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Age-associated ultrastructural changes in the aortic intima of rats with diet-induced hypercholesterolemia
101
Atherosclerosis, 79 (1989) 101-111
Publishers Ireland, Ltd.
Elsevier Scientific
ATHERO
04371
Age-associated ultrastructural changes in the aortic intima of rats with diet-induced hypercholesterolemia Hiroaki Nakamura
‘, Naotaka Izumiyama 2, Ken-i& and Kohichiro Ohtsubo 2
Nakamura
2
’Laborator?, of Electron Microscopy, Saitama Medical School, Saitama 350-04 (Japan). and .’Department of Clinical Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173 (Japan) (Received 3 February. 1989) (Revised. received 22 May, 1989) (Accepted 24 May, 1989)
Summary The ultrastructure of the aortic intima and serum lipid levels in Fischer 344 rats were examined at the ages of 12, 18 and 24 months after feeding the animals an atherogenic diet (2% cholesterol, 0.25% sodium cholate, 5% beef fat) for 6 months. Structural changes in the intima were noticeable only at the age of 24 months. In control rats, the endothelial cells were irregular in shape and each had a well-developed Golgi complex and a few lipid droplets. Simultaneously, reticular, basal lamina-like material and electron-dense granules of extracellular liposomes accumulated in the subendothelium. In fat-fed rats, these structural changes were more conspicuous in association with hypercholesterolemia, and numerous monocytes with lipid droplets were attached to the endothelium, occasionally invading into the subendothelium. Slight foam cell lesions were evident in the intima. The finding that older rats were more susceptible to the atherogenic diet suggests that atherogenesis in the rat is promoted by intrinsic age-associated changes in the aortic intima.
Key words:
Aging;
Atherosclerosis;
Hypercholesterolemia;
Introduction Hypercholesterolemia factor associated with
is the most important risk atherosclerosis [l]. Using
Correspondence to: Hiroaki Nakamura, Ph.D., Laboratory of Electron Microscopy, Saitama Medical School, 38 Morohongo, Moroyama-cho, Iruma-gun, Saitama 350-04, Japan. 0021-9150/89/$03.50
Q 1989 Elsevier Scientific
Publishers
Ireland,
Aortic
intima;
Rat;
Electron
microscopy
several animal models, it has been demonstrated that diet-induced hypercholesterolemia, which may produce primary endothelial injury, promotes monocyte adhesion to the endothelium, followed by their invasion into the intima and modulation into lipid-laden macrophages, the accumulation of which finally forms fatty streaks in the aorta [2-61. On the other hand, it is generally recognized from histopathological studies of human arteries that the extent and severity of atherosclerosis are Ltd.
102 closely associated with aging [7]. However, few experimental studies have directly examined whether there is a relationship between the progression of atherosclerosis and aging [S]. For example, the rabbit is widely used in studies of diet-induced atherosclerosis because of the relative rapidity of lesion formation in this animal [6]. However, in the rabbit, the association of atherogenesis with aging is difficult to study, because no reliable life table data are available [9]. In contrast, the rat has been relatively well examined throughout its life span [lo] with regard to age-associated changes in the aortic wall structure [ll-141 as well as serum lipid levels [10,14-161. There have been many ultrastructural investigations on the pathogenesis of atherosclerotic lesions in rats fed a cholesterol diet [17-221, but few have referred to the relationship between aging and the development of atherosclerosis. The occurrence of spontaneous aortic atherosclerosis has been found in old rats by light microscopy in association with an elevated serum cholesterol concentration [23, 241. In this study, we report that aged rats of the Fischer 344 strain fed an atherogenic diet, showed marked differences from younger rats in the ultraTABLE
structure of the aortic intima as well as in serum lipid concentration. Materials and methods Male rats of the Fischer 344 strain, aged 6, 12 and 18 months, purchased from Charles River Inc. (Japan) were used (Table 1). All rats were housed 2 or 3 per cage (plastic cage with sawdust bedding), and kept in a temperature-controlled clean room (21-23OC) with free access to their respective diets and water. They were fed either MF pellet (Oriental Yeast Co., Japan) as a control group, or the same basal diet containing 2% cholesterol, 0.25% sodium cholate and 5% beef fat as a fat-fed group. No thiouracil was added. After 1 and 6 months of feeding, blood was withdrawn into heparinized syringes from severed tail vessels under light ether anesthesia after an overnight fast. Serum was separated by centrifugation at 2000 rpm for 10 min and stored at - 70°C until assay. Total cholesterol and triglyceride concentrations in serum were measured spectrophotometrically using Sigma kits Nos. 325 and 405. After 6 months of feeding, rats aged 12, 18 or 24 months were anesthetised with ether and killed
1
CHANGES IN MEAN BODY WEIGHT, IN THE FISCHER 344 RATS
SERUM TOTAL CHOLESTEROL
AND TRIGLYCERIDE
LEVELS WITH AGING
All values are means k SD. Age (months)
Experimental
Body weight
Total cholesterol
Triglyceride
group (n)
(9)
(mg/dl)
(mg/dI)
6+1* 6 12+1 6 18+1 6
Control (4) (4) (4) (4) (4) (4)
415.3 f 490.0+ 487.8 + 520.3+ 515.8 f 521.3 +
29.9 32.0 a 8.4 b 3.9 a 26.3 17.6
73.5+ 109.s+ 90.0+ 106.35 113.5* 163.Ok
11.7 9.5 7.3 8.7 15.7 44.6
6+1* 6 12+1 6 18+1 6
Fat-fed (6) (6) (6) (6) (6) (5)
429.3 f 519.0+ 495.2 f 529.0* 521.2 f 515.8 f
17.6 24.1 a 21.7 b 21.8 a 35.8 30.7
140.8+ 200.8 + 161.8 f 247.0 k 236.5 f 1138.85
13.6 14.1 29.6 52.8 53.9 538.6
* a b c
a b a
123.3 + 262.Ok 179.5f 226.3 + 164.3 + 234.3 +
= += = ac b,c a,b.c
167.7 209.3 155.7 286.3 207.2 724.3
a
Age at the start of the experiment plus feeding period. Significant increase from 1 to 6 months in the same age group (P < 0.05). Significantly different from the mean in the younger age group after 1 or 6 months (P < 0.05). Significantly different from the mean in the control at corresponding ages after 1 or 6 months (P < 0.05).
f f f + f f
21.2 77.0 a 16.5 b 70.5 49.7 32.9 45.5 23.9 26.6 44.3 a 57.8 369.9 a
103 by perfusion with 2.5% glutaraldehyde (0.1 M cacodylate buffer, pH 7.2) at a hydrostatic pressure of 120 cm for 20 min. The thoracic aorta was cut transversely into short rings, fixed in the same fresh fixative for 2 h at 4°C rinsed 3 times with the same buffer containing 8% sucrose, post-fixed with 1% osmium tetroxide in the same buffer for 3 h at 4” C, dehydrated in a graded ethanol series, and flat-embedded in Polybed 812. Thick sections (1 pm) were stained with toluidine blue for light microscopy. Ultrathin sections were stained with uranyl acetate followed by lead citrate for transmission electron microscopy. After dehydration, some of the above samples were critical-point dried, and ion sputter-coated with platinum for scanning electron microscopy. The number of monocytes attached to the endo-
thelium per 1 mm* was counted at 5 randomly selected areas in 4 rats of each age group. Data including body weight, lipid concentration and monocyte number were analyzed by Student’s t-test. Results Body weight and serum lipids The body weights, total serum cholesterol and triglyceride levels of the control and fat-fed rats are shown in Table 1. The body weights of younger rats at the ages of 6 and 12 months increased significantly from 1 to 6 months in both the control and fat-fed groups, whereas no body weight differences between the control and fat-fed rats were present in any age group. The total cholesterol
Fig. 1. Light micrographs of age-related changes in the aortic intima of rats fed a fatty diet for 6 months. (a), (b) and (c): control rats aged 12. 18 and 24 months; (d), (e) and (f): fat-fed rats at corresponding ages, respectively. Monocytes attach to the endothelium of fat-fed rats aged 18 and 24 months (arrows). x 900.
104 level increased significantly from 1 to 6 months at all ages in both the control and fat-fed groups. Especially at the age of 18 months, the level increased by 49.5 mg/ml in the control rats (average: 163, max.: 195, min.: 112 mg/dl at age 24 months) and by 902.3 mg/ml in the fat-fed rats (average: 1138.8, max.: 1632, min.: 501 mg/dl at age 24 months) after 6 months. Significant differences between the levels in the control and fat-fed groups were found at all ages (Table 1). High triglyceride levels were present only in fat-fed rats at the age of 24 months (Table 1). Light microscopy
With aging, the subendothelial space of the thoracic aorta tended to expand in the control as well as the fat-fed groups (Fig. 1). Monocytes were frequently attached to the endothelium and foam cells were present in the subendothelium of the fat-fed rats at the age of 24 months (Fig. lf), although they were rare at 18 months (Fig. le). The monocytes were filled with lipid droplets at the age of 24 months, but were free of droplets at 18 months. The numbers of these cells varied in the tissue portion or in rats, apparently in associa-
tion with serum total cholesterol levels. However, no atherosclerotic lesions such as fatty streaks and fibrous plaques were apparent in the aorta at any age in either the control or fat-fed rats. Transmission
electron microscopy
Ultrastructural changes with aging were apparent in the thoracic aorta at the age of 24 months. In control rats aged 24 months, the endothelial cells were irregular in shape, and each had a well developed Golgi complex, lysosomes and a few lipid droplets. Basal lamina-like material consisting of a coarse reticulum filled the subendothelium (Fig. 2). Massive clusters of electron-dense granules were present focally near the internal elastic lamina (Fig. 2). In fat-fed rats aged 24 months, the endothelial cells were occasionally swollen, and frequently contained several lipid droplets and a large Golgi complex (Fig. 3). In the subendothelium, variously sized electron-dense granules (similar to those described above) and vesicles accumulated, and the reticular basal lamina-like material was more condensed in the fat-fed rats than in the controls (Fig. 3). Neither collagen fibrils nor elastic fibers were increased in
Fig. 2. The aortic intima of a control rat aged 24 months. End, endothelial cell; BLM, reticular basal lamina-like material; EDG, electron-dense granules; Lp, lipid droplets; Ly, lysosomes; IEL, internal elastic lamina. X 14000.
Fig. 3. The aortic
intima
of a fat-fed rat aged 24 months. End, endothelial cell; Lp, lipid droplets; GC, Golgi reticular basal lamina-like materials; EDG, electron-dense granules. x 1OOCO.
the subendothelium of the fat-fed rats in comparison with those of control rats at any age (Figs. 2 and 3). In fat-fed rats at the age of 24 months, monocytes were frequently attached to endothelial cells containing a few lipid droplets (Fig. 4a) or to them without lipid droplets. The monocytes with various numbers of lysosomes always contained various-sized lipid vacuoles, and extended cell pro jections toward the endothelium (Fig. 4a). The features of these monocytes were consistent with lipid-laden macrophages in blood [2,3]. These lipid-laden macrophages occasionally invaded through endothelial cell junctions into the subendothelium (Fig. 4b). From the examination of serial sections, these invading macrophages appeared to push aside the endothelial cells (Fig. 4b). In fat-fed rats at the age of 24 months, foam cells of various sites containing several amounts of
complex;
BLM,
lipid droplets, were present either singly or in clusters in the subendothelium (Fig. 5a). The endothelium covering these areas was thin but intact, and in some regions covered large foam cells with an extremely thin layer of cytoplasm. Lipid droplets in the foam cells were not membrane bound and appeared less osmiophilic with a continuous osmiophilic rim (Fig. 5a). Large foam cells frequently had membranous globules and deposits within the cytoplasm, suggesting breakdown of the large amorphous lipid droplets (Fig. 5a). Lipidladen macrophages were occasionally present in the subendothelium of the aorta in fat-fed rats aged 18 months (Fig. 5b). The subendothelial cells differed from the foam cells in older rats, because they had more lysosomes and fewer lipid droplets. Scanning electron microscopy The luminal surfaces of the thoracic aorta at the ages of 12 and 18 months in both the control
106
Fig. 4. Monocytes attached to the aortic endothelium of fat-fed rats aged 24 months. (a) Cell with microvilli containing large lipid droplets (Lp) and a small amount of lysosomes (Ly). An endothelial cell contains lipid droplets (arrows). x 6500. (b) Cell with lipid droplets (Lp) invading between endothelial cells (arrows), extending an elongated projection into the subendothelium. x 6500.
Fig. 5. Foam cells in the aortic subendothelium of fat-fed rats. (a) At the age of 24 months, a large foam cell containing variously sized lipid droplets (Lp) is covered at the htminal side by a thin layer of cytoplasm of an endothelial cell (arrows). EDG, electron-dense granules. x 6500. (b) At the age of 18 months, a lipid-laden macrophage contains a small number of lysosomes (Ly) and lipid droplets (Lp). X 10000.
Fig. 6. SEM view of luminal surfaces of the aortae. (a) Endothelial cells of a fat-fed rat aged 18 months are smooth and intact with cell fringes (arrows) and microvilli (Mv). X 10000. (b) Endothelial surface of a control aged 24 months is somewhat swollen, and cell fringes and microvilli are not clear. x 10000.
and fat-fed rats, were covered by smooth and intact endothelial cells with clear cell fringes (Fig. 6a). In the control rats aged 24 months, however, these endothelial cell fringes were irregular and obscure, and occasionally there were small focal areas of the endothelium protruding into the lumen (Fig. 6b). In the fat-fed rats aged 24 months, the endothelial surface was very irregular and widely extruded into the lumen in many parts (Fig. 7). In addition, monocytes with microvilli were frequently attached to the endothelium. However, SEM proved that the endothelium was relatively intact and not denuded of cells (Fig. 7). Focal clusters of small globular protrusions were present on the endothelial cells at 24 months (Fig. 7) suggesting that these were the lipid droplets observed in the endothelial cells by TEM as shown in Fig. 3. Monocytes were spherical to tadpole-like in shape, and present singly or in clusters of 3 to 15 cells on the endothelium. Quantitatively, monocytes adhering to the endothelial cells were
significantly more numerous in fat-fed rats at the age of 24 months as compared with those at the age of 18 months or in the control rats (Table 2). There was no significant difference in monocyte
TABLE
2
CHANGES IN NUMBERS OF MONOCYTES ON THE ENDOTHELIUM WITH AGING OF RATS FED A CONTROL OR A FATTY DIET FOR 6 MONTHS All values areas).
are means+SD
(n = 4 rats,
total
endothelium
n = 20 counted
Experimental
Cell number/mm2
groups (n)
12 months
18 months
24 months
(n = 20)
Control (4) Fat-fed (4)
3.3 + 2.3 3.4*2.1
4.4 f 3.2 9.6 f 4.5 a.b
9.2k4.8 a 28.5 f 9.3 a,b
a Significantly different from the mean at younger age in the same experimental group (P < 0.05). b Significantly different from the mean in the controls at the corresponding age (P < 0.05).
108
Fig. 7. SEM view of a luminal surface of the aorta of a fat fed rat aged 24 months. Endothelial surface is expanded irregularly with small globular protrusions extending into the lumen (arrows). Monocytes with microvilli are present (large arrows). X 14500.
numbers between the controls aged 24 months and the fat-fed rats aged 18 months (P > 0.05). Discussion This study revealed that a fat-rich diet accelerated the initiation of atherosclerotic lesions to the endothrough adhesion of monocytes thelium and their migration into the subendothelium, forming foam cell lesions in the thoracic aorta of 24-month-old rats. The severity and extent of the lesions were correlated with the degree of elevation of serum cholesterol levels. These findings are consistent with those of other experimental studies on atherosclerosis in pigs [2,3], rabbits [6], pigeons [5] and monkeys [4] as well as rats [20,21]. In hypercholesterolemia, monocyte
adhesion to endothelial cells, which has been demonstrated both in vivo [2-6,20,21] and in vitro [25,26], is likely to play an important role in the initiation and evolution of atherosclerosis. However, there have been different views on the morphology of the attached monocytes found in rats fed atherogenic diets. In accordance with our observations, O’Neal et al. [17,1X] and Balint et al. [19] have reported that monocytes containing lipid droplets (termed circulating lipophages) appear in blood along with elevated plasma lipid levels, and migrate from the blood to the subendothelium of the aorta. On the other hand, Clowes et al. [20] and Joris et al. [21] have shown that monocytes without lipid droplets become attached to the endothehal cells, and migrate into the subendothelium, subsequently developing into foam
109 cells of varying sizes in the aortic intima. The mechanisms of monocyte transformation to foam cells have been investigated in several studies [27-301. These studies on experimental animals with hyperlipidemia have suggested that modification of the native LDL by some factors induces the transformation of macrophages into foam cells in vitro. The presence of lipid-laden macrophages in blood, as shown in our study of fat-fed rats, suggests a possibility that macrophage modification has already occurred in hypercholesterolemic blood, and that the modified cells migrate, actively taking up more lipid, to become foam cells in the subendothelium. In our study, no advanced atheromatous lesions such as fatty streaks and fibrous plaques had developed in the aorta of any rat. O’Neal et al. [17,18] have reported similar results in rats fed a high-fat diet on the basis of electron microscopy. Several authors, however, have succeeded in producing fatty streaks in the rat aorta by feeding a diet of cholesterol and sodium cholate along with thiouracil [19-211. Thiouracil is an antithyroid drug, which inhibits the formation of thyroid hormone [33]. When a high-cholesterol diet is given concomitantly with thiouracil, the plasma cholesterol level shows a marked increase to a mean of 1085 mg/dl in Sprague-Dawley rats weighing 300-350 g after 6 months [20], or to a mean of 900 mg/dl in Wistar rats weighing 225-250 g after 336 months [21]. In our study without thiouracil, Fischer 344 rats aged 24 months showed a high level of serum cholesterol with a mean level of 1138.8 mg/dl after feeding with a cholesterol and fat diet for 6 months (Table l), but no fatty streaks occurred in the aorta. An antithyroid drug such as thiouracil can directly damage the elastic fibers of the aortic wall [34]. Thus, factors other than a high plasma lipid level may be necessary for progression of atherosclerotic lesions in rats. Several electron microscopic studies on hypercholesterolemic animals [2,3,5,20,21], apart from ours, have demonstrated that monocyte adhesion occurs on morphologically intact endothelial cells without any evidence of areas denuded of cells. In addition, we found that ultrastructural changes occurred in the aortic intima of aged rats (Figs. 2 and 3). In the control rats aged 24 months, the aortic endothelial cells contained a few lipid
droplets and well developed Golgi complex, and electron-dense granules had accumulated in the subendothelium. Simultaneously, monocyte adhesion to the endothelium was more frequent at the age of 24 months than at 18 months (Table 2). These structural changes were more conspicuous in the fat-fed rats at 24 months, corresponding to the elevation of serum cholesterol. Similar endothelial lipid droplets have been reported in hypercholesterolemic rabbits [6] and rats [17,21], and also in normal rats aged 2-3 years [ll]. Joris et al. [21] have noted that myelin figures arising from the endothelial surface are commonly present in the aorta of rats fed a cholesterol diet. However, we did not find any such structure on the endothelium in this study. Electron-dense granules in the aortic subendothelium are reported to be extracellular liposomes containing apolipoprotein B and unesterified cholesterol [31]. They increase in rabbits with diet-induced hyperlipidemia [31,32] and in normal rats with aging [11,12]. Age-dependent changes in total cholesterol levels in serum have been reported for several species, including man and rat. Generally in normal rats, body weight increases rapidly until 12 months of age and remains stable thereafter, whereas total cholesterol levels gradually increase until the age of 12 months, then increase steeply [10,15,16,22]. Our data for body weight and total cholesterol in the control rats were similar to those in the above reports (Table 1). High levels of total cholesterol in serum only developed at the age of 24 months in fat-fed rats (Table 2) whereas the younger rats tolerated the fatty diet. Serum cholesterol levels may therefore rise in association with several factors, such as alterations of hepatic lipid metabolism [lo] and intestinal lipid absorption [35] with aging. Hypercholesterolemia may have caused the ultrastructural changes in the aortic intima seen in our study. However, there are a few reports suggesting that diet-induced atherosclerosis is due to intrinsic age-associated changes in the arterial wall rather than elevation of plasma cholesterol [8,36]. In a study of diet-induced atherosclerosis in cynomolgus monkeys with aging [8], adult animals develop extensive atherosclerotic plaques in the coronary artery, whereas juveniles have focal fatty streaks, despite the similar plasma lipid levels at
110 both ages. In atherosclerosis-susceptible and -resistant strains of pigeons, showing no significant differences in plasma cholesterol level, blood pressure or thyroid activity, the cytoskeletal organization of aortic endothelial cells changes with aging and the cytoskeletal distribution of susceptible birds is different from that of resistant ones [36]. In our present study of Fischer 344 rat, hypercholesterolemia and foam cell lesions in the aorta occurred only at the age of 24 months after feeding the animals with the fatty diet for 6 months. In contrast, the younger rats were resistant to this. Despite their milder degree, we found ultrastructural changes in the aortic intima as well as an increase of monocyte adhesion to the endothelium in the control rats aged 24 months. These results show that atherosclerosis progression in the rat is closely associated with aging. The question of whether the structural and functional changes in the endothelial cells apparent with aging cause or promote the monocyte adhesion and lipoprotein permeation during atherogenesis remains to be clarified. Acknowledgements The authors express their gratitude to Dr. Hiroaki Okabe, Medical School, Kumamoto University, for the analysis of serum lipids, and to Miss Noriko Murai for her technical assistance with electron microscopy. This study was supported in part by the Project on Blood Vessel and Aging of the Tokyo Metropolitan Institute of Gerontology. References Pooling Project Research Group, Relationship of blood pressure, serum cholesterol, smoking habit, relative weight and ECG abnormalities to incidence of major coronary events: final report of pooling project, J. Chron. Dis., 31 (1978) 201. Gerrity, R.G., The role of the monocyte in atherogenesis. I. Transition of blood-borne monocytes into foam cells in fatty lesions, Am. J. Pathol., 103 (1981) 181. Gerrity, R.G., The role of the monocyte in atherogenesis. II. Migration of foam cells from atherosclerotic lesions. Am. J. Pathol., 103 (1981) 191. Faggiotto, A., Ross, R. and Harker, L., Studies of hypercholesterolemia in the nonhuman primate. I. Changes that
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10 Story, J.A., Tepper. S.A. and Kritchevsky, D., Age-related changes in the lipid metabolism of Fischer 344 rats, Lipids, 11 (1976) 623. 11 Gerrity, R.G. and Cliff, W.J., The aortic tunica intima in young and aging rats. Exp. Mol. Pathol., 16 (1972) 382. 12 Schwartz, S.M. and Benditt. E.P., Postnatal development on the aortic subendothelium in rats, Lab. Invest., 26 (1972) 778. 13 Gerrity, R.G. and Cliff, W.J., The aortic tunica media of the developing rat. I. Quantitative stereologic and biochemical analysis, Lab. Invest., 32 (1975) 585. 14 Guyton, J.R., Lindsay, K.L. and Dao, D.T., Comparison of aortic intima and inner media in young adult versus aging rats. Stereology in a polarized system, Am. J. Pathol., 111 (1983) 234. changes in rat 15 Lacko, A.G. and Davis, J.L., Age-related and primate plasma cholesterol metabolism, J. Am. Geriatr. Sot., 27 (1979) 212. H.A. and Yu, B.P.. 16 Liepa, G.U., Masoro, E.J., Bertrand, Food restriction as a modulator of age-related changes in serum lipids, Am. J. Physiol., 238 (1980) E253. 17 Still, W.J.S. and O’Neal, R.M., Electron microscopic study of experimental atherosclerosis in the rat. Am. J. Pathol., 40 (1962) 21. 18 Suzuki, M. and O’Neal, R.M., Circulating lipophages, serum lipids, and atherosclerosis in rats, Arch. Pathol., 83 (1967) 169. 19 Bglint, A., Veress, B., Nagy, Z. and Jellinek, H., Role of lipophages in the development of rat atheroma, Atherosclerosis, 15 (1972) 7. M.J., Regres20 Clowes, A.W., Breslow, J.L. and Kamovsky, sion of myointimal thickening following carotid endothelial injury and development of aortic foam cell lesions in long term hypercholesterolemic rats, Lab. Invest.. 36 (1977) 73. J.J.. Krolikowski, F.J. and 21 Joris, I., Zand, T., Nunnari. Majno, G., Studies on the pathogenesis of atherosclerosis. I. Adhesion and emigration of mononuclear cells in the aorta of hypercholesterolemic rats, Am. J. Pathol., II3 (1983) 341.
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Atherosclerosis, 79 (1989) 101-111
Publishers Ireland, Ltd.
Elsevier Scientific
ATHERO
04371
Age-associated ultrastructural changes in the aortic intima of rats with diet-induced hypercholesterolemia Hiroaki Nakamura
‘, Naotaka Izumiyama 2, Ken-i& and Kohichiro Ohtsubo 2
Nakamura
2
’Laborator?, of Electron Microscopy, Saitama Medical School, Saitama 350-04 (Japan). and .’Department of Clinical Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173 (Japan) (Received 3 February. 1989) (Revised. received 22 May, 1989) (Accepted 24 May, 1989)
Summary The ultrastructure of the aortic intima and serum lipid levels in Fischer 344 rats were examined at the ages of 12, 18 and 24 months after feeding the animals an atherogenic diet (2% cholesterol, 0.25% sodium cholate, 5% beef fat) for 6 months. Structural changes in the intima were noticeable only at the age of 24 months. In control rats, the endothelial cells were irregular in shape and each had a well-developed Golgi complex and a few lipid droplets. Simultaneously, reticular, basal lamina-like material and electron-dense granules of extracellular liposomes accumulated in the subendothelium. In fat-fed rats, these structural changes were more conspicuous in association with hypercholesterolemia, and numerous monocytes with lipid droplets were attached to the endothelium, occasionally invading into the subendothelium. Slight foam cell lesions were evident in the intima. The finding that older rats were more susceptible to the atherogenic diet suggests that atherogenesis in the rat is promoted by intrinsic age-associated changes in the aortic intima.
Key words:
Aging;
Atherosclerosis;
Hypercholesterolemia;
Introduction Hypercholesterolemia factor associated with
is the most important risk atherosclerosis [l]. Using
Correspondence to: Hiroaki Nakamura, Ph.D., Laboratory of Electron Microscopy, Saitama Medical School, 38 Morohongo, Moroyama-cho, Iruma-gun, Saitama 350-04, Japan. 0021-9150/89/$03.50
Q 1989 Elsevier Scientific
Publishers
Ireland,
Aortic
intima;
Rat;
Electron
microscopy
several animal models, it has been demonstrated that diet-induced hypercholesterolemia, which may produce primary endothelial injury, promotes monocyte adhesion to the endothelium, followed by their invasion into the intima and modulation into lipid-laden macrophages, the accumulation of which finally forms fatty streaks in the aorta [2-61. On the other hand, it is generally recognized from histopathological studies of human arteries that the extent and severity of atherosclerosis are Ltd.
102 closely associated with aging [7]. However, few experimental studies have directly examined whether there is a relationship between the progression of atherosclerosis and aging [S]. For example, the rabbit is widely used in studies of diet-induced atherosclerosis because of the relative rapidity of lesion formation in this animal [6]. However, in the rabbit, the association of atherogenesis with aging is difficult to study, because no reliable life table data are available [9]. In contrast, the rat has been relatively well examined throughout its life span [lo] with regard to age-associated changes in the aortic wall structure [ll-141 as well as serum lipid levels [10,14-161. There have been many ultrastructural investigations on the pathogenesis of atherosclerotic lesions in rats fed a cholesterol diet [17-221, but few have referred to the relationship between aging and the development of atherosclerosis. The occurrence of spontaneous aortic atherosclerosis has been found in old rats by light microscopy in association with an elevated serum cholesterol concentration [23, 241. In this study, we report that aged rats of the Fischer 344 strain fed an atherogenic diet, showed marked differences from younger rats in the ultraTABLE
structure of the aortic intima as well as in serum lipid concentration. Materials and methods Male rats of the Fischer 344 strain, aged 6, 12 and 18 months, purchased from Charles River Inc. (Japan) were used (Table 1). All rats were housed 2 or 3 per cage (plastic cage with sawdust bedding), and kept in a temperature-controlled clean room (21-23OC) with free access to their respective diets and water. They were fed either MF pellet (Oriental Yeast Co., Japan) as a control group, or the same basal diet containing 2% cholesterol, 0.25% sodium cholate and 5% beef fat as a fat-fed group. No thiouracil was added. After 1 and 6 months of feeding, blood was withdrawn into heparinized syringes from severed tail vessels under light ether anesthesia after an overnight fast. Serum was separated by centrifugation at 2000 rpm for 10 min and stored at - 70°C until assay. Total cholesterol and triglyceride concentrations in serum were measured spectrophotometrically using Sigma kits Nos. 325 and 405. After 6 months of feeding, rats aged 12, 18 or 24 months were anesthetised with ether and killed
1
CHANGES IN MEAN BODY WEIGHT, IN THE FISCHER 344 RATS
SERUM TOTAL CHOLESTEROL
AND TRIGLYCERIDE
LEVELS WITH AGING
All values are means k SD. Age (months)
Experimental
Body weight
Total cholesterol
Triglyceride
group (n)
(9)
(mg/dl)
(mg/dI)
6+1* 6 12+1 6 18+1 6
Control (4) (4) (4) (4) (4) (4)
415.3 f 490.0+ 487.8 + 520.3+ 515.8 f 521.3 +
29.9 32.0 a 8.4 b 3.9 a 26.3 17.6
73.5+ 109.s+ 90.0+ 106.35 113.5* 163.Ok
11.7 9.5 7.3 8.7 15.7 44.6
6+1* 6 12+1 6 18+1 6
Fat-fed (6) (6) (6) (6) (6) (5)
429.3 f 519.0+ 495.2 f 529.0* 521.2 f 515.8 f
17.6 24.1 a 21.7 b 21.8 a 35.8 30.7
140.8+ 200.8 + 161.8 f 247.0 k 236.5 f 1138.85
13.6 14.1 29.6 52.8 53.9 538.6
* a b c
a b a
123.3 + 262.Ok 179.5f 226.3 + 164.3 + 234.3 +
= += = ac b,c a,b.c
167.7 209.3 155.7 286.3 207.2 724.3
a
Age at the start of the experiment plus feeding period. Significant increase from 1 to 6 months in the same age group (P < 0.05). Significantly different from the mean in the younger age group after 1 or 6 months (P < 0.05). Significantly different from the mean in the control at corresponding ages after 1 or 6 months (P < 0.05).
f f f + f f
21.2 77.0 a 16.5 b 70.5 49.7 32.9 45.5 23.9 26.6 44.3 a 57.8 369.9 a
103 by perfusion with 2.5% glutaraldehyde (0.1 M cacodylate buffer, pH 7.2) at a hydrostatic pressure of 120 cm for 20 min. The thoracic aorta was cut transversely into short rings, fixed in the same fresh fixative for 2 h at 4°C rinsed 3 times with the same buffer containing 8% sucrose, post-fixed with 1% osmium tetroxide in the same buffer for 3 h at 4” C, dehydrated in a graded ethanol series, and flat-embedded in Polybed 812. Thick sections (1 pm) were stained with toluidine blue for light microscopy. Ultrathin sections were stained with uranyl acetate followed by lead citrate for transmission electron microscopy. After dehydration, some of the above samples were critical-point dried, and ion sputter-coated with platinum for scanning electron microscopy. The number of monocytes attached to the endo-
thelium per 1 mm* was counted at 5 randomly selected areas in 4 rats of each age group. Data including body weight, lipid concentration and monocyte number were analyzed by Student’s t-test. Results Body weight and serum lipids The body weights, total serum cholesterol and triglyceride levels of the control and fat-fed rats are shown in Table 1. The body weights of younger rats at the ages of 6 and 12 months increased significantly from 1 to 6 months in both the control and fat-fed groups, whereas no body weight differences between the control and fat-fed rats were present in any age group. The total cholesterol
Fig. 1. Light micrographs of age-related changes in the aortic intima of rats fed a fatty diet for 6 months. (a), (b) and (c): control rats aged 12. 18 and 24 months; (d), (e) and (f): fat-fed rats at corresponding ages, respectively. Monocytes attach to the endothelium of fat-fed rats aged 18 and 24 months (arrows). x 900.
104 level increased significantly from 1 to 6 months at all ages in both the control and fat-fed groups. Especially at the age of 18 months, the level increased by 49.5 mg/ml in the control rats (average: 163, max.: 195, min.: 112 mg/dl at age 24 months) and by 902.3 mg/ml in the fat-fed rats (average: 1138.8, max.: 1632, min.: 501 mg/dl at age 24 months) after 6 months. Significant differences between the levels in the control and fat-fed groups were found at all ages (Table 1). High triglyceride levels were present only in fat-fed rats at the age of 24 months (Table 1). Light microscopy
With aging, the subendothelial space of the thoracic aorta tended to expand in the control as well as the fat-fed groups (Fig. 1). Monocytes were frequently attached to the endothelium and foam cells were present in the subendothelium of the fat-fed rats at the age of 24 months (Fig. lf), although they were rare at 18 months (Fig. le). The monocytes were filled with lipid droplets at the age of 24 months, but were free of droplets at 18 months. The numbers of these cells varied in the tissue portion or in rats, apparently in associa-
tion with serum total cholesterol levels. However, no atherosclerotic lesions such as fatty streaks and fibrous plaques were apparent in the aorta at any age in either the control or fat-fed rats. Transmission
electron microscopy
Ultrastructural changes with aging were apparent in the thoracic aorta at the age of 24 months. In control rats aged 24 months, the endothelial cells were irregular in shape, and each had a well developed Golgi complex, lysosomes and a few lipid droplets. Basal lamina-like material consisting of a coarse reticulum filled the subendothelium (Fig. 2). Massive clusters of electron-dense granules were present focally near the internal elastic lamina (Fig. 2). In fat-fed rats aged 24 months, the endothelial cells were occasionally swollen, and frequently contained several lipid droplets and a large Golgi complex (Fig. 3). In the subendothelium, variously sized electron-dense granules (similar to those described above) and vesicles accumulated, and the reticular basal lamina-like material was more condensed in the fat-fed rats than in the controls (Fig. 3). Neither collagen fibrils nor elastic fibers were increased in
Fig. 2. The aortic intima of a control rat aged 24 months. End, endothelial cell; BLM, reticular basal lamina-like material; EDG, electron-dense granules; Lp, lipid droplets; Ly, lysosomes; IEL, internal elastic lamina. X 14000.
Fig. 3. The aortic
intima
of a fat-fed rat aged 24 months. End, endothelial cell; Lp, lipid droplets; GC, Golgi reticular basal lamina-like materials; EDG, electron-dense granules. x 1OOCO.
the subendothelium of the fat-fed rats in comparison with those of control rats at any age (Figs. 2 and 3). In fat-fed rats at the age of 24 months, monocytes were frequently attached to endothelial cells containing a few lipid droplets (Fig. 4a) or to them without lipid droplets. The monocytes with various numbers of lysosomes always contained various-sized lipid vacuoles, and extended cell pro jections toward the endothelium (Fig. 4a). The features of these monocytes were consistent with lipid-laden macrophages in blood [2,3]. These lipid-laden macrophages occasionally invaded through endothelial cell junctions into the subendothelium (Fig. 4b). From the examination of serial sections, these invading macrophages appeared to push aside the endothelial cells (Fig. 4b). In fat-fed rats at the age of 24 months, foam cells of various sites containing several amounts of
complex;
BLM,
lipid droplets, were present either singly or in clusters in the subendothelium (Fig. 5a). The endothelium covering these areas was thin but intact, and in some regions covered large foam cells with an extremely thin layer of cytoplasm. Lipid droplets in the foam cells were not membrane bound and appeared less osmiophilic with a continuous osmiophilic rim (Fig. 5a). Large foam cells frequently had membranous globules and deposits within the cytoplasm, suggesting breakdown of the large amorphous lipid droplets (Fig. 5a). Lipidladen macrophages were occasionally present in the subendothelium of the aorta in fat-fed rats aged 18 months (Fig. 5b). The subendothelial cells differed from the foam cells in older rats, because they had more lysosomes and fewer lipid droplets. Scanning electron microscopy The luminal surfaces of the thoracic aorta at the ages of 12 and 18 months in both the control
106
Fig. 4. Monocytes attached to the aortic endothelium of fat-fed rats aged 24 months. (a) Cell with microvilli containing large lipid droplets (Lp) and a small amount of lysosomes (Ly). An endothelial cell contains lipid droplets (arrows). x 6500. (b) Cell with lipid droplets (Lp) invading between endothelial cells (arrows), extending an elongated projection into the subendothelium. x 6500.
Fig. 5. Foam cells in the aortic subendothelium of fat-fed rats. (a) At the age of 24 months, a large foam cell containing variously sized lipid droplets (Lp) is covered at the htminal side by a thin layer of cytoplasm of an endothelial cell (arrows). EDG, electron-dense granules. x 6500. (b) At the age of 18 months, a lipid-laden macrophage contains a small number of lysosomes (Ly) and lipid droplets (Lp). X 10000.
Fig. 6. SEM view of luminal surfaces of the aortae. (a) Endothelial cells of a fat-fed rat aged 18 months are smooth and intact with cell fringes (arrows) and microvilli (Mv). X 10000. (b) Endothelial surface of a control aged 24 months is somewhat swollen, and cell fringes and microvilli are not clear. x 10000.
and fat-fed rats, were covered by smooth and intact endothelial cells with clear cell fringes (Fig. 6a). In the control rats aged 24 months, however, these endothelial cell fringes were irregular and obscure, and occasionally there were small focal areas of the endothelium protruding into the lumen (Fig. 6b). In the fat-fed rats aged 24 months, the endothelial surface was very irregular and widely extruded into the lumen in many parts (Fig. 7). In addition, monocytes with microvilli were frequently attached to the endothelium. However, SEM proved that the endothelium was relatively intact and not denuded of cells (Fig. 7). Focal clusters of small globular protrusions were present on the endothelial cells at 24 months (Fig. 7) suggesting that these were the lipid droplets observed in the endothelial cells by TEM as shown in Fig. 3. Monocytes were spherical to tadpole-like in shape, and present singly or in clusters of 3 to 15 cells on the endothelium. Quantitatively, monocytes adhering to the endothelial cells were
significantly more numerous in fat-fed rats at the age of 24 months as compared with those at the age of 18 months or in the control rats (Table 2). There was no significant difference in monocyte
TABLE
2
CHANGES IN NUMBERS OF MONOCYTES ON THE ENDOTHELIUM WITH AGING OF RATS FED A CONTROL OR A FATTY DIET FOR 6 MONTHS All values areas).
are means+SD
(n = 4 rats,
total
endothelium
n = 20 counted
Experimental
Cell number/mm2
groups (n)
12 months
18 months
24 months
(n = 20)
Control (4) Fat-fed (4)
3.3 + 2.3 3.4*2.1
4.4 f 3.2 9.6 f 4.5 a.b
9.2k4.8 a 28.5 f 9.3 a,b
a Significantly different from the mean at younger age in the same experimental group (P < 0.05). b Significantly different from the mean in the controls at the corresponding age (P < 0.05).
108
Fig. 7. SEM view of a luminal surface of the aorta of a fat fed rat aged 24 months. Endothelial surface is expanded irregularly with small globular protrusions extending into the lumen (arrows). Monocytes with microvilli are present (large arrows). X 14500.
numbers between the controls aged 24 months and the fat-fed rats aged 18 months (P > 0.05). Discussion This study revealed that a fat-rich diet accelerated the initiation of atherosclerotic lesions to the endothrough adhesion of monocytes thelium and their migration into the subendothelium, forming foam cell lesions in the thoracic aorta of 24-month-old rats. The severity and extent of the lesions were correlated with the degree of elevation of serum cholesterol levels. These findings are consistent with those of other experimental studies on atherosclerosis in pigs [2,3], rabbits [6], pigeons [5] and monkeys [4] as well as rats [20,21]. In hypercholesterolemia, monocyte
adhesion to endothelial cells, which has been demonstrated both in vivo [2-6,20,21] and in vitro [25,26], is likely to play an important role in the initiation and evolution of atherosclerosis. However, there have been different views on the morphology of the attached monocytes found in rats fed atherogenic diets. In accordance with our observations, O’Neal et al. [17,1X] and Balint et al. [19] have reported that monocytes containing lipid droplets (termed circulating lipophages) appear in blood along with elevated plasma lipid levels, and migrate from the blood to the subendothelium of the aorta. On the other hand, Clowes et al. [20] and Joris et al. [21] have shown that monocytes without lipid droplets become attached to the endothehal cells, and migrate into the subendothelium, subsequently developing into foam
109 cells of varying sizes in the aortic intima. The mechanisms of monocyte transformation to foam cells have been investigated in several studies [27-301. These studies on experimental animals with hyperlipidemia have suggested that modification of the native LDL by some factors induces the transformation of macrophages into foam cells in vitro. The presence of lipid-laden macrophages in blood, as shown in our study of fat-fed rats, suggests a possibility that macrophage modification has already occurred in hypercholesterolemic blood, and that the modified cells migrate, actively taking up more lipid, to become foam cells in the subendothelium. In our study, no advanced atheromatous lesions such as fatty streaks and fibrous plaques had developed in the aorta of any rat. O’Neal et al. [17,18] have reported similar results in rats fed a high-fat diet on the basis of electron microscopy. Several authors, however, have succeeded in producing fatty streaks in the rat aorta by feeding a diet of cholesterol and sodium cholate along with thiouracil [19-211. Thiouracil is an antithyroid drug, which inhibits the formation of thyroid hormone [33]. When a high-cholesterol diet is given concomitantly with thiouracil, the plasma cholesterol level shows a marked increase to a mean of 1085 mg/dl in Sprague-Dawley rats weighing 300-350 g after 6 months [20], or to a mean of 900 mg/dl in Wistar rats weighing 225-250 g after 336 months [21]. In our study without thiouracil, Fischer 344 rats aged 24 months showed a high level of serum cholesterol with a mean level of 1138.8 mg/dl after feeding with a cholesterol and fat diet for 6 months (Table l), but no fatty streaks occurred in the aorta. An antithyroid drug such as thiouracil can directly damage the elastic fibers of the aortic wall [34]. Thus, factors other than a high plasma lipid level may be necessary for progression of atherosclerotic lesions in rats. Several electron microscopic studies on hypercholesterolemic animals [2,3,5,20,21], apart from ours, have demonstrated that monocyte adhesion occurs on morphologically intact endothelial cells without any evidence of areas denuded of cells. In addition, we found that ultrastructural changes occurred in the aortic intima of aged rats (Figs. 2 and 3). In the control rats aged 24 months, the aortic endothelial cells contained a few lipid
droplets and well developed Golgi complex, and electron-dense granules had accumulated in the subendothelium. Simultaneously, monocyte adhesion to the endothelium was more frequent at the age of 24 months than at 18 months (Table 2). These structural changes were more conspicuous in the fat-fed rats at 24 months, corresponding to the elevation of serum cholesterol. Similar endothelial lipid droplets have been reported in hypercholesterolemic rabbits [6] and rats [17,21], and also in normal rats aged 2-3 years [ll]. Joris et al. [21] have noted that myelin figures arising from the endothelial surface are commonly present in the aorta of rats fed a cholesterol diet. However, we did not find any such structure on the endothelium in this study. Electron-dense granules in the aortic subendothelium are reported to be extracellular liposomes containing apolipoprotein B and unesterified cholesterol [31]. They increase in rabbits with diet-induced hyperlipidemia [31,32] and in normal rats with aging [11,12]. Age-dependent changes in total cholesterol levels in serum have been reported for several species, including man and rat. Generally in normal rats, body weight increases rapidly until 12 months of age and remains stable thereafter, whereas total cholesterol levels gradually increase until the age of 12 months, then increase steeply [10,15,16,22]. Our data for body weight and total cholesterol in the control rats were similar to those in the above reports (Table 1). High levels of total cholesterol in serum only developed at the age of 24 months in fat-fed rats (Table 2) whereas the younger rats tolerated the fatty diet. Serum cholesterol levels may therefore rise in association with several factors, such as alterations of hepatic lipid metabolism [lo] and intestinal lipid absorption [35] with aging. Hypercholesterolemia may have caused the ultrastructural changes in the aortic intima seen in our study. However, there are a few reports suggesting that diet-induced atherosclerosis is due to intrinsic age-associated changes in the arterial wall rather than elevation of plasma cholesterol [8,36]. In a study of diet-induced atherosclerosis in cynomolgus monkeys with aging [8], adult animals develop extensive atherosclerotic plaques in the coronary artery, whereas juveniles have focal fatty streaks, despite the similar plasma lipid levels at
110 both ages. In atherosclerosis-susceptible and -resistant strains of pigeons, showing no significant differences in plasma cholesterol level, blood pressure or thyroid activity, the cytoskeletal organization of aortic endothelial cells changes with aging and the cytoskeletal distribution of susceptible birds is different from that of resistant ones [36]. In our present study of Fischer 344 rat, hypercholesterolemia and foam cell lesions in the aorta occurred only at the age of 24 months after feeding the animals with the fatty diet for 6 months. In contrast, the younger rats were resistant to this. Despite their milder degree, we found ultrastructural changes in the aortic intima as well as an increase of monocyte adhesion to the endothelium in the control rats aged 24 months. These results show that atherosclerosis progression in the rat is closely associated with aging. The question of whether the structural and functional changes in the endothelial cells apparent with aging cause or promote the monocyte adhesion and lipoprotein permeation during atherogenesis remains to be clarified. Acknowledgements The authors express their gratitude to Dr. Hiroaki Okabe, Medical School, Kumamoto University, for the analysis of serum lipids, and to Miss Noriko Murai for her technical assistance with electron microscopy. This study was supported in part by the Project on Blood Vessel and Aging of the Tokyo Metropolitan Institute of Gerontology. References Pooling Project Research Group, Relationship of blood pressure, serum cholesterol, smoking habit, relative weight and ECG abnormalities to incidence of major coronary events: final report of pooling project, J. Chron. Dis., 31 (1978) 201. Gerrity, R.G., The role of the monocyte in atherogenesis. I. Transition of blood-borne monocytes into foam cells in fatty lesions, Am. J. Pathol., 103 (1981) 181. Gerrity, R.G., The role of the monocyte in atherogenesis. II. Migration of foam cells from atherosclerotic lesions. Am. J. Pathol., 103 (1981) 191. Faggiotto, A., Ross, R. and Harker, L., Studies of hypercholesterolemia in the nonhuman primate. I. Changes that
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