlled dietary conditions), Arch. Path., 1962, 74: 312. 16 PORTMAN, O. W., M. ALEXANDER AND C. A. MARUFFO, Nutritional control of arterial lipid composition in squirrel monkeys. Major ester classes and types of phospholipids, J. Nutr., 1967, 91: 35. 17 ANDROS, S. B. AND O. W. PORTMAN, Comparative studies of spontaneous and experimental atherosclerosis in primates. In: R. N. T-W-FIENNES (Ed.), Some Recent Developments in Comparative Medicine, Academic Press, London, 1966, pp. 161-177. 18 PORTMAN, O. W. AND S. B. ANDRUS, Comparative evaluation of three species of New World monkeys for studies of dietary factors, tissue lipids and atherogenesis, J. Nutr., 1965, 87: 429. 19 HEGSTED, D. M., R. C. MILLS, C. A. ]~LVEHJEM AND E. n. HART, Choline in the nutrition of chicks, J. biol. Chem., 1941, 138: 459. 20 MALINOW, M. R., J. A. MOGUILEVSKY, ]3. E. LEMA, G. E. BUR AND A. ERENFRYD, T e s t i c u l a r action and structural relationships of compounds with antiatherogenic activity, Circular. Res., 1961, 9: 204. 21 MANUAL OF HISTOLOGIC AND SPECIAL STAINING TECHNIQUES, Armed Forces Institute of Pathology, Washington, D.C., 1957. 22 SABATINI, D. D., K. BENSCH AND R. J. ]3ARRNETT, Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation, J. Cell Biol., 1963, 17: 19. 23 TRUMP, B. F., E. A. SMUCKLER AND E. P. BENDITT, A method for staining epoxy sections for light microscopy, J. Ultrastruct. Res., 1961, 5: 343. 24 PARKER, F., An electron microscope study of coronary arteries, Amer. J. Anat., 1958, 103: 247. 25 MOORE, D. H. AND ]-I. RUSKA, The fine structure of capillaries and small arteries, J. biophys, biochem. Cytol., 1957, 3: 457. 26 FRENCH, J. E., Endothelial structure and function. In: R. J. JONES (Ed.), Evolution of the Atherosclerotic Plaque, Part l, Univ. Chicago Press, Chicago, 1963, pp. 15-33. 27 GEER, J. C., Fine structure of canine experimental atherosclerosis, A mer. J. Path., 1965, 47 : 241. 28 PARKER, F. AND G. F. ODLAND, A correlative histochemieal, biochemical and electron microscopic study of experimental atherosclerosis in the rabbit aorta with speeiai reference to the myo-intimal cell, Amer. J. Path., 1966, 48: 197. 29 PARKER, F. AND G. F. ODLAND, A light microscopic, histochemical and electron microscopic study of experimental atherosclerosis in rabbit coronary artery and a comparison with rabbit aorta atherosclerosis, Amer. J. Path., 1966, 48: 451. 30 MIDDLETON, C. C., AND H. B. LOFLAND, Aggravation of atherosclerosis in squirrel monkeys (Saimiri sciurea) by diet, Fed. Proc., 1965, 24: 311. 31 MIDDLETON, C. C., T. B. CLARKSON, H. ]3. LOFLAND AND R. W. PRICHARD, Atherosclerosis in the squirrel monkey. Naturally occurring lesions of the aorta and coronary arteries, Arch. Path., 1964, 78: 16. 32 PARKER, F., An electron microscopic study of experimental atherosclerosis, Amer. J. Path., 1960, 36: 19. 33 ]3ALIS, J. U., M. D. HAUST AND R. i~. MORE, Electron-microscopic studies in human atherosclerosis. Cellular elements in aortic fatty streaks, Exp. tool. Path., 1964, 3:511. 34 STILL, W. J. S., An electron microscope study of cholesterol atherosclerosis in the rabbit, Exp. tool. Path., 1963, 2: 491.
J. Atheroscler. Res., 1968, 8:237-247
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
N U T R I T I O N A L CONTROL OF CORONARY A R T E R Y A T H E R O S C L E R O S I S IN T H E S Q U I R R E L M O N K E Y
C. A. MARUFFO AND O. W. PORTMAN Departments of Pathology and Primate Nutrition, Oregon Regional Primate Research Center, Beaverton, and Departments of Pathology and Biochemistry, University of Oregon Medical School, Portland, Oreg. ( U.S.d.)
(Revised, received August 23rd, 1967)
SUMMARY
A study was made of the coronary arteries from squirrel monkeys which were fed one of two diets, one which induced aortic atherosclerosis and one which did not, for 3, 6, and 8 months. A third series of monkeys was fed the atherogenic diet for 3 months followed b y 3 or 5 months on the control diet. Atherosclerotic changes consisting of extensive intra- and extracellular lipid accumulation in the intima and media without significant cell proliferation occurred in the coronary arteries of squirrel monkeys after various intervals on a diet containing butter plus additional cholesterol. Correlations of coronary artery lesions with the terminal plasma cholesterol and aorta and esterified cholesterol concentrations were statistically significant. In animals transferred from the atherogenic diet to the basal diet, a lesion characteristic of the early plaque was seen: lipid-poor and rich in PAS-positive material. Muscle cells of the media at the base of the plaque showed ultrastructural alterations. Cytoplasmic changes in the endothelial cells were relatively subtle: a few heterogeneous lipid droplets, an increase in pinocytotic vacuoles, a prominent endoplasmic reticulum and large lysosomes. Fragmentation of the elastic lamellae and calcium deposition in the inner media were often observed.
INTRODUCTION
Numerous experiments have been performed in monkeys in order to gain an insight into the pathogenesis of atherosclerosis. Although some of the experimentallyinduced lesions cannot be considered identical to their naturally-occurring counterparts in humans, studies of experimental atherosclerosis have advanced our knowledge and understanding of the development of the condition in man. J. Atheroscler. Res., 1968, 8:237-247
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c.A.
M A R U F F O , O. W. P O R T M A N
Several different procedures have been used to obtain arterial lesions in primates: endocrine manipulationsa, 2, pyridoxine deficiency3-6, magnesium deficiency7, and high cholesterol and/or high fat intakes s-15. We have recently described the sequence of early nutritionally induced changes in the composition of the major lipid classes and subclasses of phospholipids from aortas of squirrel monkeys and the potential for regression of lipid-rich lesions with dietary alterations a6. This communication describes a histological study of coronary atherosclerosis induced in those squirrel monkeys as well as the ultrastructural detail of the arterial lesions. The effect of diet on the induction and progression, as well as on the regression, of coronary artery lesions is described. MATERIALS AND METHODS
Eighty-seven sexually mature and apparently healthy female squirrel monkeys were used. A detailed report of the experimental procedures has already been presentedl6; a brief summary of those procedures follows. These animals were obtained from two sources. One importer obtained Brazilian squirrel monkeys (Saimiri madeirae juruanus) from a collecting station in Leticia, Colombia; the other obtained squirrel monkeys (Saimiri boliviensis nigric@s) from Iquitos, Peru. In all of the experiments the animals from each origin were evenly distributed between groups. Although squirrel monkeys from both sources were similar in many of the measurements made, the Brazilian squirrels appeared to be somewhat more susceptible to dietary increases in arterial lipids and also healthier than the squirrel monkeys from Peru. They were quarantined at the Oregon Regional Primate Research Center for 60 days, treated with antihelminthics, and X-rayed. Hematological studies and tuberculin testing were repeated at varying intervals.
Experimental diets used The two semipurified diets used most extensively produced the fewest and the most aortic sudanophilic lesions during earlier studies of squirrel monkeysaT, 18. The first of the diets provided 8% (16% of calories) corn oil and contained no added cholesterol; the second contained 25.2% butter (45% of calories) and contained cholesterol at 0.1 g/kcal. Casein was supplied at 20% of the diet and a salt mixture at 4% (ref. 19). The following quantities of vitamins were supplied per kg of diet: vitamin A acetate 12,500 units, a-tocopherol 0.1 g, crystalline vitamin Da 100 #g (4000 units), menadione 0.04 g, ascorbic acid 0.5 g, inositol 1.0 g, choline chloride 5.0 g, niacin 0.049 g, riboflavin 0.01 g, thiamine 0.01 g, pyridoxine 0.01 g, calcium pantothenate 0.03 g, biotin 0.2 rag, folie acid 1.0 rag, and vitamin B12 0.02 rag. The diets were made to 100~ by the addition of sucrose.
Preparation of arterial tissue The animals were sacrificed at three different intervals. Usually 5 animals from
J. Alheroscler. Res., 1968, 8:237 247
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239
each group were available at each sacrifice period. The dietary treatments and durations are indicated in Table 1. At 3 months a group of control animals (corn oil) and a group of high butter were sacrificed. At 6 months three groups of animals were used: one control (corn oil), one high butter, and a third group composed of animals which had been on the high butter diet for the first 3 months and had transferred to the corn oil (control) diet for the last 3 months before the sacrifice. At 8 months,the same experimental pattern was repeated: a control group (corn oil), a high butter group, and a group on the high butter diet during the first 3 months and on the control diet (corn oil) during the last 5 months before termination of the experiment. TABLE
1
EXPERIMENTAL DIET SERIES
Group
Diet
Control, 3 months corn oil 3 months high butter Control, 6 months corn oil 6 months high butter 3 months high butter + 3 months corn oil Control, 8 months corn oil 8 months high butter 3 months high butter + 5 months corn oil
The monkeys were sacrificed after Sernylan (phencyclidine (1-(1-phenylcyclohexyl) piperidine hydrochloride)) was administered by opening the thoracic and ab dominal cavities with a mento-pubic incision and withdrawing blood from the abdominal aorta at its bifurcation. A complete autopsy was performed on each animal. The heart was fixed in 10 % buffered formalin and cut into 5 transverse blocks parallel to the A-V groove. From each block frozen sections (10 20 # thick) were stained with Sudan IV and hematoxylin in order to grade microscopically the coronary atherosclerotic lesions, which were classified according to the extent of sundanophilia and cell proliferation (plaques). For each monkey, all graded arteries (0.5-3.0 in ascending order of severity of involvement) were added and results expressed as mean coronary score per 100 coronary arterial cross-sections 2~ The remaining portions of the blocks were embedded in paraffin, and several sections were stained with the following techniques21: hematoxylin and eosin, Gomori's aldehyde fuchsin for elastic fibers, periodic acid-Schiff (PAS) preceded by treatment of hydrated sections for 10 rain with saliva, and Von Kossa for calcium. For light microscopy of thin sections and for electron microscopy (6 animals), tissue from the anterior aspect of the heart (below the A-V groove) was cut in crosssection under cold phosphate-buffered osmium tetroxide into 1-2 mm cubes which were fixed at approximately 4~ for 60-90 min. In addition, several 1 mm slices of myocardium were fixed in 2 % glutaraldehyde 22 for 90 rain, washed overnight in phosphate buffer (sucrose 4.5 %), cut into 1 mm cubes and postfixed in osmiumtetroxJ. Atheroscler. Res., 1968, 8:237-247
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c . A . MARUFFO, O. W. PORTMAN
ide for 60-90 min at approximately 4~ The blocks were then dehydrated through graded alcohols and embedded in araldite; sections were cut with glass and diamond knives on an L K B Ultrotome. From each block several 0.5-1/z sections were mounted on glass slides, stained with 1% toluidine blue 23 and examined with a light microscope. Ultrathin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with RCA-3F and Philips EM200 electron microscopes. RESULTS
General observations All the animals were regarded as young adults of comparable age, as estimated by long bone development, eruption of the third molar, and wear of the canine teeth. The body weights were similar in all groups of animals 16 and remained essentially static throughout the experiment. No skeletal alterations were observed. Only 3 cases of peritoneal filariasis (achanthocheilonema) were found. A few animals of the high butter group showed mild f a t t y metamorphosis of the liver. Coronary atherosclerosis. Effect of diet and time on extent of lesions The results of the grading of coronary atherosclerotic lesions and the statistical tests of significance are presented in Table 2. After 3 months the monkeys fed the butter-containing diet had significantly higher coronary artery disease scores than monkeys of the control group. After 6 and 8 months on these dietary regimens the extent of coronary artery lesions was much more severe in the monkeys fed the butter diet than was the case at 3 months. In groups 5 and 8, which were fed the lesion-
TABLE 2 THE EFFECT
OF DIET AND DURATION
OF FEEDING
ON THE SEVERITY
OF CORONARY
ATHEROSCLEROSIS
See t e x t for m e a n i n g of the c o r o n a r y score which is equivalent to the p r o d u c t of the p r o p o r t i o n of involved c o r o n a r y a r t e r y sections and the m e a n e x t e n t of involvement. Meanings of g r o u p designations are indicated in Table 1. Group
No. of arterial crosssections observed
Mean coronary score a
~ S.E. mean
1 2
462 530
0 12
0 2.2
4 5 6 7 8
438 536 513 484 395
52 10 2 61 23
12.8 1.2 1.6 3.0 4.2
Significance b
i
I
] ]
[
a The m e a n c o r o n a r y score for 6 squirrel m o n k e y s m a i n t a i n e d on a diet of P u r i n a M o n k e y Chow for 2 m o n t h s and sacrificed before initiation of t h e feeding e x p e r i m e n t was 2 ~ 1.3. b G r o u p m e a n s which are connected b y a line are statistically different ( S t u d e n t ' s t test) at the P = 0.025 level or less. j . Atheroscler. Res., 1968, 8 : 2 3 7 - 2 4 7
NUTRITIONALCONTROL OF CORONARYARTERY ATHEROSCLEROSISIN MONKEY
241
inducing diet for 3 months followed by 3 and 5 months on the control diet, the coronary artery scores appeared to stabilize or increase only slightly after the time of diet cross-over. Correlation of coronary atherosclerosis with aortic atherosclerosis and the serum cholesterol level A previous study 17 showed that aortic sudanophilia correlated extremely well with the total and esterified cholesterol concentration of the inner layers of the aorta. The correlation coefficients (r) of the coronary artery score with the aortic cholesterol ester and aortic total cholesterol concentrations for 45 pairs of values were 0.399 and 0.547 respectively. The correlation coefficient for the coronary artery score with the terminal plasma cholesterol level was 0.428. The probabilities of these correlations occurring by chance is less than 0.01. Light microscopic observations. Control animals Groups 1, 3, and 6, which were on a corn oil diet during 3, 6, and 8 months, showed only a minimal amount of intimal sudanophilia; these lipids were mainly extracellular, located beneath the endothelium. There were no alterations of the internal elastic lamina, no increase in mucopolysaccharides or calcium deposition. Besides these small foci of subendothelial lipids, several plaques were observed in these groups of control animals. These lesions were lipid-poor and occupied approximately 10-20 % of the arterial lumen (Fig. 2); the internal elastic lamina at the base of the plaque was seldom disrupted; the proliferation consisted mainly of elongated cells (spindle cells) and a snlall increase of PAS-positive material; no calcium precipitation was demonstrated in these lesions. Experimental animals Groups 2, 4, and 7, which were on a high butter diet during 3, 6, and 8 months, showed a clear response to the atherogenic diet with numerous severe arterial lesions. The most prevalent type of atherosclerotic plaque found in these animals was different from the ones observed in the previously described control groups. I t consisted of a large accumulation of lipids in the intima and media of the arteries. The lipid accumulation, which involved only the inner media at 3 months, sometimes occupied almost the whole thickness of the media after 8 months on the high butter diet (Fig. 1). The lesions were observed all along the course of the coronary arteries, but the maximal narrowing always occurred near the base of the heart. No significant cell proliferation was present in these plaques and calcium deposition was minimal. In groups 5 and 8 which were fed the high butter diet for 3 months followed b y 3 and 5 months on the corn oil diet, the lesions remained with microscopic features similar to those of arteries from group 2. The animals of groups 5 and 8 which were transferred to the corn oil diet after 3 months had significantly lower coronary artery disease scores than the groups which were maintained on the butter diet throughout the experimental period. J. Atheroscler. Res., 1968, 8:237-247
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c . A . MARUFFO, O. W. PORTMAN
Fig. 1. Severe lipid-sudanophilic deposition (S) can be o b s e r v e d t h r o u g h o u t a cross-frozen s e c t i o n of a c o r o n a r y arteriole. M o n k e y g r o u p 7 (8 m o n t h s h i g h b u t t e r ) . Stained w i t h S u d a n IV a n d h e m a t oxylin. • 350. Fig. 2. L a r g e s p o n t a n e o u s atherosclerotic p l a q u e in a c o r o n a r y a r t e r y of a c o n t r o l a n i m a l . Mild s u d a n o p h i l i a (arrows) is o b s e r v e d in t h e m e d i a a t t h e base of t h e plaque. F r o z e n section, s t a i n e d with S u d a n IV a n d h e m a t o x y l i n . • 220.
j . Atheroscler. Res., 1968, 8 : 2 3 7 247
NUTRITIONAL CONTROL OF CORONARY ARTERY ATHEROSCLEROSIS IN MONKEY
243
Fig. 3. Early atherosclerotic lesion in a coronary artery from ~ m o n k e y of group 8 (3 m o n t h s high b u t t e r + 5 m o n t h s corn oil). Subendothelial and medial increase of PAS-positive material (M) is observed; t h e tunica elastics has varying thickness t h r o u g h o u t t h e plaque (arrow). Aralditee m b e d d e d , PAS and 1 % toluidine blue stained. • 1600. Fig. 4. Electron micrograph of a portion of t h e lesion shown in Fig. 3. The absence of lipids is a p p a r e n t in this early plaque; t h e negative image of a calcium particle, lost in the process of sectioning (Ca), is observed close to a site of mild disruption (arrow) of t h e elastics (EL). M: mucopolysaccharides; L: lumen; C: collagen fibers. Araldite-embedded, uranyl a c e t a t e and lead citrate stain. Fixed in glutaraldehyde, postfixed in OsO4. • 7000.
J. Atheroscler. Res., 1968, 8 : 2 3 7 - 2 4 7
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c . A . MARUFFO, O. W. PORTMAN
Coronary arteries of the latter groups (5 and 8) showed, with light microscopy of araldite-embedded myocardium, another type of lesion which was infrequent in the corn oil controls or high butter groups (Fig. 3). This lesion consisted of a thickening of the intima which included large amounts of PAS-positive material in the subendothelial spaces and inner media; these lesions were Sudan negative although minute granules of calcium were detected beneath the internal elastic lamina.
Electron microscopic observations A comprehensive description of the ultrastructure of the normal coronary artery 24, arterioles, and capillaries25, 2~ has been reported in other species; no special features were observed in the present monkeys. In the high butter groups, the alterations observed in the coronary arteries were similar to those described in previous electron microscopic studies of other species u7-29. Large amounts of lipids of varying electron density were observed in endothelial cells, in irregular subendothelial cells (not identified as smooth muscle cells), and in smooth muscle cells of the media; the lipids appeared as homogeneous cytoplasmic inclusions limited by a single agranular membrane. The extraceUular lipids had a higher electron density than the previously described inclusions and evidence of phagocytosis was observed at the periphery of the plaques. These lipid inclusions were coarsely granular and also limited by a single membrane. The alterations of the media in experimental coronary atherosclerosis were severe and consisted mainly of fragmentation of the elastic lamina and cytoplasmic modifications of the smooth muscle cells within the media; they showed lipid deposition with abundant "myelin figures", dilatation of the cisterna of the endoplasmic reticulum and large numbers of free ribosomes. The lesions observed in tile groups of animals (groups 5 and 8) which were transferred from the atherogenic to the basal diet were of two different types. One was the lesion already described in the high butter groups but containing less lipid. The other was a lipid-free, early lesion that was not detected in frozen sections but was frequently observed in thick sections of araldite-embedded myocardium. Cytoplasmic changes in the endothelial cells were relatively subtle and consisted of very few heterogeneous lipid droplets without limiting membranes. There was also an increase in pinocytotic vacuoles and a prominent endoplasmic reticulum; large lysosomes were frequently seen. Tile subendothelial space was considerably enlarged and contained mucopolysaccharides as shown by its positivity to the PAS reaction at light microscopy (araldite-embedded) and by an electron density consistent with, but not definitive for, mucopolysaccharide (Fig. 4); some globular material was also present in this area. The internal elastic membrane was often fragmented and the media at the base of tile plaque showed calcium deposition and proliferation of collagen fibers. The smooth muscle cells did not contain vacuoles; however, they showed a prominent endoplasmic reticulum and free RNA particles.
j . Atherosder. Res., 1968, 8 : 2 3 7 - 2 4 7
N U T R I T I O N A L C O N T R O L OF C O R O N A R Y A R T E R Y A T H E R O S C L E R O S I S I N M O N K E Y
245
DISCUSSION
There have been relatively few controlled studies of the relationship of diet to coronary atherosclerosis in primates. The present study demonstrates that the semipurified diet associated with atherosclerosis in squirrel monkeys is also results in increased coronary atherosclerosis. After initiation of atherosclerosis in the coronary artery b y feeding the butter-containing diet, further progression of the process was stopped or partially inhibited by transferring the animals to the corn oil-containing control diet. Actual regression in the extent of lesions was not demonstrated in the diet cross-over group within the duration of these experiments, although the coronary arteries from the cross-over group had certain unique histological characteristics. There are a few descriptions of experimentally-induced atherosclerosis in squirrel monkeys. MIDDLETON AND LOFLAND30 reported a potentiating effect of high protein-high cholesterol intakes on coronary and aortic atherosclerosis in this species. ANDRUS AND PORTMAN17,18 reported that squirrel monkeys resembled other nonhuman primates in their serum cholesterol responses to four different semipurified diets but were more susceptible to induced sudanophilia than the other species tested. MALINOW et al. 14 observed aortic and coronary atherosclerosis induced in a large series of squirrel monkeys by diets which included margarine, butter, and cholesterol. These reports have shown the susceptibility of this species to the induction of experimental aortic and coronary atherosclerosis; however, it is clear in our animals as well as in those studied b y MIDDLETON et al. 31 that naturally-occurring lesions have a quite different pattern from those produced by special conditions such as the feeding of diets which contain high levels of butter and/or cholesterol. In the present study we have observed in the control groups lesions which were well circumscribed and proliferative with a moderate amount of intracellular medial sudanophilia at the base of the lesion; on the other hand, the lesions of the experimental animals were diffuse and had abundant intra- and extracellular lipids. With the electron microscope we have not been able to demonstrate smooth muscle cells within discrete experimental atherosclerotic plaques, as has been reported in aortas of rabbits fed diets with cholesterol for 3 months 32 and in the aortas of dogs fed cholesterol for 4 months 2~, as well as in the aortas of squirrel monkeys I7. Tile types of cells observed in these lesions were characterized b y varying degree of electron density, a round contour, and b y the absence of myofilaments; similar cells have been described in human aorta f a t t y streaks 33 and in experimental aortic atherosclerosis in rabbits a4. I t is possible as PARKER AND ODLAND29 suggest that the cells in these lesions m a y have derived from smooth muscle cells. The alterations of smooth muscle cells in the media adjacent to lesions were marked and in m a n y respects conformed to the description of PARKER AND ODLAND. Nevertheless, ANDRUS AND PORTMAN1~ have also noted the relative absence of musculo-elastic lesions and mature atherosclerosis in the coronary arteries of squirrel monkeys. The early plaques observed in the animals of the "diet cross-over groups" were characterized b y abundant extracellular PAS-positive material and b y dense granular j . Atheroscler. Res., 1968, 8:237-247
246
c . A . MARUFFO, O. W. PORTMAN
c o m p o n e n t s in tile p l a q u e e x t r a c e l lu la r space as r e v e a l e d b y electron microscopy; b o t h o b s e r v a t i o n s v e r y likely indicate th e presence of m u co p o l y sacch ar i d es in increased q u a n t i t i e s or in m a t e r i a l m o r e r e a d il y d e m o n s t r a t e d histologically. D i s r u p t i o n of t h e elastic lamellae, calcium deposition, a n d collagen fiber proliferation were o t h e r comp o n e n t s of these early lesions. T h e s e v e r i t y of c o r o n a r y atherosclerosis in m o n k e y s which were r e t u r n e d to t h e c o n t r o l diets after h a v i n g been fed t h e high b u t t e r diet was essentially stabilized b y t h e diet cross-over. Monkeys which were fed t h e atherogenic diet for the full e x p e r i m e n t a l period (8 months) h a d progressively more severe c o r o n a r y atherosclerosis w i t h time. T h e r e was on t h e o th e r h a n d no evidence of a decrease in t h e e x t e n t of atherosclerosis d u r i n g 3 or 5 m o n t h s after cross-over to th e basal diet. I t seems desirable to i n v e s t i g a t e longer periods of feeding t h e lesion-inducing diet an d p a r t i c u l a r l y longer i n t e r v a l s on t he basal diet in these cross-over e x p e r i m e n t s . ACKNOWLEDGEMENTS This s t u d y was s u p p o r t e d b y a g r a n t - i n - a i d from t h e N a t i o n a l I n s t i t u t e s of H e a l t h ( H E 09744), Bethesda, Md. P u b l i c a t i o n No. 295 from the Oregon R e g i o n a l P r i m a t e R e s e a r c h Center, s u p p o r t e d in p a r t b y G r a n t No. F R 00163 f r o m t h e N a t i o n a l I n s t i t u t e s of H e a l t h .
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