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Probucol and atherosclerosis in the Watanabe heritable hyperlipidemic rabbit — long-term antiatherogenic effect and effects on established plaques
92 (1992) 131-140 0 1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved 0021-9150/92/$05.00 Printed and Published in Ireland
Atherosclerosis,
131
ATHERO 04760
Probucol and atherosclerosis in the Watanabe heritable hyperlipidemic rabbit - long-term antiatherogenic effect and effects on established plaques Yutaka Nagano *, Tadashi Nakamura2, Yuji Matsuzawa2, Mikimaro Cho ‘, Yukihiko &da3 and Toru Kita’ ‘Department of Geriatric Medicine. Faculty of Medicine, Kyoro University. ‘Second Deparrmenr of Internal Medicine, School of Medicine, Osaka University, Osaka 553 and ‘Third Division, Department of Internal Medicine, Faculty of Medicine, Kyoro Universily. Kyoto 606 (Japan)
(Received 26 June, 1991) (Revised, received 8 October, 1991) (Accepted 25 October, 1991)
Summary
We performed two studies to investigate the effect of probucol on atherogenesis in vivo in the Watanabe heritable hyperlipidemic (WHHL) rabbit. In the first study (Study A), probucol was administered to 2-month-old WHHL rabbits, to evaluate its long-term effect. When killed at about 1.5 years of age, the percentage area of aorta covered with atherosclerotic plaque in probucol-treated rabbits was markedly less than that seen in non-treated rabbits (23.0 f 11.4”/0vs. 87.7 & 8. I%, M f S.D., P < 0.001). In the second study (study B), administration of probucol was commenced with 8-month-old WHHL rabbits to investigate whether the drug was effective for limiting atherosclerosis in rabbits in which plaques had already developed. When killed after 6 months of treatment, the percentage area of aorta covered with plaque was 38.1 i 12.1% in treated rabbits and 82.7 f 22.6% in non-treated rabbits (P < 0.02). Microscopic observations of lesions also supported the effect of probucol. Probucol treatment resulted in a change not only in the size but also the composition of lesions. Thus, probucol was effective in preventing atherosclerosis in long-term studies at both early and late stages. Key words: Foam cells; Macrophages; Antioxidants;
Introduction
Familial hypercholesterolemia (FH) is one of the most common inherited diseases. It is caused Correspondence
io:
Y. Nagano,
Department
of Geriatric
Medicine,Faculty of Medicine, Kyoto University, 54 Shogoin Kawaharacho Sakyo-ku, Kyoto 606, Japan.
Regression
by an abnormality of the receptor for low density lipoprotein (LDL). Patients with FH are usually affected with ischemic heart disease because of the development of premature atherosclerosis [ 11. Therefore, it is very important to prevent this process. In atheroma or in cutaneous and tendon xan-
132 thomas, lipid-storing foam cells have been recognized as a characteristic feature. Most of the foam cells observed in the early stages of atherosclerotic lesions have been shown to originate from monocyte-derived macrophages [2,3]. Involvement of lipid peroxides in atherogenesis has been reported [4-61, and probucol, a drug originally developed as an antioxidant, is effective in vivo for regression of xanthomas of FH patients [7]. Probucol also inhibits oxidative modification of LDL in vitro [8]. In view of these observations, we recently performed a study to evaluate the effect of probucol on atherosclerotic lesions in vivo in WHHL rabbits. The result revealed a significant protective effect of probucol against the progression of atherosclerosis when the drug was given at the age of 2 months, and this effect could be explained by its antioxidative action [9,10]. Similar results were also reported by Carew et al. [ll]. The data in these studies and other recent studies strongly suggest the existence of oxidized LDL in vivo [ 12,131, which cause foam cell transformation of macrophages. Therefore, inhibition of oxidative modification of LDL is a very important step for prevention of atherosclerosis. The effect of probucol as an antioxidative and antiatherogenic drug on WHHL rabbits is likely to be similar to that in FH patients, because both of them have an abnorma!ity of LDL receptor, which causes hypercholesterolemia and rapid progression of at erosclerosis. It should be noted also that P FH patients have to be treated for life. Therefore, in the present study we undertook a long-term treatment of WHHL rabbits with probucol. Since treatment of FH patients is not always started in childhood, we also investigated the effect of probucol on atherosclerotic lesions when the drug was started after definite aortic lesions had developed in WHHL rabbits. Materials and methods Animals and drug administration
Homozygous WHHL rabbits were bred in Kobe or in Kyoto by mating heterozygous or homozygous female WHHL rabbits with homozygous male WHHL rabbits. They were fed rabbit laboratory chow RM-1 (Oriental Yeast Co.) before the administration of probucol [ 141.
We performed two studies to evaluate the effect of probucol in WHHL rabbits. In study A, in which we investigated the long-term effect of probucol, 1 g/day of the drug was administered to 2-month-old WHHL rabbits (n = 6), and was continued until the rabbits were about 18 months old. In study B, in which we investigated the effect of administration of probucol to WHHL rabbits after they already had developed atherosclerotic lesions, the same amount of the drug was started in 8-month-old WHHL rabbits (n = 4) and the administration was continued for a further 6 months. We used 8-month-old rabbits because our previous study clearly showed that untreated WHHL rabbits of that age revealed definite lesions in their aortae [9]. In both studies, control WHHL rabbits (n = 7 for study A and n = 4 for study B) were fed the same amount of the standard rabbit chow. Two of the control rabbits were used for both studies. Probucol was simply mixed with the raw material of the chow without organic solvents. Blood was sampled after overnight fasting. Measurement of aortic plaque
In study A, control group rabbits were killed between the age of 14 and 19 months (mean 16.7 months) and the probucol group rabbits were 17 or 19 months (mean 18 months). In study B, all rabbits were killed at the age of 14 months. Sodium pentobarbital (100 mg) was administered intravenously for anesthesia. The entire thoracic aorta was isolated from each rabbit, opened longitudinally, and en face photographs of the inner surface were taken. We measured the percentages of intimal surface area involved with atheromatous plaque according to the method that we have described previously [9]. Briefly, photographs of aortae were copied to graph paper with magnification (x 2), and the total and lesioned area of aorta were measured. The entire thoracic aorta was divided into two portions (aortic arch and descending portion) at the level of the first intercostal artery. Percentages of the lesioned area were also measured for each part. Preparations for histological examinations
After photographs were taken, aliquots of aorta which contained the lesioned area were obtained
133 from the orifice of the second intercostal arteries. They were fixed with 10% formaldehyde solution of phosphate buffered saline (pH = 7.4), or frozen immediately for lipid staining studies.
In study B, specimens were obtained from lesions around the orifice of the celiac artery of both groups of rabbits. They were fixed with 2% glutaraldehyde, posttixed with osmic tetroxide, and prepared for electron microscopic observation with standard procedures.
from Ono pharmaceutical Co. (Osaka, Japan). The cholesterol level in each lipoprotein subfraction was measured after separation of lipoprotein with ultracentrifugation according to the method previously described [ 161. Plasma level of probucol was measured by HPLC as described elsewhere u71. Statistical analyses were performed with Student’s t-test. Changes in data of plasma level of lipids in the same group of rabbits were analyzed with paired t-test, and data between different groups of rabbits with unpaired t-test.
Analysis of lipid composition in plaques
Results
Electron microscopy
An aliquot of aortic specimen was obtained from the lesioned area of each rabbit just distal to the left subclavian artery. All samples were completely covered with plaques. Tissue adhering to the outer surface of the aorta was completely removed, and the samples were homogenized with a Polytron homogenizer. Total lipid was extracted by the method described by Folch et al. [ 151. The amount of cholesterol was analyzed using gas chromatography with 5 alpha-cholestane as an internal standard. Other assays
Plasma levels of lipids were measured by enzymatic methods using a commercial kit obtained
Study A
In Table 1, plasma level of lipids and probucol are shown. Plasma levels of cholesterol and triglycerides fell significantly during the experimental period (P < 0.05). When we compare the final level of plasma cholesterol between probucol-treated and control rabbits, the results of the former were slightly higher than those of the latter. This suggests that the changes were caused not by the effects of probucol as a cholesterol lowering agent, but mainly by the aging of the rabbits [ 181. Plasma HDL cholesterol level of probucol-treated rabbits decreased significantly during the experimental period (13 f 2 to 6 f 2
TABLE 1 PLASMA LIPID DATA OF STUDY A Samples were obtained from each rabbit just before and at the end of the study. Subfractions of lipoprotein cholesterol were determined after sequential ultracentrifugations of plasma. Mean f S.D.
Total cholesterol VLDL cholesterol LDL cholesterol HDL cholesterol Triglyceride
(mg/dl)
Probucol
Wmll
(mg/dl) (mg/dl) (mg/dll (mg/dll
initial final initial final initial final initial final initial final final
Control group (n = 7)
Probucol group tn = 6)
575 f II3 392 zt 96 228 f 78 38 f 30 325 f 52 328 f IO1 22zk 4 17* 4 739 f 86 251 + 24 not detectable
555 * 83 444* 74 224 f 53 l24* 66 314 f 64 314 f 97 13* 2 6* 2 913 f 301 494 f 282 42* 9
134 PROBUCOL GROUP
CONTROL GROUP
C-3,SmoF
C-5,lBmeF
C-7,lBnoF
Fig. 1. Traced figure of the atherosclerotic lesions on thoracic aorta in the rabbits of study A. Lesioned areas are shown in black.
(mg/dl), P < O.Ol), but that of the control rabbits did not (22 i 4 to 17 f 4, P > 0.05). Plasma concentration of probucol was undetectable in the control animals, and 42 f 9 pg/ml in the treated animals. This was almost the same level as that in patients treated with standard dosage of this drug
PI. In Fig. 1, lesioned areas in the aorta of each rabbit are shown in black. This figure clearly shows the effect of probucol for the inhibition of atherogenesis in aortas of the WHHL rabbits in the long-term study. We divided each aorta into two portions at the level of the first intercostal artery; the proximal ‘aortic arch’ and the distal ‘descending thoracic aorta’. The percentage area covered by atherosclerotic plaque was definitely less in the probucol-treated group of rabbits in both the aortic arch and the descending aorta (P < 0.001) (Table 2). When estimated with the mean percentage of the lesioned area, probucol was more effective in the descending thoracic aorta than in the aortic arch. The r-value calculated from a comparison of the percentage lesioned area of the aortic arch in treated and non-treated rabbits was 6.63, whereas that of the descending thoracic aorta was 10.33, suggesting that the effect of pro-
bucol was most noticeable in the descending thoracic aorta. Most lesions in the probucol-treated rabbits were observed in the ascending aorta. In the descending aorta, lesions were limited to the portions just distal to the orifices of branch arteries. In contrast, lesions of the control group of rabbits seemed to occur anywhere on the aorta, and without any predilection for branch sites. Figure 2 shows typical histological features of TABLE 2 PERCENT SURFACE AREA OF AORTA INVOLVED WITH ATHEROMATOUS PLAQUE IN CONTROL AND PROBUCOL-TREATED WHHL RABBITS OF STUDY A Percent area of aorta involved with plaques
(np’“=“rs,
Probucol group (n = 6)
94.1 ?? 3.8 82.6 f 14.5 87.7 f 8.7
41.9 f 19.2* 11.3 ?? 9.3* 23.0 zt 12.5*
Control
Aortic arch (%) Descending aorta (“/) Total thoracic aorta (o/o) Mean f S.D., *P < 0.001.
135
NORMAL RABBIT
WHHL CONTROL
WHHL PRCmcOL
Fig. 2. Aortic specimens of normal, probucol-treated WHHL and control WHHL rabbits. Specimens of WHHL rabbits were obtained from study A. The samples were prepared at the level of the intercostal artery and stained with Sudan III. ( x 30) I: lumen. iel: internal elastic lamina.
the aortic lesions of rabbits stained with Sudan III. We examined 6 samples of 6 probucol-treated rabbits, and 5 samples of 5 control rabbits. Samples were obtained at the level of the second intercostal arteries, When we counted the number of samples in which lipid droplets were observed beneath the internal elastic lamina with Sudan III staining, we found that 5 out of 5 of the samples from the control rabbits were positive, while only 1 in 6 of the samples obtained from the probucol-treated rabbits was stained with Sudan III beneath the internal elastic lamina. We also measured the distance between the internal elastic lamina and the top of the plaque under microscopy. The thickness was 480 f 50 pm in control group, and 280 f 120 pm in probucol-treated group (P < 0.01). We also present a microscopic appearance of an agematched (17 months) normal Japanese White rabbit, in which there is no atheromatous lesion. Table 3 shows the amount of cholesterol extracted from plaques of the aortic arch. Both free and total cholesterol mass, normalized for the wet weight of aortic tissue, were significantly less in lesions of probucol-treated rabbits, suggesting that the mass of lesions was definitely less in the probucol-treated animals also.
Study B
No significant changes were observed in lipid levels of either group of rabbits during the study period (Table 4). They did not differ significantly between the two groups either at the beginning or at the end of the study period. Aortic specimens from most of the control rabbits were almost completely covered with atherosclerotic lesions at the level of sampling (Fig. 3). However, probucol TABLE 3 CHOLESTEROL CONTENT IN ATHEROSCLEROTIC LESIONS OF RABBITS IN STUDY A Samples were obtained from atherosclerotic aortae of the rabbits in study A. Data are reported after normalizing for the wet weight of aortic tissue. WHHL rabbits
Free cholesterol Cholesteryl ester Total cholesterol
Control group (n = 7)
Probucol group (n = 6)
15.5 + 3.6 8.2 zt 3.6 23.7 f 2.5
3.4 ?? 1.7* 4.7 f 2.0 8.1 f 3.6*
(mg/p tissue, mean f S.D., *P < 0.01.)
136 TABLE 4 PLASMA LIPID DATA OF STUDY B Samples were obtained from each rabbit just before and at the end of the study. Subfractions of lipoprotein cholesterol were determined after. sequential ultracentrifugations of plasma. Mean f S.D. Probucol group
Control group Total cholesterol VLDL cholesterol LDL cholesterol HDL cholesterol Triglyceride
@W-W
initial final initial final initial final initial final initial final final
(mg/dl) (mg/dl) (mg/dl) (mg/dl)
Probucol
f&ml)
371 zt 85 399 zt 84 25 f I6 57 f 23 337 f 95 330 f 71 9* 3 l2* 4 224 zt 57 242 zt 43 not detectable
PROBUCOL
CONTROLGROUP
P-l
14mo M
P-2
14mo M
14mo M
P-3
14mo M
14mo M
P-4
14mo M
14mo M
C-2
14mo M
C-3
C-4
d
53 53 x9 58 61 37 3
II*
2
448 ?? x0 263 f II2 30* II
pearances of lesions in both groups of rabbits in study B. Small aliquots of lesions, which were completely covered with plaques, were obtained from lesions around the celiac artery. The number of foam cells in each sample was counted, and the percentages of smooth muscle cell (SMC)-derived foam cells among them were calculated. SMCderived foam cells were identified by their intracellular muscle fibers. Although the percentages of SMC-derived foam cells were 61.2 f 8.5% in samples of control rabbits, 90.1 f 7.0% of foam cells were SMC-derived in those of probucoltreated rabbits (P < 0.01). Typical photographs of lesions in both groups are shown in Fig. 4.
treatment diminished the extent of the lesions in spite of the fact that the treatment was started so late at 8 months of age. This is the age by which some lesions had already developed [9]. Statistically significant differences were observed between the probucol-treated and control rabbits in the percentage area of aortic intima covered with plaques (Table 5). In Table 6, the amount of extracted cholesterol from the lesioned area of the aortic arch is shown. The mean value of each fraction was less in the probucol-treated group, although the difference was not significant. We also studied electron microscopic ap-
C-l
369 f 345 f x7* 72 f 271 f 263 f II*
GROUP
Fig. 3. Traced figure of the atherosclerotic lesions on thoracic aorta in the rabbits of study B. Lesioned
arcas arc shown in black.
137 TABLE 5
TABLE 6
PERCENT SURFACE AREA OF AORTA INVOLVED WITH ATHEROMATOUS PLAQUE IN CONTROL AND PROBUCOL-TREATED WHHL RABBITS OF STUDY B
CHOLESTEROL CONTENT IN ATHEROSCLEROTIC LESIONS OF RABBITS IN STUDY B
Percent area of aorta involved with plaques
Aortic arch (%) Descending aorta (%) Total thoracic aorta (%)
Control group (n = 4)
Probucol group (?I = 4)
89.4 f 15.1 15.2 f 33.3 82.7 •t 22.6
58.2 f 11.6* 26.3 f 18.4** 38.1 f 12.l*
Mean f SD.
Samples were obtained from atherosclerotic aortae of the rabbits in study B. Data are reported after normalizing for the wet weight of aortic tissue. mg/g tissue, Mean f S.D. WHHL rabbits
Free cholesterol Cholesteryl ester Total cholesterol
Control group (n = 4)
Probucol group (n = 4)
13.3 f 3.8 5.0 f 0.5 18.4 f 3.8
9.2 f 3.7 4.3 f 2.5 13.5 f 6.1
fP < 0.02; **p < 0.05.
Discussion In study A, our data clearly showed that probucol protected the aorta of WHHL rabbits against the progression of atherosclerosis in the long term. It is also of interest whether or not there exists any differences in the microscopic
CONTROL
characteristics of atherosclerotic lesions between the two groups of rabbits, and the results are described in detail elsewhere [ 191. In short, lesions of probucol-treated rabbits consisted of much less macrophage-derived foam cells compared with those of control rabbits. In study B, it was demonstrated that probucol
PROBUCOL-TREATED
Fig. 4. Typical electron microscopic appearances of atherosclerotic plaques obtained from lesions around celiac artery of rabbits in study B. Left: Control rabbits, Right: Probucol-treated rabbits. Bars represent IO pm.
138 could prevent atherogenesis in WHHL rabbits even when the administration was started after atherosclerotic plaques had already developed. The average percentage lesioned area of 38.1 * 12.1% for the 14 months probucol-treated rabbits was similar or even a little less than that of 8-month-old control WHHL rabbits (54.2 f 18.8%), which we reported in our previous study [9]. Moreover, the fact that probucol causes regression of cutaneous and tendon xanthomas in FH patients [7] raises the possibility that regression of atherosclerosis might have occurred in aorta of probucol-treated rabbits of study B. In this study, we have shown also that probucol could stimulate elimination of macrophage-derived foam cells (Fig. 4). This not only confirms the previous report [19], but suggests the strong possibility of the regression of plaques with probucol. In both studies, there were similar tendencies between data of percentages of aortic surface area with plaques and the amount of cholesterol in plaques in each group of rabbits (Tables 2 and 3, 5 and 6). These data demonstrate that rabbits which show less surface area with plaque accumulate less cholesterol in lesions. Figure 2 also supports this observation; the lesions in the control group of rabbits were much thicker than those in the probucol-treated group of rabbits. The results in study A suggest that a continuous antiatherogenic effect of probucol can be expected in human FH patients when administration is started in childhood. Regarding the results in study B, it is conceivable that prevention of the progression of the lesions might also be expected even when the administration of the drug is begun after aortic lesions have developed. However, since probucol did not completely suppress the formation of atherosclerotic lesions, it is necessary to consider the putative mechanisms of initiation of atherosclerosis in areas where lesions occurred in spite of probucol treatment. We noted that most of the atheromatous plaques in the descending aorta of probucol-treated rabbits in study A were limited to the distal portion of branch arteries, while those of control group rabbits occurred almost anywhere on the entire surface of the descending aorta. Roach et al. have reported that, in cholesterol-fed rabbits, the initial aortic atherosclerotic lesions occurred focally [20];
they were seen distal to the orifices of branch arteries of the descending thoracic aorta, and diffusely in the ascending aorta and aortic arch. Carew et al. have demonstrated that, even in normal rabbits fed standard chow, the areas of aorta that are destined to become lesioned, degrade and store increased amounts of administered LDL [21]. Such areas appear identical to those reported by Roach et al. A common factor for the initiation of atherogenesis that may explain these observations, including our study A, is mechanical stress. In the absence of hypercholesterolemia, the mechanical stress does not result in atheromatous lesions. Age-matched normal Japanese white rabbits, whose aortae have been exposed to similar mechanical stress, develop no atheroma at all as shown in Fig. 2. However, when the mechanical stress is accompanied with hypercholesterolemia, initial lesions of atherosclerotic plaque occur. Recently, Thubrikar et al. reported that, if shear stress could be avoided, it might be possible to inhibit the formation of atherosclerotic lesions [22]. The influence of hypertension on atherogenesis was also investigated by Chobanian et al. who demonstrated that hypertension markedly stimulates atherogenesis in WHHL rabbits [23]. Because it is impossible to reduce shear stress in normotensive rabbits or men, the logical approach to atherosclerosis which could not be prevented by probucol would be to attain sufficient reduction of plasma cholesterol levels. In study on humans it has been reported that the cholesterol-lowering effect of probucol is not as strong as that of other drugs such as 3-hydroxy-3-methylglutaryl (HMG)CoA reductase inhibitors or anion exchange resins in a human study [24]. In our study, probucol also did not decrease plasma and LDL cholesterol levels in homozygous WHHL rabbits compared with control WHHL rabbits. Accordingly, it is possible that lesions which occurred despite probucol therapy might be diminished, if plasma levels of cholesterol could be kept lower. Recently, pravastatin (CS-514) a potent competitive inhibitor of HMG-CoA reductase, was reported to be effective for the prevention of coronary atherosclerosis in WHHL rabbits in association with a significant reduction of the plasma level of cholesterol [25]. Our present data show the antiatherogenic
139 effect of probucol on WHHL rabbits, an animal model for human FH patients. However, combination therapy with other drugs or methods that are known to lower plasma cholesterol level markedly, including HMG-CoA reductase inhibitors [26], anion exchange resins [27,28], or plasmapheresis [29], may be more effective in prevention of atherosclerosis in human patients with FH or severe hypercholesterolemia.
6
7
8
Acknowledgments 9
This work was supported partly by research grants of the Ministry of Education, Science and Culture of Japan (nos. 01304063, 01619503, 0204408 l), a research grant for intractable diseases and a grant for cardiovascular diseases (2A-1) from the Japanese Ministry of Health and Welfare, the Cell Science Research Foundation, CIBAGEIGY Foundation for the Promotion of Science, and the Japanese Foundation of Metabolism and Diseases. The authors thank Alan Chait and Kevin O’Brien for critical comments on this manuscript; Kenji Ishii, Noriaki Kume, Hideo Otani and Hidenori Arai for useful discussions; Yoshio Watanabe for providing some WHHL rabbits in Hitonobu Tomoike, Motoomi this study; Nakamura, Mitsutaka Sawada and Osamu Midorikawa for their collaboration; Makio Fujioka for excellent technical assistance in electron microscopy; Kyosuke Yoshitomi for providing probucol; Toshihiro Shigezumi for taking care of the rabbits.
IO
Glavind, J., Hartmann, S., Clemmesen, J., Jessen, K.E. and Dam, H., Studies on the role of lipoperoxides in human pathology. 11. The presence of peroxidized lipids in the atherosclerotic aorta, Acta. Pathol. Microbial. Stand., 30 (1952) I. Yamamoto, A., Matsuzawa, Y., Kishino. B.. Hayashi, R., Hirobe, K. and Kikkawa, T., Effects of probucol on homozygous cases of familial hypercholesterolemia. Atherosclerosis, 4X (1983) 157. Parthasarathy, S., Young, S.G., Witztum, J.L., Pittman. R.C. and Steinberg, D.. Probucol inhibits oxidative modification of low density lipoprotein, J. Clin. Invest.. 77 (1986) 641. Kita, T., Nagano, Y., Yokode. M., Ishii. K., Kume. N.. Ooshima, A., Yoshida, H. and Kawai. C.. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 592X. Nagano, Y.. Kita. T., Yokode, M.. Ishii. K.. Kume, N.. Otani. H., Arai, H. and Kawai, C.. Probucol does not affect lipoprotein metabolism in macrophages of Watanabe heritable hyperlipidemic rabbits, Arteriosclerosis, 9 (1989) 453.
II
Carew, T.E., Schwenke, D.C. and Steinberg, D., Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: Evidence that antioxidants in vivo
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I2
13
Palinski. W., Rosenfeld, M.E.. Yli-Herttuala, S.. Gurtner, G.C., Socher, S.S., Butler, S.W.. Parthasarathy, S.. Carcw, T.E.. Steinberg, D. and Witztum, J.L.. Low density lipoprotein undergoes oxidative modilication in viva. Proc. Natl. Acad. Sci. U.S.A., X6 ( 1989) 1372. Yli-Herttuala, S., Palinski, W., Rosenfeld, M.E., Parthasarathy, S.. Carew, T.E., Butler. S.. Witztum. J.L. and Steinberg, D., Evidence for the presence of oxidatively modified
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14
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Atherosclerosis,
131
ATHERO 04760
Probucol and atherosclerosis in the Watanabe heritable hyperlipidemic rabbit - long-term antiatherogenic effect and effects on established plaques Yutaka Nagano *, Tadashi Nakamura2, Yuji Matsuzawa2, Mikimaro Cho ‘, Yukihiko &da3 and Toru Kita’ ‘Department of Geriatric Medicine. Faculty of Medicine, Kyoro University. ‘Second Deparrmenr of Internal Medicine, School of Medicine, Osaka University, Osaka 553 and ‘Third Division, Department of Internal Medicine, Faculty of Medicine, Kyoro Universily. Kyoto 606 (Japan)
(Received 26 June, 1991) (Revised, received 8 October, 1991) (Accepted 25 October, 1991)
Summary
We performed two studies to investigate the effect of probucol on atherogenesis in vivo in the Watanabe heritable hyperlipidemic (WHHL) rabbit. In the first study (Study A), probucol was administered to 2-month-old WHHL rabbits, to evaluate its long-term effect. When killed at about 1.5 years of age, the percentage area of aorta covered with atherosclerotic plaque in probucol-treated rabbits was markedly less than that seen in non-treated rabbits (23.0 f 11.4”/0vs. 87.7 & 8. I%, M f S.D., P < 0.001). In the second study (study B), administration of probucol was commenced with 8-month-old WHHL rabbits to investigate whether the drug was effective for limiting atherosclerosis in rabbits in which plaques had already developed. When killed after 6 months of treatment, the percentage area of aorta covered with plaque was 38.1 i 12.1% in treated rabbits and 82.7 f 22.6% in non-treated rabbits (P < 0.02). Microscopic observations of lesions also supported the effect of probucol. Probucol treatment resulted in a change not only in the size but also the composition of lesions. Thus, probucol was effective in preventing atherosclerosis in long-term studies at both early and late stages. Key words: Foam cells; Macrophages; Antioxidants;
Introduction
Familial hypercholesterolemia (FH) is one of the most common inherited diseases. It is caused Correspondence
io:
Y. Nagano,
Department
of Geriatric
Medicine,Faculty of Medicine, Kyoto University, 54 Shogoin Kawaharacho Sakyo-ku, Kyoto 606, Japan.
Regression
by an abnormality of the receptor for low density lipoprotein (LDL). Patients with FH are usually affected with ischemic heart disease because of the development of premature atherosclerosis [ 11. Therefore, it is very important to prevent this process. In atheroma or in cutaneous and tendon xan-
132 thomas, lipid-storing foam cells have been recognized as a characteristic feature. Most of the foam cells observed in the early stages of atherosclerotic lesions have been shown to originate from monocyte-derived macrophages [2,3]. Involvement of lipid peroxides in atherogenesis has been reported [4-61, and probucol, a drug originally developed as an antioxidant, is effective in vivo for regression of xanthomas of FH patients [7]. Probucol also inhibits oxidative modification of LDL in vitro [8]. In view of these observations, we recently performed a study to evaluate the effect of probucol on atherosclerotic lesions in vivo in WHHL rabbits. The result revealed a significant protective effect of probucol against the progression of atherosclerosis when the drug was given at the age of 2 months, and this effect could be explained by its antioxidative action [9,10]. Similar results were also reported by Carew et al. [ll]. The data in these studies and other recent studies strongly suggest the existence of oxidized LDL in vivo [ 12,131, which cause foam cell transformation of macrophages. Therefore, inhibition of oxidative modification of LDL is a very important step for prevention of atherosclerosis. The effect of probucol as an antioxidative and antiatherogenic drug on WHHL rabbits is likely to be similar to that in FH patients, because both of them have an abnorma!ity of LDL receptor, which causes hypercholesterolemia and rapid progression of at erosclerosis. It should be noted also that P FH patients have to be treated for life. Therefore, in the present study we undertook a long-term treatment of WHHL rabbits with probucol. Since treatment of FH patients is not always started in childhood, we also investigated the effect of probucol on atherosclerotic lesions when the drug was started after definite aortic lesions had developed in WHHL rabbits. Materials and methods Animals and drug administration
Homozygous WHHL rabbits were bred in Kobe or in Kyoto by mating heterozygous or homozygous female WHHL rabbits with homozygous male WHHL rabbits. They were fed rabbit laboratory chow RM-1 (Oriental Yeast Co.) before the administration of probucol [ 141.
We performed two studies to evaluate the effect of probucol in WHHL rabbits. In study A, in which we investigated the long-term effect of probucol, 1 g/day of the drug was administered to 2-month-old WHHL rabbits (n = 6), and was continued until the rabbits were about 18 months old. In study B, in which we investigated the effect of administration of probucol to WHHL rabbits after they already had developed atherosclerotic lesions, the same amount of the drug was started in 8-month-old WHHL rabbits (n = 4) and the administration was continued for a further 6 months. We used 8-month-old rabbits because our previous study clearly showed that untreated WHHL rabbits of that age revealed definite lesions in their aortae [9]. In both studies, control WHHL rabbits (n = 7 for study A and n = 4 for study B) were fed the same amount of the standard rabbit chow. Two of the control rabbits were used for both studies. Probucol was simply mixed with the raw material of the chow without organic solvents. Blood was sampled after overnight fasting. Measurement of aortic plaque
In study A, control group rabbits were killed between the age of 14 and 19 months (mean 16.7 months) and the probucol group rabbits were 17 or 19 months (mean 18 months). In study B, all rabbits were killed at the age of 14 months. Sodium pentobarbital (100 mg) was administered intravenously for anesthesia. The entire thoracic aorta was isolated from each rabbit, opened longitudinally, and en face photographs of the inner surface were taken. We measured the percentages of intimal surface area involved with atheromatous plaque according to the method that we have described previously [9]. Briefly, photographs of aortae were copied to graph paper with magnification (x 2), and the total and lesioned area of aorta were measured. The entire thoracic aorta was divided into two portions (aortic arch and descending portion) at the level of the first intercostal artery. Percentages of the lesioned area were also measured for each part. Preparations for histological examinations
After photographs were taken, aliquots of aorta which contained the lesioned area were obtained
133 from the orifice of the second intercostal arteries. They were fixed with 10% formaldehyde solution of phosphate buffered saline (pH = 7.4), or frozen immediately for lipid staining studies.
In study B, specimens were obtained from lesions around the orifice of the celiac artery of both groups of rabbits. They were fixed with 2% glutaraldehyde, posttixed with osmic tetroxide, and prepared for electron microscopic observation with standard procedures.
from Ono pharmaceutical Co. (Osaka, Japan). The cholesterol level in each lipoprotein subfraction was measured after separation of lipoprotein with ultracentrifugation according to the method previously described [ 161. Plasma level of probucol was measured by HPLC as described elsewhere u71. Statistical analyses were performed with Student’s t-test. Changes in data of plasma level of lipids in the same group of rabbits were analyzed with paired t-test, and data between different groups of rabbits with unpaired t-test.
Analysis of lipid composition in plaques
Results
Electron microscopy
An aliquot of aortic specimen was obtained from the lesioned area of each rabbit just distal to the left subclavian artery. All samples were completely covered with plaques. Tissue adhering to the outer surface of the aorta was completely removed, and the samples were homogenized with a Polytron homogenizer. Total lipid was extracted by the method described by Folch et al. [ 151. The amount of cholesterol was analyzed using gas chromatography with 5 alpha-cholestane as an internal standard. Other assays
Plasma levels of lipids were measured by enzymatic methods using a commercial kit obtained
Study A
In Table 1, plasma level of lipids and probucol are shown. Plasma levels of cholesterol and triglycerides fell significantly during the experimental period (P < 0.05). When we compare the final level of plasma cholesterol between probucol-treated and control rabbits, the results of the former were slightly higher than those of the latter. This suggests that the changes were caused not by the effects of probucol as a cholesterol lowering agent, but mainly by the aging of the rabbits [ 181. Plasma HDL cholesterol level of probucol-treated rabbits decreased significantly during the experimental period (13 f 2 to 6 f 2
TABLE 1 PLASMA LIPID DATA OF STUDY A Samples were obtained from each rabbit just before and at the end of the study. Subfractions of lipoprotein cholesterol were determined after sequential ultracentrifugations of plasma. Mean f S.D.
Total cholesterol VLDL cholesterol LDL cholesterol HDL cholesterol Triglyceride
(mg/dl)
Probucol
Wmll
(mg/dl) (mg/dl) (mg/dll (mg/dll
initial final initial final initial final initial final initial final final
Control group (n = 7)
Probucol group tn = 6)
575 f II3 392 zt 96 228 f 78 38 f 30 325 f 52 328 f IO1 22zk 4 17* 4 739 f 86 251 + 24 not detectable
555 * 83 444* 74 224 f 53 l24* 66 314 f 64 314 f 97 13* 2 6* 2 913 f 301 494 f 282 42* 9
134 PROBUCOL GROUP
CONTROL GROUP
C-3,SmoF
C-5,lBmeF
C-7,lBnoF
Fig. 1. Traced figure of the atherosclerotic lesions on thoracic aorta in the rabbits of study A. Lesioned areas are shown in black.
(mg/dl), P < O.Ol), but that of the control rabbits did not (22 i 4 to 17 f 4, P > 0.05). Plasma concentration of probucol was undetectable in the control animals, and 42 f 9 pg/ml in the treated animals. This was almost the same level as that in patients treated with standard dosage of this drug
PI. In Fig. 1, lesioned areas in the aorta of each rabbit are shown in black. This figure clearly shows the effect of probucol for the inhibition of atherogenesis in aortas of the WHHL rabbits in the long-term study. We divided each aorta into two portions at the level of the first intercostal artery; the proximal ‘aortic arch’ and the distal ‘descending thoracic aorta’. The percentage area covered by atherosclerotic plaque was definitely less in the probucol-treated group of rabbits in both the aortic arch and the descending aorta (P < 0.001) (Table 2). When estimated with the mean percentage of the lesioned area, probucol was more effective in the descending thoracic aorta than in the aortic arch. The r-value calculated from a comparison of the percentage lesioned area of the aortic arch in treated and non-treated rabbits was 6.63, whereas that of the descending thoracic aorta was 10.33, suggesting that the effect of pro-
bucol was most noticeable in the descending thoracic aorta. Most lesions in the probucol-treated rabbits were observed in the ascending aorta. In the descending aorta, lesions were limited to the portions just distal to the orifices of branch arteries. In contrast, lesions of the control group of rabbits seemed to occur anywhere on the aorta, and without any predilection for branch sites. Figure 2 shows typical histological features of TABLE 2 PERCENT SURFACE AREA OF AORTA INVOLVED WITH ATHEROMATOUS PLAQUE IN CONTROL AND PROBUCOL-TREATED WHHL RABBITS OF STUDY A Percent area of aorta involved with plaques
(np’“=“rs,
Probucol group (n = 6)
94.1 ?? 3.8 82.6 f 14.5 87.7 f 8.7
41.9 f 19.2* 11.3 ?? 9.3* 23.0 zt 12.5*
Control
Aortic arch (%) Descending aorta (“/) Total thoracic aorta (o/o) Mean f S.D., *P < 0.001.
135
NORMAL RABBIT
WHHL CONTROL
WHHL PRCmcOL
Fig. 2. Aortic specimens of normal, probucol-treated WHHL and control WHHL rabbits. Specimens of WHHL rabbits were obtained from study A. The samples were prepared at the level of the intercostal artery and stained with Sudan III. ( x 30) I: lumen. iel: internal elastic lamina.
the aortic lesions of rabbits stained with Sudan III. We examined 6 samples of 6 probucol-treated rabbits, and 5 samples of 5 control rabbits. Samples were obtained at the level of the second intercostal arteries, When we counted the number of samples in which lipid droplets were observed beneath the internal elastic lamina with Sudan III staining, we found that 5 out of 5 of the samples from the control rabbits were positive, while only 1 in 6 of the samples obtained from the probucol-treated rabbits was stained with Sudan III beneath the internal elastic lamina. We also measured the distance between the internal elastic lamina and the top of the plaque under microscopy. The thickness was 480 f 50 pm in control group, and 280 f 120 pm in probucol-treated group (P < 0.01). We also present a microscopic appearance of an agematched (17 months) normal Japanese White rabbit, in which there is no atheromatous lesion. Table 3 shows the amount of cholesterol extracted from plaques of the aortic arch. Both free and total cholesterol mass, normalized for the wet weight of aortic tissue, were significantly less in lesions of probucol-treated rabbits, suggesting that the mass of lesions was definitely less in the probucol-treated animals also.
Study B
No significant changes were observed in lipid levels of either group of rabbits during the study period (Table 4). They did not differ significantly between the two groups either at the beginning or at the end of the study period. Aortic specimens from most of the control rabbits were almost completely covered with atherosclerotic lesions at the level of sampling (Fig. 3). However, probucol TABLE 3 CHOLESTEROL CONTENT IN ATHEROSCLEROTIC LESIONS OF RABBITS IN STUDY A Samples were obtained from atherosclerotic aortae of the rabbits in study A. Data are reported after normalizing for the wet weight of aortic tissue. WHHL rabbits
Free cholesterol Cholesteryl ester Total cholesterol
Control group (n = 7)
Probucol group (n = 6)
15.5 + 3.6 8.2 zt 3.6 23.7 f 2.5
3.4 ?? 1.7* 4.7 f 2.0 8.1 f 3.6*
(mg/p tissue, mean f S.D., *P < 0.01.)
136 TABLE 4 PLASMA LIPID DATA OF STUDY B Samples were obtained from each rabbit just before and at the end of the study. Subfractions of lipoprotein cholesterol were determined after. sequential ultracentrifugations of plasma. Mean f S.D. Probucol group
Control group Total cholesterol VLDL cholesterol LDL cholesterol HDL cholesterol Triglyceride
@W-W
initial final initial final initial final initial final initial final final
(mg/dl) (mg/dl) (mg/dl) (mg/dl)
Probucol
f&ml)
371 zt 85 399 zt 84 25 f I6 57 f 23 337 f 95 330 f 71 9* 3 l2* 4 224 zt 57 242 zt 43 not detectable
PROBUCOL
CONTROLGROUP
P-l
14mo M
P-2
14mo M
14mo M
P-3
14mo M
14mo M
P-4
14mo M
14mo M
C-2
14mo M
C-3
C-4
d
53 53 x9 58 61 37 3
II*
2
448 ?? x0 263 f II2 30* II
pearances of lesions in both groups of rabbits in study B. Small aliquots of lesions, which were completely covered with plaques, were obtained from lesions around the celiac artery. The number of foam cells in each sample was counted, and the percentages of smooth muscle cell (SMC)-derived foam cells among them were calculated. SMCderived foam cells were identified by their intracellular muscle fibers. Although the percentages of SMC-derived foam cells were 61.2 f 8.5% in samples of control rabbits, 90.1 f 7.0% of foam cells were SMC-derived in those of probucoltreated rabbits (P < 0.01). Typical photographs of lesions in both groups are shown in Fig. 4.
treatment diminished the extent of the lesions in spite of the fact that the treatment was started so late at 8 months of age. This is the age by which some lesions had already developed [9]. Statistically significant differences were observed between the probucol-treated and control rabbits in the percentage area of aortic intima covered with plaques (Table 5). In Table 6, the amount of extracted cholesterol from the lesioned area of the aortic arch is shown. The mean value of each fraction was less in the probucol-treated group, although the difference was not significant. We also studied electron microscopic ap-
C-l
369 f 345 f x7* 72 f 271 f 263 f II*
GROUP
Fig. 3. Traced figure of the atherosclerotic lesions on thoracic aorta in the rabbits of study B. Lesioned
arcas arc shown in black.
137 TABLE 5
TABLE 6
PERCENT SURFACE AREA OF AORTA INVOLVED WITH ATHEROMATOUS PLAQUE IN CONTROL AND PROBUCOL-TREATED WHHL RABBITS OF STUDY B
CHOLESTEROL CONTENT IN ATHEROSCLEROTIC LESIONS OF RABBITS IN STUDY B
Percent area of aorta involved with plaques
Aortic arch (%) Descending aorta (%) Total thoracic aorta (%)
Control group (n = 4)
Probucol group (?I = 4)
89.4 f 15.1 15.2 f 33.3 82.7 •t 22.6
58.2 f 11.6* 26.3 f 18.4** 38.1 f 12.l*
Mean f SD.
Samples were obtained from atherosclerotic aortae of the rabbits in study B. Data are reported after normalizing for the wet weight of aortic tissue. mg/g tissue, Mean f S.D. WHHL rabbits
Free cholesterol Cholesteryl ester Total cholesterol
Control group (n = 4)
Probucol group (n = 4)
13.3 f 3.8 5.0 f 0.5 18.4 f 3.8
9.2 f 3.7 4.3 f 2.5 13.5 f 6.1
fP < 0.02; **p < 0.05.
Discussion In study A, our data clearly showed that probucol protected the aorta of WHHL rabbits against the progression of atherosclerosis in the long term. It is also of interest whether or not there exists any differences in the microscopic
CONTROL
characteristics of atherosclerotic lesions between the two groups of rabbits, and the results are described in detail elsewhere [ 191. In short, lesions of probucol-treated rabbits consisted of much less macrophage-derived foam cells compared with those of control rabbits. In study B, it was demonstrated that probucol
PROBUCOL-TREATED
Fig. 4. Typical electron microscopic appearances of atherosclerotic plaques obtained from lesions around celiac artery of rabbits in study B. Left: Control rabbits, Right: Probucol-treated rabbits. Bars represent IO pm.
138 could prevent atherogenesis in WHHL rabbits even when the administration was started after atherosclerotic plaques had already developed. The average percentage lesioned area of 38.1 * 12.1% for the 14 months probucol-treated rabbits was similar or even a little less than that of 8-month-old control WHHL rabbits (54.2 f 18.8%), which we reported in our previous study [9]. Moreover, the fact that probucol causes regression of cutaneous and tendon xanthomas in FH patients [7] raises the possibility that regression of atherosclerosis might have occurred in aorta of probucol-treated rabbits of study B. In this study, we have shown also that probucol could stimulate elimination of macrophage-derived foam cells (Fig. 4). This not only confirms the previous report [19], but suggests the strong possibility of the regression of plaques with probucol. In both studies, there were similar tendencies between data of percentages of aortic surface area with plaques and the amount of cholesterol in plaques in each group of rabbits (Tables 2 and 3, 5 and 6). These data demonstrate that rabbits which show less surface area with plaque accumulate less cholesterol in lesions. Figure 2 also supports this observation; the lesions in the control group of rabbits were much thicker than those in the probucol-treated group of rabbits. The results in study A suggest that a continuous antiatherogenic effect of probucol can be expected in human FH patients when administration is started in childhood. Regarding the results in study B, it is conceivable that prevention of the progression of the lesions might also be expected even when the administration of the drug is begun after aortic lesions have developed. However, since probucol did not completely suppress the formation of atherosclerotic lesions, it is necessary to consider the putative mechanisms of initiation of atherosclerosis in areas where lesions occurred in spite of probucol treatment. We noted that most of the atheromatous plaques in the descending aorta of probucol-treated rabbits in study A were limited to the distal portion of branch arteries, while those of control group rabbits occurred almost anywhere on the entire surface of the descending aorta. Roach et al. have reported that, in cholesterol-fed rabbits, the initial aortic atherosclerotic lesions occurred focally [20];
they were seen distal to the orifices of branch arteries of the descending thoracic aorta, and diffusely in the ascending aorta and aortic arch. Carew et al. have demonstrated that, even in normal rabbits fed standard chow, the areas of aorta that are destined to become lesioned, degrade and store increased amounts of administered LDL [21]. Such areas appear identical to those reported by Roach et al. A common factor for the initiation of atherogenesis that may explain these observations, including our study A, is mechanical stress. In the absence of hypercholesterolemia, the mechanical stress does not result in atheromatous lesions. Age-matched normal Japanese white rabbits, whose aortae have been exposed to similar mechanical stress, develop no atheroma at all as shown in Fig. 2. However, when the mechanical stress is accompanied with hypercholesterolemia, initial lesions of atherosclerotic plaque occur. Recently, Thubrikar et al. reported that, if shear stress could be avoided, it might be possible to inhibit the formation of atherosclerotic lesions [22]. The influence of hypertension on atherogenesis was also investigated by Chobanian et al. who demonstrated that hypertension markedly stimulates atherogenesis in WHHL rabbits [23]. Because it is impossible to reduce shear stress in normotensive rabbits or men, the logical approach to atherosclerosis which could not be prevented by probucol would be to attain sufficient reduction of plasma cholesterol levels. In study on humans it has been reported that the cholesterol-lowering effect of probucol is not as strong as that of other drugs such as 3-hydroxy-3-methylglutaryl (HMG)CoA reductase inhibitors or anion exchange resins in a human study [24]. In our study, probucol also did not decrease plasma and LDL cholesterol levels in homozygous WHHL rabbits compared with control WHHL rabbits. Accordingly, it is possible that lesions which occurred despite probucol therapy might be diminished, if plasma levels of cholesterol could be kept lower. Recently, pravastatin (CS-514) a potent competitive inhibitor of HMG-CoA reductase, was reported to be effective for the prevention of coronary atherosclerosis in WHHL rabbits in association with a significant reduction of the plasma level of cholesterol [25]. Our present data show the antiatherogenic
139 effect of probucol on WHHL rabbits, an animal model for human FH patients. However, combination therapy with other drugs or methods that are known to lower plasma cholesterol level markedly, including HMG-CoA reductase inhibitors [26], anion exchange resins [27,28], or plasmapheresis [29], may be more effective in prevention of atherosclerosis in human patients with FH or severe hypercholesterolemia.
6
7
8
Acknowledgments 9
This work was supported partly by research grants of the Ministry of Education, Science and Culture of Japan (nos. 01304063, 01619503, 0204408 l), a research grant for intractable diseases and a grant for cardiovascular diseases (2A-1) from the Japanese Ministry of Health and Welfare, the Cell Science Research Foundation, CIBAGEIGY Foundation for the Promotion of Science, and the Japanese Foundation of Metabolism and Diseases. The authors thank Alan Chait and Kevin O’Brien for critical comments on this manuscript; Kenji Ishii, Noriaki Kume, Hideo Otani and Hidenori Arai for useful discussions; Yoshio Watanabe for providing some WHHL rabbits in Hitonobu Tomoike, Motoomi this study; Nakamura, Mitsutaka Sawada and Osamu Midorikawa for their collaboration; Makio Fujioka for excellent technical assistance in electron microscopy; Kyosuke Yoshitomi for providing probucol; Toshihiro Shigezumi for taking care of the rabbits.
IO
Glavind, J., Hartmann, S., Clemmesen, J., Jessen, K.E. and Dam, H., Studies on the role of lipoperoxides in human pathology. 11. The presence of peroxidized lipids in the atherosclerotic aorta, Acta. Pathol. Microbial. Stand., 30 (1952) I. Yamamoto, A., Matsuzawa, Y., Kishino. B.. Hayashi, R., Hirobe, K. and Kikkawa, T., Effects of probucol on homozygous cases of familial hypercholesterolemia. Atherosclerosis, 4X (1983) 157. Parthasarathy, S., Young, S.G., Witztum, J.L., Pittman. R.C. and Steinberg, D.. Probucol inhibits oxidative modification of low density lipoprotein, J. Clin. Invest.. 77 (1986) 641. Kita, T., Nagano, Y., Yokode. M., Ishii. K., Kume. N.. Ooshima, A., Yoshida, H. and Kawai. C.. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 592X. Nagano, Y.. Kita. T., Yokode, M.. Ishii. K.. Kume, N.. Otani. H., Arai, H. and Kawai, C.. Probucol does not affect lipoprotein metabolism in macrophages of Watanabe heritable hyperlipidemic rabbits, Arteriosclerosis, 9 (1989) 453.
II
Carew, T.E., Schwenke, D.C. and Steinberg, D., Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: Evidence that antioxidants in vivo
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I2
13
Palinski. W., Rosenfeld, M.E.. Yli-Herttuala, S.. Gurtner, G.C., Socher, S.S., Butler, S.W.. Parthasarathy, S.. Carcw, T.E.. Steinberg, D. and Witztum, J.L.. Low density lipoprotein undergoes oxidative modilication in viva. Proc. Natl. Acad. Sci. U.S.A., X6 ( 1989) 1372. Yli-Herttuala, S., Palinski, W., Rosenfeld, M.E., Parthasarathy, S.. Carew, T.E., Butler. S.. Witztum. J.L. and Steinberg, D., Evidence for the presence of oxidatively modified
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14
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