Atherosclerosis, 96 (1992) 43-52 0 1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved. 0021-9150/92/%05.00
43
Printed and Published in Ireland
ATHERO 04873
Inheritability of atherosclerosis and the role of lipoproteins as risk factors in the development of atherosclerosis in WHHL rabbits: Risk factors related to coronary atherosclerosis are different from those related to aortic atherosclerosis Masashi Shiomi, Takashi Ito, Motonari
Shiraishi and Yoshio Watanabe
Institute for Experimental Animals, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650 f Japan)
(Received 2 December, 1991) (Revised, received 6 May, 1992) (Accepted 1 June, 1992)
Summary Inheritability of atherosclerosis and the influences of serum lipids on atherosclerosis were examined by following its progression in selectively bred WHHL rabbits. Our studies indicate (1) coronary atherosclerosis is clearly inherited from parents by offspring whereas inheritability of aortic atherosclerosis is uncertain; (2) coronary stenosis is positively correlated to serum cholesterol level, although the correlation coefficient is markedly low: in contrast, no relationship between serum lipid levels and aortic atherosclerosis was observed; (3) cholesterol-rich VLDL showed atherogenicity in aorta, but not in coronary arteries; (4) an unknown lipoprotein detected by 3.6% polyacrylamide gel electrophoresis was related to coronary atherosclerosis, although no relationship between the unknown lipoprotein and aortic atherosclerosis was observed. These findings suggest that there are two types of genetic factors involved in atherosclerosis, one of which is unique to coronary atherosclerosis whereas the other is related to only aortic atherosclerosis.
Key words: Coronary atherosclerosis;
Inheritability;
Introduction It is well known that environmental and genetic factors are responsible for development of atherosclerosis. Serum cholesterol level, which is directly related to the development of coronary Correspondence to: Masashi Shiomi, Ph.D., Institute for Exper-
imental Animals, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
cholesterol rich VLDL; Midband; WHHL rabbits
atherosclerosis [l-4], is genetically determined and increases in response to a number of environmental stimuli. While it may prove difficult to exclude environmental factors in human studies, animal models are well suited to genetic studies of the disease as environmental conditions can be controlled. WHHL rabbits show hypercholesterolemia from birth due to genetic deficiency of low density
44
lipoprotein (LDL) receptors [5-71 and consequently, atherosclerosis is spontaneously developed in the aorta, coronary arteries and other arteries [8,9]. Therefore, WHHL rabbits are useful for analyzing the relationship between genetic factors and atherosclerosis. However, at the time when the WHHL rabbit strain was established (designated stage I), the incidence of coronary atherosclerosis was extremely low. To increase the incidence of coronary atherosclerosis in WHHL rabbits, selective breeding was carried out [lo]. Although the incidence of coronary atherosclerosis was increased by the selective breeding, the coronary stenoses were still relatively mild (designated stage II). To advance the development of coronary atherosclerosis further in the rabbit strain, we carried out further selective breeding (designated stage III). Through this selective breeding, we examined the inheritability of atherosclerosis and the lipid factors which affect atherosclerosis. In previous studies on the effect of serum cholesterol reduction on atherosclerosis in WHHL rabbits [ 11,121, coronary atherosclerosis was significantly suppressed but the effect of serum cholesterol reduction was not clear in aortic atherosclerosis. In addition, histopathological differences can be observed between coronary and aortic atherosclerosis. Therefore, we consider that coronary atherosclerosis is different from aortic atherosclerosis. However, there have been no previous reports which closely examine the difference between the two types of atherosclerosis. In this study, we examined the difference between coronary and aortic atherosclerosis in terms of inheritability and of its risk factors related to development. Materials and Methods Animals All WHHL rabbits used in this experiment were bred and raised at Kobe University. Coronary atherosclerosis was assessed in 245 animals obtained by the first selective breeding between 1981 and 1984 (stage II) and 338 animals obtained by the second selective breeding between 1985 and 1990 (stage III). After the first selective breeding, we found a rabbit line with severe coronary stenosis in
the pedigree of this strain at stage II. The rabbits in this line were selected as new breeding stocks to increase the severity of coronary atherosclerosis. WHHL rabbits in stage III were derived from these breeding stocks. Each WHHL rabbit was given 120 g of commercial rabbit chow (Type GC4, Oriental Yeast Co., Japan). Water was available ad libitum. The animals were accommodated individually in metal cages kept at room temperature (22” f 2°C). All animal experiments were conducted in conformity with the Guideline to Animal Experiment Kobe University School of Medicine. Estimation of atherosclerotic lesion The hearts and aortas were obtained from both dead animals and animals killed by exsanguination of the carotid artery under sodium pentobarbitalinduced anesthesia. Since Keohane et al. [ 131 reported that relative arterial dimensions remained relatively constant in both perfused and nonperfused formalin-fixed hearts, we examined sections prepared from both immersion-fixed and perfusion-fixed hearts. After 1989 paraffin blocks of hearts were prepared by the method shown in Fig. 1 in order to cut the coronary arteries transversely. The upper quarter of the heart was cut into four blocks. Block I, containing the original portion of both left and right coronary arteries, was serially sectioned at 200~pm intervals. Blocks II, III and IV were serially sectioned at 500-pm intervals. Blocks V, VI and VII were sectioned at l-mm intervals. Each section was stained with elastic van Gieson. Paraffin blocks prepared before 1990 by another method [lO,l l] were sectioned again using the present method. Coronary stenosis was determined as follows: (intimal lesion surrounded by internal elastic area/area lamina) x 100 in perfusion-fixed samples. In immersion-fixed samples, the area surrounded by internal elastic lamina was calculated by the method reported by Pesonen et al. [14]. Since the original portion of coronary arteries in the sections prepared before 1990 were transected obliquely, data from these portions were deleted. In this study the most severe stenosis was adopted as the representative stenosis in the rabbit. Lesion area in the aorta was determined according to the method described by Watanabe et al. [ 111.The percentage of lesion area in the aorta was
45 Orifice Orificx
0,
pulmanary
of
aorta artery
Bloclc-I BIocK-II
BIOCK- IV-
BlocK-III
BlocK-V
BIocK-VI
BlocK-VII
Fig. 1. Method for the preparation of paraffin blocks for histopathological analysis of coronary atherosclerosis in WHHL rabbits. Block I includes the origin portion of both left and right coronary arteries. Block II includes left septal artery. Block III includes left circumflex artery. Block IV includes left anterior descending artery and right coronary artery. Blocks V-VII include above four arteries. Each block was embedded in paraffin. Extramural major coronary arteries were serially and transversely sectioned at 200 pm intervals in Block I, 500 pm intervals in Blocks II-IV and 1000 pm intervals in Blocks V-VII.
using L8-60M ultracentrifuge equipped with SW55Ti rotor (Beckman Instruments Inc., CA): very low density lipoprotein (VLDL, d < 1.006 g/ml), intermediate density lipoprotein (IDL, d = 1.006-1.019 g/ml), LDL (d = 1.019-1.063 g/ml) and high density lipoprotein (HDL, d = 1.063-1.210 g/ml). Polyacrylamide gel electrophoresis (3.6%) of whole serum was carried out following a slight modification to the method described by Narayan et al. [16]. The sera were pre-stained with Sudan black-B and then applied to the top of the gel. Electrophoresis was performed at constant voltage of 100 V using Tris-glycine buffer (pH 8.3). Total cholesterol, triglyceride and phospholipid levels were measured enzymatically [ Ill. Since serum lipid levels in WHHL rabbits decrease with ageing, we calculated average lifetime lipid level in each animal by dividing the lipid level integrated through the lifetime by age in months at death or euthanasia to examine the relationship with atherosclerosis. As a result of the analysis using 583 WHHL rabbits, no gender-related difference was observed with regard to serum lipid levels (data not shown). Therefore, we used data from both genders. Statistical analysis
determined as follows: (lesion area/surface luminal area) x 100. All parameters of the atherosclerotic lesions were measured by a color image analyzer, SP-500 (Olympus Co., Japan). In the 338 WHHL rabbits examined, we found no gender-related difference with regard to the degree of both coronary and aortic atherosclerosis (data not shown). Therefore, data from both genders were used.
Data were represented as mean f S.E. Statistical analysis was carried out by the MannWhitney U-test for percentage of lesion area in aorta and coronary stenosis and Student’s t-test for serum lipid levels. The frequency was analyzed by x2-test. The correlation coefficient was calculated as Pearson moment correlation coefficient. Results
Measurement of serum lipid levels andfractionation of lipoproteins
Progression of coronary atherosclerosis in WHHL rabbits by selective breeding
Blood samples were periodically taken after overnight fasting at weaning age (1 month), mature age (6 months), 12 months and 24 months and at just before euthanasia. Density of serum was prepared according to the method described by Hatch and Lees [ 151 and lipoprotein was fractionated by ultracentrifugation at 2-month intervals from 2 months of age to 12 months of age
Coronary stenosis was significantly increased by selective breeding (Fig. 2). At stage III the coronary stenosis was 15.0% f 3.9% in newly-weaned animals and progressed to 38.9% f 6.7% at a mature age (5-6 months) and to 53.3% f 2.6% at 11-13 months of age. At above 17 months of age, coronary stenosis was over 70%. The degree of coronary stenosis was 1.5-5 times higher in stage
80
60
2
4
6
8
10
12
14
16
18
20
22
24
Month of age Fig. 2. Progression of coronary atherosclerosis in WHHL rabbits by selective breeding. 0, before selective breeding (stage II); 0, after selective breeding (stage III); bars, mean f SE; **P < 0.01, ***P < O.OOS,‘***P < 0.001 by Mann-Whitney U-test
III animals than in stage II animals. With an increase of coronary stenosis, the incidence of coronary lesion was significantly increased (data not shown). At stage III, the incidence of coronary atherosclerosis was 78% even in newly-weaned rabbits, and at above 4 months of age all rabbits suffered from coronary atherosclerosis. Aortic atherosclerosis in WHHL stage
rabbits at each
Figure 3 shows the percentage of the lesion area in the aorta at each stage. Although there was almost no difference in the percentage of the lesion area of the aorta between stage I and II (data not shown), the lesions of stage III animals were significantly increased compared to the stage II animals at less than 10 months of age. At above 9 months of age, the percentage of lesion area of stage III animals was about 10% larger than that in stage II animals.
discover what degree of coronary stenosis is inheritable (Table 1). Rabbits were divided into three groups, i.e., parents with severe coronary stenosis (above average stenoses at stage III), parents with mild stenosis (under average coronary stenoses at stage II) and parents with intermediate lesions. The frequency of the offspring with severe coronary lesion was 49% in the severepair group, 27% in the intermediate-pair group and 18% in the mild-pair group. The difference between the severe-pair group and the mild-pair group was significant. In addition, the frequency of the offspring with mild lesion was 24% in the severe-pair group, 47% in the intermediate-pair group and 71% in the mild-pair group. The differences between each group were also significant. However, there was no relationship in aortic atherosclerosis between parents and the offspring (data not shown).
Inheritability of coronary atherosclerosis
Relationship sclerosis
between
serum
lipid and athero-
The relationship between coronary stenosis in parents and their offspring was examined to
The average lifetime cholesterol level was significantly increased but the average lifetime
47
2
4
6
I
1
1
1
8
10
12
14
16
,
1
I
18
20
22
24
Month of age Fig. 3. Percentage of lesion area in aorta of WHHL rabbits before (0) and after (0) selective breeding. Bars. mean f S.E.; **P < 0.01, ***P< 0.005 by Mann-Whitney U-test
TABLE 1 RELATIONSHIP IN WHHL RABBITS BETWEEN SEVERITY OF CORONARY STENOSIS IN PARENTS AND THAT OF THEIR OFFSPRING Severe lesion indicates above average stenosis in stage III animals, mild lesion indicates below average stenosis in stage II animals and moderate lesion indicates coronary stenosis between severe and mild lesions. Severe pair indicates parents with severe lesions, mild pair indicates parents with mild lesions and intermediate pair indicates parents with intermediate lesions. Parents
Severity of coronary stenosis in offspring Severe
Severe pair 49% (16/33)* Intermediate 27% ( 15155) pair Mild pair 18% (5/28)
Modefate
Mild
27% (9/33)
24% (8/33)t+*
26% (14/55) 47% (26155); 11% (3/28) 71% (20/28)
Significantly different from mild pair group by X2-test: ?? P < 0.05, **P< 0.01. Significantly different from intermediate pair group by x2 test: tP < 0.05.
triglyceride level was significantly decreased with progression of coronary atherosclerosis induced by selective breeding (data not shown). These results suggest that the serum lipid level affects atherosclerosis. Therefore, the relationship between average lifetime serum lipid levels and coronary atherosclerosis was examined using stage III WHHL rabbits (Table 2). WHHL rabbits were divided into three groups on the basis of coronary stenosis. In the group with severe coronary stenosis, the average lifetime cholesterol level (838 f 25 mg/dl) was significantly higher than that in the mild coronary stenosis group (755 f 24 mg/dl). Conversely, the average lifetime triglyceride level in the severe coronary stenosis group (273 f 19 mg/dl) was significantly decreased compared to the mild stenosis group (386 f 30 mg/dl). However, the correlation coefficients were low, namely, 0.274 (P < 0.05, n = 81) between coronary stenosis and the average lifetime cholesterol level and -0.261 (P < 0.05, n = 81) between coronary stenosis and the average lifetime triglyceride level. On the other hand, there was no significant correlation between the percentage of the lesion area in the aorta and the average lifetime lipid levels.
48 TABLE 2 AVERAGE LIFETIME LIPID LEVELS OF WHOLE SERUM AT EACH SEVERITY OF CORONARY STENOSIS IN STAGE III WHHL RABBITS AGED 8-10 MONTHS Data are represented as mean f SE. Average lipid levela
Total cholesterol (mgdl) Triglyceride (mgdl)
Severity of coronary stenosis < 30% (n = 23)
30-60% (n = 32)
> 600/o (n = 26)
155
f 24
836 f 26t
838 f 25t
386 + 30
343 f 21
273 f 19$**
aAverage lipid level was calculated by dividing integrated serum lipid levels over the lifetime by age in months at death. Significantly different from under 30% of stenosis group by Student’s r-test: tP < 0.05, $P < 0.005. Significantly different from rabbits group with coronary stenosis between 30?/ and 60% by Student’s t-test: *P c 0.05.
Relationship between atherosclerosis
lipoprotein
lipids
and
To examine the effect of lipoprotein fractions on atherosclerosis, 20 WHHL rabbits were used. Table 3 shows the correlation coefficient between lipoprotein lipid composition and the atherosclerotic lesion. Significant correlation coefficient
was only observed between percentage of lesion area in aorta and cholesterol/triglyceride ratio (r = 0.555, P < 0.05) triglyceridelphospholipid ratio (-0.448, P < O.OS),or triglyceride/total lipid ratio (r = -0.495, P < 0.05) in VLDL fraction. A midband was observed between the LDLband and VLDL-band when 3.6% polyacrylamide gel electrophoresis of WHHL whole sera was performed (Fig. 4). Table 4 shows the relationship between this midband and coronary stenosis. The frequency of rabbits with above average stenosis in stage III animals was 67% in the midband group and 14% in the non-midband group. In addition, the frequency of rabbits with under-average stenosis in stage II animals was 16% in the midband group and 71% in the non-midband group. However, there was no significant relationship between the midband and the percentage of the lesion area in the aorta. Discussion The aim of this study is to elucidate the inheritability of atherosclerosis, serum lipid factors related to atherosclerosis, and the difference in risk factors between coronary atherosclerosis and aortic atherosclerosis. Through selective breeding, coronary atherosclerosis markedly progressed (Fig. 2) and the inheritability of severe lesions from parents by their
VLDL midband
LDL
(+) Fig. 4. Polyacrylamide gel electrophotogram of WHHL rabbits sera. Whole serum was pre-stained with Sudan black-B. Unknown lipoprotein band is observed between the VLDL-band and LDL-band.
COEFFICIENT
BETWEEN LIPOPROTEIN
LIPID COMPOSITION
AND SEVERITY OF ATHEROSCLEROSIS
IN WHHL RABBITS
Aorta Coronary Aorta Coronary Aorta Coronary Aorta Coronary Aorta 0.120 0.160 0.266 0.179 0.082 -0.024 -0.119 0.130 0.277 0.193
‘Ch, total cholesterol; TG, triglyceride; PL, phospholipid; *P < 0.05.
HDL
LDL
IDL
VLDL
Whole serum
Ch 0.138 0.084 0.255 0.129 0.078 -0.048 -0.037 0.277 0.085 -0.087
PL
TL, total lipid.
-0.007 -0.024 0.081 0.072 -0.03 1 -0.078 -0.184 -0.098 0.101 -0.018
TG
Lipoprotein lipida
0.106 0.197 0.555* 0.346 0.125 0.245 -0.024 0.128 -0.103 0.052
Ch/TG -0.114 0.195 0.032 0.093 -0.125 0.130 -0.150 -0.125 0.260 0.357
ChiPL
0.064
0.217 0.402 0.156 0.040 0.206 -0.067 0.034 0.106 0.300
-0.095
CbrrL
-0.070 -0.448* -0.232 -0.161 -0.198 -0.052 -0.187 0.069 -0.050
TGlPL
-0.050 -0.076 -0.495’ -0.267 -0.131 -0.203 -0.026 -0.287 0.054 -0.082
TG/-TL
The severity of aortic atherosclerosis was represented as percentage of surface lesion area and that of coronary lesion was represented as coronary stenosis. Lipoprotein lipid levels were determined by dividing lipoprotein lipid levels integrated through the lifetime by age in months at death. Twenty rabbits were used in this analysis.
CORRELATION
TABLE 3
50
TABLE 4 THE FREQUENCY OF MIDBAND DETECTED BY 3.6% POLYACRYLAMIDE GEL ELECTROPHORESIS OF WHOLE SERUM AT EACH SEVERITY OF CORONARY ATHEROSCLEROSIS Midbanda
Severity of coronary stenosis Severe
Moderate
67% (39/58)****t 17% (19158) 46% (21/46)** 22% (1O/46) 14% (2/14) 14% (2/14)
Mild 16% (9/58)**pt 33% (15/46)* 71% (10/14)
aSee Fig. 4. Midband (+) indicates clearly observed, (*) indicates slightly observed and (-) indicates not observed. Severity of coronary stenosis, see Table 1. Significantly different from midband (-) group by X2-test: *P < 0.05, **P< 0.01, ***P< 0.005. Significantly different from midband (h) group by x2-test: +P < 0.05.
offspring was observed (Table 1). However, progression of aortic atherosclerosis was relatively low (Fig. 3) and inheritability of aortic atherosclerosis was unclear. In addition, the correlation coefficient between coronary stenosis and the percent of the lesion area in the aorta was low (I = 0.240, P c 0.05, n = 73 at age from 8 to 10 months). These results suggest that genetic factors related to coronary atherosclerosis differ from those related to aortic atherosclerosis. Coronary atherosclerosis was positively related to the average lifetime serum cholesterol level and negatively related to the average lifetime triglyceride level (Table 2) in spite of the low correlation coefficient. Similar results were observed through two selective breedings from stage I to stage II and from stage II to stage III (data not shown). However, the decrease of serum triglyceride level in rabbits with severe coronary atherosclerosis most likely results from an increase in the ratio of cholesterol/triglyceride in the VLDL fraction (Table 3). Therefore, it is considered that only the serum cholesterol level is related to coronary atherosclerosis. On the other hand, the relationship between serum lipids and aortic atherosclerosis was not observed (data not shown). In humans the significant relationship between serum or plasma cholesterol levels and coronary heart disease is reported by many
epidemiological studies [l-4]. However, there is no consensus that serum or plasma triglyceride levels relate to coronary heart disease. When serum cholesterol levels in WHHL rabbits were reduced by hypolipidemic drug(s), the progression of coronary atherosclerosis was clearly suppressed whereas aortic atherosclerosis was not [ 11,121. In addition, aortic atherosclerosis was always developed at above 400 mg/dl of the average lifetime serum cholesterol level in WHHL rabbits aged 8-10 months (stage I) but the incidence of coronary atherosclerosis increased when their average lifetime serum cholesterol level exceeded 500 mg/dl in rabbits of the same age (stage II). These results suggest that the serumcholesterol level necessary for the development of coronary atherosclerosis is high compared with that for the development of aortic atherosclerosis. This may indicate that serum cholesterol levels play a more significant role in the development of coronary atherosclerosis than in the development of aortic atherosclerosis. Cholesterol-rich VLDL was associated with an increased severity of lesions in aorta (Table 3). Wakasugi et al. report that the ratio of cholesterol/triglyceride in VLDL fraction was about 0.29 in normal rabbits and 0.74 in WHHL rabbits at stage I [ 171. In rabbits at stage III, this ratio increased to 1.8 f 0.14 (n = 20). Therefore, the ratio of cholesterol/triglyceride in VLDL increased in parallel with breeding-induced atherosclerosis. A similar finding has been observed by Kita et al. [18] and Ishii et al. [19]. They reported that VLDL with a high mass ratio of cholesterol/protein is atherogenic. They observed that the incubation of mouse peritoneal macrophages with a cholesterol-rich VLDL fraction from WHHL rabbits stimulated cholesteryl ester synthesis 10.5-fold more than in the relatively cholesteryl ester depleted VLDL [19]. They also pointed out that the amount of apolipoprotein E (apo E) in the cholesterol-rich VLDL was larger than that in the relatively cholesterol-depleted VLDL. There is some evidence that apo E is the recognition factor of receptors for &VLDL [20,21]. Therefore, they consider that the cholesterol-rich VLDL may be taken up by macrophages through its &VLDL receptors [19]. The atherogenicity of cholesterol-rich VLDL in
51
our study is most likely a result of the same mechanism as that induced by cholesterol-rich VLDL reported by Ishii et al. [19]. In this study, atherogenicity of cholesterol-rich VLDL was clearly observed in aortas, but was unclear in coronary arteries. Although the reason for this difference is unknown, a histopathological difference in atherosclerotic lesions between aortas and coronary arteries may be involved. Although LDL is a representative atherogenic lipoprotein, no correlation was found between LDL and atherosclerosis in this study. In atherosclerotic lesion in WHHL aorta, a large amount of peroxidized LDL was observed [22-241. Therefore, LDL is certainly one of the atherogenic lipoproteins. The average LDLcholesterol level through a lifetime of WHHL rabbits used in this analysis was 403 f 15 mg/dl. At such high LDL cholesterol levels, there should be no difference in the atherogenicity among LDL in Table 3. An unknown lipoprotein as shown by the detection of a midband when 3.6% polyacrylamide gel electrophoresis is performed (Fig. 4) significantly relates to the coronary atherosclerosis (Table 4). However, a relationship between the midband and the aortic atherosclerosis was not observed. These results suggest that the midband is a specific lipoprotein which affects only coronary atherosclerosis. After fractionation by ultracentrifugation, this midband was observed at density between 1.019 and 1.050. Therefore, this midband is probably not lipoprotein(a). In the current study using WHHL rabbits, inheritability of coronary atherosclerosis was clearly observed but this phenomenon was unclear in aortic atherosclerosis. In addition, we showed two types of factors related to atherosclerosis; one affects only coronary atherosclerosis and the other affects only aortic atherosclerosis. Acknowledgments The authors are indebted to Miss Nao Kawamura and Mr. Toshiaki Tamura for their excellent technical assistance. We are also grateful to Mr. Christopher P. Autry for his editorial advice. This work was supported in part by research grants from the Ministry of Education, Science
and Culture 63113008).
of
Japan
(No.
61480436
and
References 1 Kannel, W.B., Castelli, W.P., Gordon, T. and Mcnamara, P.M., Serum cholesterol, lipoproteins and the risk of coronary heart disease, The Framingham Study. Ann. Intern. Med., 74 (1971) 1. 2 Keys, A., Coronary heart disease in seven countries, Circulation 41 (1970) 1. 3 Lipid Research Clinics Program, The lipid research clinics coronary primary prevention trial results, I. Reduction in incidence of coronary heart disease, J. Am. Med. Assoc., 251 (1984) 351. 4 Lipid Research Clinics Program, The lipid research clinics coronary primary prevention trial results, II. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering, J. Am. Med. Assoc., 251 (1984) 365. 5 Tanzawa, K., Shimada, Y., Kuroda, M., Tsujita, Y., Arai, M. and Watanabe, Y., WHHL-rabbit: a low density lipoprotein receptor-deficient animal model for familial hypercholesterolemia, FEBS Lett., 118 (1980) 8 1. 6 Kita, T., Brown, MS., Watanabe, Y. and Goldstein, J.L., Deficiency of low density lipoprotein receptors in liver and adrenal gland of the WHHL rabbit, an animal model of familial hypercholesterolemia, Proc. Nat]. Acad. Sci. USA, 78 (1981) 2268. 7 Shiomi, M., Ito, T. and Watanabe, Y., Increase in hepatic low density lipoprotein receptor activity during pregnancy in Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia, B&him. Biophys. Acta, 917 (1987) 92. 8 Watanabe, Y., Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHL rabbit): incidence and development of atherosclerosis and xanthoma, Atherosclerosis, 36 (1980) 261. 9 Buja, L.M., Kita, T., Goldstein, J.L., Watanabe, Y. and Brown, M.S., Cellular pathology of progressive atherosclerosis in the WHHL rabbit, an animal model of familial hypercholesterolemia, Arteriosclerosis, 3 (1983) 87. 10 Watanabe, Y., Ito, T. and Shiomi, M., The effect of selective breeding in the development of coronary atherosclerosis in WHHL rabbits, an animal model for familial hypercholesterolemia, Atherosclerosis, 56 (1985) 71. 11 Watanabe, Y., Ito, T., Shiomi, M., Tsujita, Y., Kuroda, M., Arai, M., Fukami, M. and Tamura, A.. Preventive effect of pravastatin sodium, a potent inhibitor of 3hydroxy-3-methylglutaryl coenzyme A reductase, on coronary atherosclerosis and xanthoma in WHHL rabbits, Biochim. Biophys. Acta, 960 (1988) 294. 12 Watanabe, Y., Ito, T., Shiomi, M., Tsujita, Y., Kuroda, M., Arai, M., Fukami, M., Fukusige, J. and Tamura, A., Suppression of established atherosclerosis and xanthomas in mature WHHL rabbits by keeping their serum
52
13
14
15 16
17
18
19
cholesterol levels extremely low: effect of pravastatin cholestyramine, with sodium in combination Atherosclerosis, 83 (1990) 69. Keohane, S.G., Adams, C.W.M. and Poston, R.N., Coronary arterial dimensions and cell populations in ageing man, Atherosclerosis, 69 (1988) 103. Pesonen, E., Martimo, P., Eng, D. and Rapola, J., Histometry of the arterial wall: a new technique with the aid of automatic data processing, LabInvest., 30 (1974) 550. Hatch, F.T. and Lees, RX, Practical methods for plasma lipoprotein analysis, Adv. Lipid Res., 6 (1968) 1. Narayan, K.A., Narayan, S. and Kummerow, R.A., Disc electrophoresis of human serum lipoproteins, Nature, 205 (1965) 246. Wakasugi, T., Mabuchi, H., Sakai, Y., Sakai, T., Yoshimura, A., Watanabe, A., Koizumi, J., Miyamoto, S., Takeda, R. and Watanabe, Y., ApoVLDL of the rabbit and hyperlipidemic heritable Watanabe cholesterol-fed rabbit, J. Lipid Res., 25 (1984) 246. Kita, T., Yokode, M., Watanabe, Y., Narumiya, S. and Kawai, C., Stimulation of cholesteryl ester synthesis in mouse peritoneal macrophages by cholesterol-rich very low density lipoproteins from the Watanabe heritable hyperlipidemic rabbit, an animal model of familial hypercholesterolemia, J. Clin. Invest., 77 (1986) 1460. Ishii, K., Kita, T., Yokode, M., Kume, N., Nagano, Y.,
20
21
22
23
24
Otani, H., Yamamura, T., Murayama, S., Morimoto, Y., Teranishi, Y. and Kawai, C., Characterization of very low density lipoprotein from Watanabe heritable hyperlipidemic rabbits, J. Lipid Res., 30 (1989) 1. Innerarity, T.L., Arnold, K.S., Weisgraber, K.H. and Mahley, R.W., Apolipoprotein E is the determinant that mediates the receptor uptake of &very low density lipoprotein by mouse macrophages, Arteriosclerosis, 6 (1986) 114. Bates, S.R., Coughlin, B.A., Mazzon, T., Borensztajn, J. and Getz, G.S., Apolipoprotein E mediates the interaction of &VLDL with macrophages, J. Lipid Res., 28 (1987) 787. Kimura, J., Nakagami, K., Amanum, K., Ohkuma, S., Yoshida, Y. and Takano, T., Monoclonal antibodies recognizing lipid-laden cells and extra cellular regions with lipid-laden cells in atherosclerotic aorta, Virchows Arch. A, 410 (1986) 159. Haberland, M.E., Fong, D. and Cheng, L., Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits, Science, 241 (1988) 215. Palinski, W., Rosenfeld, M.E., Yla-Herttuala, S., Gurtner, G.C., Socher, S.S., Bulter, SW., Parthasarathy, S., Carew, T.E., Steinberg, D. and Witztum, J.L., Low density lipoprotein undergoes oxidative modification in vivo, Proc. Natl. Acad. Sci. USA, 86 (1989) 1372.
43
Printed and Published in Ireland
ATHERO 04873
Inheritability of atherosclerosis and the role of lipoproteins as risk factors in the development of atherosclerosis in WHHL rabbits: Risk factors related to coronary atherosclerosis are different from those related to aortic atherosclerosis Masashi Shiomi, Takashi Ito, Motonari
Shiraishi and Yoshio Watanabe
Institute for Experimental Animals, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650 f Japan)
(Received 2 December, 1991) (Revised, received 6 May, 1992) (Accepted 1 June, 1992)
Summary Inheritability of atherosclerosis and the influences of serum lipids on atherosclerosis were examined by following its progression in selectively bred WHHL rabbits. Our studies indicate (1) coronary atherosclerosis is clearly inherited from parents by offspring whereas inheritability of aortic atherosclerosis is uncertain; (2) coronary stenosis is positively correlated to serum cholesterol level, although the correlation coefficient is markedly low: in contrast, no relationship between serum lipid levels and aortic atherosclerosis was observed; (3) cholesterol-rich VLDL showed atherogenicity in aorta, but not in coronary arteries; (4) an unknown lipoprotein detected by 3.6% polyacrylamide gel electrophoresis was related to coronary atherosclerosis, although no relationship between the unknown lipoprotein and aortic atherosclerosis was observed. These findings suggest that there are two types of genetic factors involved in atherosclerosis, one of which is unique to coronary atherosclerosis whereas the other is related to only aortic atherosclerosis.
Key words: Coronary atherosclerosis;
Inheritability;
Introduction It is well known that environmental and genetic factors are responsible for development of atherosclerosis. Serum cholesterol level, which is directly related to the development of coronary Correspondence to: Masashi Shiomi, Ph.D., Institute for Exper-
imental Animals, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
cholesterol rich VLDL; Midband; WHHL rabbits
atherosclerosis [l-4], is genetically determined and increases in response to a number of environmental stimuli. While it may prove difficult to exclude environmental factors in human studies, animal models are well suited to genetic studies of the disease as environmental conditions can be controlled. WHHL rabbits show hypercholesterolemia from birth due to genetic deficiency of low density
44
lipoprotein (LDL) receptors [5-71 and consequently, atherosclerosis is spontaneously developed in the aorta, coronary arteries and other arteries [8,9]. Therefore, WHHL rabbits are useful for analyzing the relationship between genetic factors and atherosclerosis. However, at the time when the WHHL rabbit strain was established (designated stage I), the incidence of coronary atherosclerosis was extremely low. To increase the incidence of coronary atherosclerosis in WHHL rabbits, selective breeding was carried out [lo]. Although the incidence of coronary atherosclerosis was increased by the selective breeding, the coronary stenoses were still relatively mild (designated stage II). To advance the development of coronary atherosclerosis further in the rabbit strain, we carried out further selective breeding (designated stage III). Through this selective breeding, we examined the inheritability of atherosclerosis and the lipid factors which affect atherosclerosis. In previous studies on the effect of serum cholesterol reduction on atherosclerosis in WHHL rabbits [ 11,121, coronary atherosclerosis was significantly suppressed but the effect of serum cholesterol reduction was not clear in aortic atherosclerosis. In addition, histopathological differences can be observed between coronary and aortic atherosclerosis. Therefore, we consider that coronary atherosclerosis is different from aortic atherosclerosis. However, there have been no previous reports which closely examine the difference between the two types of atherosclerosis. In this study, we examined the difference between coronary and aortic atherosclerosis in terms of inheritability and of its risk factors related to development. Materials and Methods Animals All WHHL rabbits used in this experiment were bred and raised at Kobe University. Coronary atherosclerosis was assessed in 245 animals obtained by the first selective breeding between 1981 and 1984 (stage II) and 338 animals obtained by the second selective breeding between 1985 and 1990 (stage III). After the first selective breeding, we found a rabbit line with severe coronary stenosis in
the pedigree of this strain at stage II. The rabbits in this line were selected as new breeding stocks to increase the severity of coronary atherosclerosis. WHHL rabbits in stage III were derived from these breeding stocks. Each WHHL rabbit was given 120 g of commercial rabbit chow (Type GC4, Oriental Yeast Co., Japan). Water was available ad libitum. The animals were accommodated individually in metal cages kept at room temperature (22” f 2°C). All animal experiments were conducted in conformity with the Guideline to Animal Experiment Kobe University School of Medicine. Estimation of atherosclerotic lesion The hearts and aortas were obtained from both dead animals and animals killed by exsanguination of the carotid artery under sodium pentobarbitalinduced anesthesia. Since Keohane et al. [ 131 reported that relative arterial dimensions remained relatively constant in both perfused and nonperfused formalin-fixed hearts, we examined sections prepared from both immersion-fixed and perfusion-fixed hearts. After 1989 paraffin blocks of hearts were prepared by the method shown in Fig. 1 in order to cut the coronary arteries transversely. The upper quarter of the heart was cut into four blocks. Block I, containing the original portion of both left and right coronary arteries, was serially sectioned at 200~pm intervals. Blocks II, III and IV were serially sectioned at 500-pm intervals. Blocks V, VI and VII were sectioned at l-mm intervals. Each section was stained with elastic van Gieson. Paraffin blocks prepared before 1990 by another method [lO,l l] were sectioned again using the present method. Coronary stenosis was determined as follows: (intimal lesion surrounded by internal elastic area/area lamina) x 100 in perfusion-fixed samples. In immersion-fixed samples, the area surrounded by internal elastic lamina was calculated by the method reported by Pesonen et al. [14]. Since the original portion of coronary arteries in the sections prepared before 1990 were transected obliquely, data from these portions were deleted. In this study the most severe stenosis was adopted as the representative stenosis in the rabbit. Lesion area in the aorta was determined according to the method described by Watanabe et al. [ 111.The percentage of lesion area in the aorta was
45 Orifice Orificx
0,
pulmanary
of
aorta artery
Bloclc-I BIocK-II
BIOCK- IV-
BlocK-III
BlocK-V
BIocK-VI
BlocK-VII
Fig. 1. Method for the preparation of paraffin blocks for histopathological analysis of coronary atherosclerosis in WHHL rabbits. Block I includes the origin portion of both left and right coronary arteries. Block II includes left septal artery. Block III includes left circumflex artery. Block IV includes left anterior descending artery and right coronary artery. Blocks V-VII include above four arteries. Each block was embedded in paraffin. Extramural major coronary arteries were serially and transversely sectioned at 200 pm intervals in Block I, 500 pm intervals in Blocks II-IV and 1000 pm intervals in Blocks V-VII.
using L8-60M ultracentrifuge equipped with SW55Ti rotor (Beckman Instruments Inc., CA): very low density lipoprotein (VLDL, d < 1.006 g/ml), intermediate density lipoprotein (IDL, d = 1.006-1.019 g/ml), LDL (d = 1.019-1.063 g/ml) and high density lipoprotein (HDL, d = 1.063-1.210 g/ml). Polyacrylamide gel electrophoresis (3.6%) of whole serum was carried out following a slight modification to the method described by Narayan et al. [16]. The sera were pre-stained with Sudan black-B and then applied to the top of the gel. Electrophoresis was performed at constant voltage of 100 V using Tris-glycine buffer (pH 8.3). Total cholesterol, triglyceride and phospholipid levels were measured enzymatically [ Ill. Since serum lipid levels in WHHL rabbits decrease with ageing, we calculated average lifetime lipid level in each animal by dividing the lipid level integrated through the lifetime by age in months at death or euthanasia to examine the relationship with atherosclerosis. As a result of the analysis using 583 WHHL rabbits, no gender-related difference was observed with regard to serum lipid levels (data not shown). Therefore, we used data from both genders. Statistical analysis
determined as follows: (lesion area/surface luminal area) x 100. All parameters of the atherosclerotic lesions were measured by a color image analyzer, SP-500 (Olympus Co., Japan). In the 338 WHHL rabbits examined, we found no gender-related difference with regard to the degree of both coronary and aortic atherosclerosis (data not shown). Therefore, data from both genders were used.
Data were represented as mean f S.E. Statistical analysis was carried out by the MannWhitney U-test for percentage of lesion area in aorta and coronary stenosis and Student’s t-test for serum lipid levels. The frequency was analyzed by x2-test. The correlation coefficient was calculated as Pearson moment correlation coefficient. Results
Measurement of serum lipid levels andfractionation of lipoproteins
Progression of coronary atherosclerosis in WHHL rabbits by selective breeding
Blood samples were periodically taken after overnight fasting at weaning age (1 month), mature age (6 months), 12 months and 24 months and at just before euthanasia. Density of serum was prepared according to the method described by Hatch and Lees [ 151 and lipoprotein was fractionated by ultracentrifugation at 2-month intervals from 2 months of age to 12 months of age
Coronary stenosis was significantly increased by selective breeding (Fig. 2). At stage III the coronary stenosis was 15.0% f 3.9% in newly-weaned animals and progressed to 38.9% f 6.7% at a mature age (5-6 months) and to 53.3% f 2.6% at 11-13 months of age. At above 17 months of age, coronary stenosis was over 70%. The degree of coronary stenosis was 1.5-5 times higher in stage
80
60
2
4
6
8
10
12
14
16
18
20
22
24
Month of age Fig. 2. Progression of coronary atherosclerosis in WHHL rabbits by selective breeding. 0, before selective breeding (stage II); 0, after selective breeding (stage III); bars, mean f SE; **P < 0.01, ***P < O.OOS,‘***P < 0.001 by Mann-Whitney U-test
III animals than in stage II animals. With an increase of coronary stenosis, the incidence of coronary lesion was significantly increased (data not shown). At stage III, the incidence of coronary atherosclerosis was 78% even in newly-weaned rabbits, and at above 4 months of age all rabbits suffered from coronary atherosclerosis. Aortic atherosclerosis in WHHL stage
rabbits at each
Figure 3 shows the percentage of the lesion area in the aorta at each stage. Although there was almost no difference in the percentage of the lesion area of the aorta between stage I and II (data not shown), the lesions of stage III animals were significantly increased compared to the stage II animals at less than 10 months of age. At above 9 months of age, the percentage of lesion area of stage III animals was about 10% larger than that in stage II animals.
discover what degree of coronary stenosis is inheritable (Table 1). Rabbits were divided into three groups, i.e., parents with severe coronary stenosis (above average stenoses at stage III), parents with mild stenosis (under average coronary stenoses at stage II) and parents with intermediate lesions. The frequency of the offspring with severe coronary lesion was 49% in the severepair group, 27% in the intermediate-pair group and 18% in the mild-pair group. The difference between the severe-pair group and the mild-pair group was significant. In addition, the frequency of the offspring with mild lesion was 24% in the severe-pair group, 47% in the intermediate-pair group and 71% in the mild-pair group. The differences between each group were also significant. However, there was no relationship in aortic atherosclerosis between parents and the offspring (data not shown).
Inheritability of coronary atherosclerosis
Relationship sclerosis
between
serum
lipid and athero-
The relationship between coronary stenosis in parents and their offspring was examined to
The average lifetime cholesterol level was significantly increased but the average lifetime
47
2
4
6
I
1
1
1
8
10
12
14
16
,
1
I
18
20
22
24
Month of age Fig. 3. Percentage of lesion area in aorta of WHHL rabbits before (0) and after (0) selective breeding. Bars. mean f S.E.; **P < 0.01, ***P< 0.005 by Mann-Whitney U-test
TABLE 1 RELATIONSHIP IN WHHL RABBITS BETWEEN SEVERITY OF CORONARY STENOSIS IN PARENTS AND THAT OF THEIR OFFSPRING Severe lesion indicates above average stenosis in stage III animals, mild lesion indicates below average stenosis in stage II animals and moderate lesion indicates coronary stenosis between severe and mild lesions. Severe pair indicates parents with severe lesions, mild pair indicates parents with mild lesions and intermediate pair indicates parents with intermediate lesions. Parents
Severity of coronary stenosis in offspring Severe
Severe pair 49% (16/33)* Intermediate 27% ( 15155) pair Mild pair 18% (5/28)
Modefate
Mild
27% (9/33)
24% (8/33)t+*
26% (14/55) 47% (26155); 11% (3/28) 71% (20/28)
Significantly different from mild pair group by X2-test: ?? P < 0.05, **P< 0.01. Significantly different from intermediate pair group by x2 test: tP < 0.05.
triglyceride level was significantly decreased with progression of coronary atherosclerosis induced by selective breeding (data not shown). These results suggest that the serum lipid level affects atherosclerosis. Therefore, the relationship between average lifetime serum lipid levels and coronary atherosclerosis was examined using stage III WHHL rabbits (Table 2). WHHL rabbits were divided into three groups on the basis of coronary stenosis. In the group with severe coronary stenosis, the average lifetime cholesterol level (838 f 25 mg/dl) was significantly higher than that in the mild coronary stenosis group (755 f 24 mg/dl). Conversely, the average lifetime triglyceride level in the severe coronary stenosis group (273 f 19 mg/dl) was significantly decreased compared to the mild stenosis group (386 f 30 mg/dl). However, the correlation coefficients were low, namely, 0.274 (P < 0.05, n = 81) between coronary stenosis and the average lifetime cholesterol level and -0.261 (P < 0.05, n = 81) between coronary stenosis and the average lifetime triglyceride level. On the other hand, there was no significant correlation between the percentage of the lesion area in the aorta and the average lifetime lipid levels.
48 TABLE 2 AVERAGE LIFETIME LIPID LEVELS OF WHOLE SERUM AT EACH SEVERITY OF CORONARY STENOSIS IN STAGE III WHHL RABBITS AGED 8-10 MONTHS Data are represented as mean f SE. Average lipid levela
Total cholesterol (mgdl) Triglyceride (mgdl)
Severity of coronary stenosis < 30% (n = 23)
30-60% (n = 32)
> 600/o (n = 26)
155
f 24
836 f 26t
838 f 25t
386 + 30
343 f 21
273 f 19$**
aAverage lipid level was calculated by dividing integrated serum lipid levels over the lifetime by age in months at death. Significantly different from under 30% of stenosis group by Student’s r-test: tP < 0.05, $P < 0.005. Significantly different from rabbits group with coronary stenosis between 30?/ and 60% by Student’s t-test: *P c 0.05.
Relationship between atherosclerosis
lipoprotein
lipids
and
To examine the effect of lipoprotein fractions on atherosclerosis, 20 WHHL rabbits were used. Table 3 shows the correlation coefficient between lipoprotein lipid composition and the atherosclerotic lesion. Significant correlation coefficient
was only observed between percentage of lesion area in aorta and cholesterol/triglyceride ratio (r = 0.555, P < 0.05) triglyceridelphospholipid ratio (-0.448, P < O.OS),or triglyceride/total lipid ratio (r = -0.495, P < 0.05) in VLDL fraction. A midband was observed between the LDLband and VLDL-band when 3.6% polyacrylamide gel electrophoresis of WHHL whole sera was performed (Fig. 4). Table 4 shows the relationship between this midband and coronary stenosis. The frequency of rabbits with above average stenosis in stage III animals was 67% in the midband group and 14% in the non-midband group. In addition, the frequency of rabbits with under-average stenosis in stage II animals was 16% in the midband group and 71% in the non-midband group. However, there was no significant relationship between the midband and the percentage of the lesion area in the aorta. Discussion The aim of this study is to elucidate the inheritability of atherosclerosis, serum lipid factors related to atherosclerosis, and the difference in risk factors between coronary atherosclerosis and aortic atherosclerosis. Through selective breeding, coronary atherosclerosis markedly progressed (Fig. 2) and the inheritability of severe lesions from parents by their
VLDL midband
LDL
(+) Fig. 4. Polyacrylamide gel electrophotogram of WHHL rabbits sera. Whole serum was pre-stained with Sudan black-B. Unknown lipoprotein band is observed between the VLDL-band and LDL-band.
COEFFICIENT
BETWEEN LIPOPROTEIN
LIPID COMPOSITION
AND SEVERITY OF ATHEROSCLEROSIS
IN WHHL RABBITS
Aorta Coronary Aorta Coronary Aorta Coronary Aorta Coronary Aorta 0.120 0.160 0.266 0.179 0.082 -0.024 -0.119 0.130 0.277 0.193
‘Ch, total cholesterol; TG, triglyceride; PL, phospholipid; *P < 0.05.
HDL
LDL
IDL
VLDL
Whole serum
Ch 0.138 0.084 0.255 0.129 0.078 -0.048 -0.037 0.277 0.085 -0.087
PL
TL, total lipid.
-0.007 -0.024 0.081 0.072 -0.03 1 -0.078 -0.184 -0.098 0.101 -0.018
TG
Lipoprotein lipida
0.106 0.197 0.555* 0.346 0.125 0.245 -0.024 0.128 -0.103 0.052
Ch/TG -0.114 0.195 0.032 0.093 -0.125 0.130 -0.150 -0.125 0.260 0.357
ChiPL
0.064
0.217 0.402 0.156 0.040 0.206 -0.067 0.034 0.106 0.300
-0.095
CbrrL
-0.070 -0.448* -0.232 -0.161 -0.198 -0.052 -0.187 0.069 -0.050
TGlPL
-0.050 -0.076 -0.495’ -0.267 -0.131 -0.203 -0.026 -0.287 0.054 -0.082
TG/-TL
The severity of aortic atherosclerosis was represented as percentage of surface lesion area and that of coronary lesion was represented as coronary stenosis. Lipoprotein lipid levels were determined by dividing lipoprotein lipid levels integrated through the lifetime by age in months at death. Twenty rabbits were used in this analysis.
CORRELATION
TABLE 3
50
TABLE 4 THE FREQUENCY OF MIDBAND DETECTED BY 3.6% POLYACRYLAMIDE GEL ELECTROPHORESIS OF WHOLE SERUM AT EACH SEVERITY OF CORONARY ATHEROSCLEROSIS Midbanda
Severity of coronary stenosis Severe
Moderate
67% (39/58)****t 17% (19158) 46% (21/46)** 22% (1O/46) 14% (2/14) 14% (2/14)
Mild 16% (9/58)**pt 33% (15/46)* 71% (10/14)
aSee Fig. 4. Midband (+) indicates clearly observed, (*) indicates slightly observed and (-) indicates not observed. Severity of coronary stenosis, see Table 1. Significantly different from midband (-) group by X2-test: *P < 0.05, **P< 0.01, ***P< 0.005. Significantly different from midband (h) group by x2-test: +P < 0.05.
offspring was observed (Table 1). However, progression of aortic atherosclerosis was relatively low (Fig. 3) and inheritability of aortic atherosclerosis was unclear. In addition, the correlation coefficient between coronary stenosis and the percent of the lesion area in the aorta was low (I = 0.240, P c 0.05, n = 73 at age from 8 to 10 months). These results suggest that genetic factors related to coronary atherosclerosis differ from those related to aortic atherosclerosis. Coronary atherosclerosis was positively related to the average lifetime serum cholesterol level and negatively related to the average lifetime triglyceride level (Table 2) in spite of the low correlation coefficient. Similar results were observed through two selective breedings from stage I to stage II and from stage II to stage III (data not shown). However, the decrease of serum triglyceride level in rabbits with severe coronary atherosclerosis most likely results from an increase in the ratio of cholesterol/triglyceride in the VLDL fraction (Table 3). Therefore, it is considered that only the serum cholesterol level is related to coronary atherosclerosis. On the other hand, the relationship between serum lipids and aortic atherosclerosis was not observed (data not shown). In humans the significant relationship between serum or plasma cholesterol levels and coronary heart disease is reported by many
epidemiological studies [l-4]. However, there is no consensus that serum or plasma triglyceride levels relate to coronary heart disease. When serum cholesterol levels in WHHL rabbits were reduced by hypolipidemic drug(s), the progression of coronary atherosclerosis was clearly suppressed whereas aortic atherosclerosis was not [ 11,121. In addition, aortic atherosclerosis was always developed at above 400 mg/dl of the average lifetime serum cholesterol level in WHHL rabbits aged 8-10 months (stage I) but the incidence of coronary atherosclerosis increased when their average lifetime serum cholesterol level exceeded 500 mg/dl in rabbits of the same age (stage II). These results suggest that the serumcholesterol level necessary for the development of coronary atherosclerosis is high compared with that for the development of aortic atherosclerosis. This may indicate that serum cholesterol levels play a more significant role in the development of coronary atherosclerosis than in the development of aortic atherosclerosis. Cholesterol-rich VLDL was associated with an increased severity of lesions in aorta (Table 3). Wakasugi et al. report that the ratio of cholesterol/triglyceride in VLDL fraction was about 0.29 in normal rabbits and 0.74 in WHHL rabbits at stage I [ 171. In rabbits at stage III, this ratio increased to 1.8 f 0.14 (n = 20). Therefore, the ratio of cholesterol/triglyceride in VLDL increased in parallel with breeding-induced atherosclerosis. A similar finding has been observed by Kita et al. [18] and Ishii et al. [19]. They reported that VLDL with a high mass ratio of cholesterol/protein is atherogenic. They observed that the incubation of mouse peritoneal macrophages with a cholesterol-rich VLDL fraction from WHHL rabbits stimulated cholesteryl ester synthesis 10.5-fold more than in the relatively cholesteryl ester depleted VLDL [19]. They also pointed out that the amount of apolipoprotein E (apo E) in the cholesterol-rich VLDL was larger than that in the relatively cholesterol-depleted VLDL. There is some evidence that apo E is the recognition factor of receptors for &VLDL [20,21]. Therefore, they consider that the cholesterol-rich VLDL may be taken up by macrophages through its &VLDL receptors [19]. The atherogenicity of cholesterol-rich VLDL in
51
our study is most likely a result of the same mechanism as that induced by cholesterol-rich VLDL reported by Ishii et al. [19]. In this study, atherogenicity of cholesterol-rich VLDL was clearly observed in aortas, but was unclear in coronary arteries. Although the reason for this difference is unknown, a histopathological difference in atherosclerotic lesions between aortas and coronary arteries may be involved. Although LDL is a representative atherogenic lipoprotein, no correlation was found between LDL and atherosclerosis in this study. In atherosclerotic lesion in WHHL aorta, a large amount of peroxidized LDL was observed [22-241. Therefore, LDL is certainly one of the atherogenic lipoproteins. The average LDLcholesterol level through a lifetime of WHHL rabbits used in this analysis was 403 f 15 mg/dl. At such high LDL cholesterol levels, there should be no difference in the atherogenicity among LDL in Table 3. An unknown lipoprotein as shown by the detection of a midband when 3.6% polyacrylamide gel electrophoresis is performed (Fig. 4) significantly relates to the coronary atherosclerosis (Table 4). However, a relationship between the midband and the aortic atherosclerosis was not observed. These results suggest that the midband is a specific lipoprotein which affects only coronary atherosclerosis. After fractionation by ultracentrifugation, this midband was observed at density between 1.019 and 1.050. Therefore, this midband is probably not lipoprotein(a). In the current study using WHHL rabbits, inheritability of coronary atherosclerosis was clearly observed but this phenomenon was unclear in aortic atherosclerosis. In addition, we showed two types of factors related to atherosclerosis; one affects only coronary atherosclerosis and the other affects only aortic atherosclerosis. Acknowledgments The authors are indebted to Miss Nao Kawamura and Mr. Toshiaki Tamura for their excellent technical assistance. We are also grateful to Mr. Christopher P. Autry for his editorial advice. This work was supported in part by research grants from the Ministry of Education, Science
and Culture 63113008).
of
Japan
(No.
61480436
and
References 1 Kannel, W.B., Castelli, W.P., Gordon, T. and Mcnamara, P.M., Serum cholesterol, lipoproteins and the risk of coronary heart disease, The Framingham Study. Ann. Intern. Med., 74 (1971) 1. 2 Keys, A., Coronary heart disease in seven countries, Circulation 41 (1970) 1. 3 Lipid Research Clinics Program, The lipid research clinics coronary primary prevention trial results, I. Reduction in incidence of coronary heart disease, J. Am. Med. Assoc., 251 (1984) 351. 4 Lipid Research Clinics Program, The lipid research clinics coronary primary prevention trial results, II. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering, J. Am. Med. Assoc., 251 (1984) 365. 5 Tanzawa, K., Shimada, Y., Kuroda, M., Tsujita, Y., Arai, M. and Watanabe, Y., WHHL-rabbit: a low density lipoprotein receptor-deficient animal model for familial hypercholesterolemia, FEBS Lett., 118 (1980) 8 1. 6 Kita, T., Brown, MS., Watanabe, Y. and Goldstein, J.L., Deficiency of low density lipoprotein receptors in liver and adrenal gland of the WHHL rabbit, an animal model of familial hypercholesterolemia, Proc. Nat]. Acad. Sci. USA, 78 (1981) 2268. 7 Shiomi, M., Ito, T. and Watanabe, Y., Increase in hepatic low density lipoprotein receptor activity during pregnancy in Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia, B&him. Biophys. Acta, 917 (1987) 92. 8 Watanabe, Y., Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHL rabbit): incidence and development of atherosclerosis and xanthoma, Atherosclerosis, 36 (1980) 261. 9 Buja, L.M., Kita, T., Goldstein, J.L., Watanabe, Y. and Brown, M.S., Cellular pathology of progressive atherosclerosis in the WHHL rabbit, an animal model of familial hypercholesterolemia, Arteriosclerosis, 3 (1983) 87. 10 Watanabe, Y., Ito, T. and Shiomi, M., The effect of selective breeding in the development of coronary atherosclerosis in WHHL rabbits, an animal model for familial hypercholesterolemia, Atherosclerosis, 56 (1985) 71. 11 Watanabe, Y., Ito, T., Shiomi, M., Tsujita, Y., Kuroda, M., Arai, M., Fukami, M. and Tamura, A.. Preventive effect of pravastatin sodium, a potent inhibitor of 3hydroxy-3-methylglutaryl coenzyme A reductase, on coronary atherosclerosis and xanthoma in WHHL rabbits, Biochim. Biophys. Acta, 960 (1988) 294. 12 Watanabe, Y., Ito, T., Shiomi, M., Tsujita, Y., Kuroda, M., Arai, M., Fukami, M., Fukusige, J. and Tamura, A., Suppression of established atherosclerosis and xanthomas in mature WHHL rabbits by keeping their serum
52
13
14
15 16
17
18
19
cholesterol levels extremely low: effect of pravastatin cholestyramine, with sodium in combination Atherosclerosis, 83 (1990) 69. Keohane, S.G., Adams, C.W.M. and Poston, R.N., Coronary arterial dimensions and cell populations in ageing man, Atherosclerosis, 69 (1988) 103. Pesonen, E., Martimo, P., Eng, D. and Rapola, J., Histometry of the arterial wall: a new technique with the aid of automatic data processing, LabInvest., 30 (1974) 550. Hatch, F.T. and Lees, RX, Practical methods for plasma lipoprotein analysis, Adv. Lipid Res., 6 (1968) 1. Narayan, K.A., Narayan, S. and Kummerow, R.A., Disc electrophoresis of human serum lipoproteins, Nature, 205 (1965) 246. Wakasugi, T., Mabuchi, H., Sakai, Y., Sakai, T., Yoshimura, A., Watanabe, A., Koizumi, J., Miyamoto, S., Takeda, R. and Watanabe, Y., ApoVLDL of the rabbit and hyperlipidemic heritable Watanabe cholesterol-fed rabbit, J. Lipid Res., 25 (1984) 246. Kita, T., Yokode, M., Watanabe, Y., Narumiya, S. and Kawai, C., Stimulation of cholesteryl ester synthesis in mouse peritoneal macrophages by cholesterol-rich very low density lipoproteins from the Watanabe heritable hyperlipidemic rabbit, an animal model of familial hypercholesterolemia, J. Clin. Invest., 77 (1986) 1460. Ishii, K., Kita, T., Yokode, M., Kume, N., Nagano, Y.,
20
21
22
23
24
Otani, H., Yamamura, T., Murayama, S., Morimoto, Y., Teranishi, Y. and Kawai, C., Characterization of very low density lipoprotein from Watanabe heritable hyperlipidemic rabbits, J. Lipid Res., 30 (1989) 1. Innerarity, T.L., Arnold, K.S., Weisgraber, K.H. and Mahley, R.W., Apolipoprotein E is the determinant that mediates the receptor uptake of &very low density lipoprotein by mouse macrophages, Arteriosclerosis, 6 (1986) 114. Bates, S.R., Coughlin, B.A., Mazzon, T., Borensztajn, J. and Getz, G.S., Apolipoprotein E mediates the interaction of &VLDL with macrophages, J. Lipid Res., 28 (1987) 787. Kimura, J., Nakagami, K., Amanum, K., Ohkuma, S., Yoshida, Y. and Takano, T., Monoclonal antibodies recognizing lipid-laden cells and extra cellular regions with lipid-laden cells in atherosclerotic aorta, Virchows Arch. A, 410 (1986) 159. Haberland, M.E., Fong, D. and Cheng, L., Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits, Science, 241 (1988) 215. Palinski, W., Rosenfeld, M.E., Yla-Herttuala, S., Gurtner, G.C., Socher, S.S., Bulter, SW., Parthasarathy, S., Carew, T.E., Steinberg, D. and Witztum, J.L., Low density lipoprotein undergoes oxidative modification in vivo, Proc. Natl. Acad. Sci. USA, 86 (1989) 1372.
