Atherosclerosis, 97 (1992) 143- 148 0 1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved. 0021-9150/92/%05.00
143
Printed and Published in Ireland
ATHERO 04927
Inhibition of intimal thickening of the carotid artery of rabbits and of outgrowth of explants of aorta by probucol Masaki Shinomiya, Kohji Shirai, Yasushi Saito and Sho Yoshida The Second Department of Internal Medicine, School of Medicine, Chiba University Chuo-ku, Chiba Ciry, 260 fJapanJ
(Received 6 May, 1992) (Revised, received 28 July, 1992) (Accepted 3 August, 1992)
Summary
The effect of administration of probucol in preventing intimal thickening of rabbit carotid artery after balloon catheter injury and the mechanism of action of the drug were studied. Groups of 6 male New Zealand-White rabbits were given normal diet (Group I), high cholesterol diet (Group II) or high cholesterol diet plus probucol (Group III) for 4 weeks. Balloon catheter injury was made in week 2 and animals were killed in week 4. No significant differences in the total cholesterol levels in Groups II and III were found in week 4. The medians of areas of the intimal layer in cross-sections of the carotid arteries of Groups I, II and III were 0.237, 0.475 and 0.309 mm2, respectively. Thus high-cholesterol diet increased the thickness of the intimal layer and probucol reduced its effect. There were no significant differences in the areas of the medial layers in these 3 groups. For the examination of the mechanism of the effect of probucol, rabbits were given chow containing 0.5% cholesterol with and without 0.5% probucol (7 rabbits each) and then the numbers of explants from their aortas showing outgrowth were compared. The plasma total cholesterol levels of these two groups were the same. The probucol concentrations in the plasma and aorta of the former group were 18.6 f 13.2 &ml and 7.3 f 5.4 &g wet tissue, respectively. The number of explants showing outgrowth on day 14 was suppressed by 34% in the probucol-treated group. These results show that administration of probucol suppresses intimal thickening of the carotid artery after injury by a balloon catheter and that its effect may be partly explained by its suppression of smooth muscle cell migration and growth.
Key words: Balloon catheter injury; Intimal thickening; Outgrowth from explants; Probucol
Introduction Correspondence to: Dr. Yasushi Saito, The Second Department
of Internal Medicine, School of Medicine, Chiba University, Chuo-ku, Chiba City, 260 Japan. Tel.: 81-43-222-7171 ex. 2167, 2168; Fax: 81-43-222-7853.
Atheromatous lesions are identified by many criteria, such as foam cell formation, proliferations of cells and extracellular matrices, and calcium deposition. As several different processes are
144
responsible for these events [l], the prevention of these lesions might be achieved by blocking some of these processes. Probucol is used as a hypolipidemic drug [2,3] to reduce the plasma cholesterol level. In rabbits given a high cholesterol diet it is also reported to prevent atheromatous plaque formation in the arterial wall without reducing the high plasma cholesterol level significantly [4]. Probucol reduced the area of atheromatous lesion in Watanabe heritable hyperlipidemic (WHHL) rabbits without reducing plasma cholesterol level [5,6]. These findings suggest that its anti-atherogenic effect is not related to the plasma cholesterol level. Probucol has been thought to act on arterial wall cells by inhibiting the release of interleukin-1 from macrophages [7] or the oxidative moditication [8,9] on LDL induced by the interaction of LDL with endothelial cells [8,9]. These effects might be closely related to its prevention of atheromatous plaque formation. There are reports that anti-PDGF antibody [lo], anti-basic FGF antibody [ll] and other drugs [12] prevent development of intimal thickening after balloon catheter injury. However, the effect of probucol on intimal thickening of the carotid artery has not yet been reported. In the present study, we examined the effect of pretreatment with probucol on the development of intimal thickening in rabbit carotid artery. As intimal thickening is mainly caused by the migration and proliferation of smooth muscle cells, to clarify the mechanism of the action of probucol, we examined the effect of probucol treatment of rabbits on outgrowth of explants of their arterial tissue and determined probucol concentration in those tissues. Materials and Methods Animal conditions Ballooning study. Groups of 6 male New Zea-
land-white rabbits weighing 2 kg were fed for 4 weeks on 100 g/day of (I) normal diet (standard chow purchased from Oriental Co., Tokyo), (II) chow containing 0.5% cholesterol (high cholesterol diet) and (III) high cholesterol diet containing 0.5% probucol. Pure probucol was a gift from Merrell Dow Pharmaceuticals.
Balloon catheter injury. Balloon catheter injury was made in week 2 and animals were killed in week 4. The right carotid artery of the rabbits was mechanically injured by passing an inflatable embolectomy catheter (Fogarty 3F catheter, American Edwards Laboratories, Santa Ana, CA) along the length of the vessel three times [ 131. This was done under sterile conditions in animals under anesthesia with pentobarbital. Measurement of thickening of intimal layer. Two weeks after the balloon catheter injury, the rabbits were killed under anesthesia by exsanguination from the left carotid artery. The right carotid artery was removed, cut into 4 portions of equal length and stored in 10% formalin solution. Preparations were made for light microscopy and stained with Hematoxylin-Eosin and van GiesonElastica stain. The areas of intimal and medial layers in cross-sections of the artery were calculated using an image scanner (Epson model GT-lOOV, Tokyo, Japan). To determine the optimum time for determination of intimal thickening in preliminary experiments, the cross-sectional areas in weeks 0, 1, 2, 3 and 4 were compared. Intimal thickening was recognized in week 1, increased until week 3 and decreased in week 4. Subsequently, intimal thickenings in week 2 was compared. Rabbits for primary explants. For experiments on primary explants of aorta, one group of 7 rabbits was given high cholesterol diet (containing 0.5% cholesterol) and another 7 rabbits was given high cholesterol plus probucol diet (containing 0.5% cholesterol and 0.5% probucol). These rabbits were killed for examination 4 weeks later. Explants of rabbit aorta. Rabbits were killed by anesthesia with sodium pentobarbital (50 mg/kg), which satisfies the ethical concerns regarding experimental animals. Blood was removed under sterile conditions with a cannula inserted into the carotid artery. With the feeding regimen used the aorta showed no microscopic indications of atherosclerotic lesions such as fatty streaks or fibrous plaques. Explantation to examine smooth muscle cell (SMC) outgrowth was carried out as reported previously [14]. The method is based on that of Fischer-Dzoga et al. [ 151. Briefly, the tissue was cut into small pieces (2 mm x 2 mm square) and placed in T-25 flasks (Corning Co., NY) with
145
1 ml of medium containing DME (Grand Island Biological Co., Grand Island, NY) supplemented with 0.67 mg/ml of sodium bicarbonate, 0.01 mg/ml of gentamicin and 10% bovine fetal serum (Gibco Laboratories, Grand Island NY) and incubated at 37OC in a C02(5%) incubator. One hundred pieces were obtained from each rabbit. McMurray et al. showed [16] that the cutting method and time between sacrifice of animals and explant of aorta affect the proportion of explants showing outgrowth. By our method, no difference was observed between groups in the adhesion of explanted specimens. Outgrowth was examined microscopically and was judged positive when one or more cells growing out of the specimen were seen. The number of explants showing outgrowth was expressed as a percentage of the total number of explants examined and the percentages reached to 100% finally in the adhered explants. Chemical analyses A volume of 0.1 ml of serum was extracted with chloroform/methanol (2:l) and the probucol concentration of the extract was determined by high performance liquid chromatography (HPLC) using a PBondapack Cis column (13 x 300 mm, Waters Co., MA) by the procedure described by Satonin and Coutant [ 171. The mobile phase was acetonitrile/3% acetic acid (88: 12). Plasma total cholesterol was determined by the method of Richmond [18]. All reagents were of experimental grade. Statistics Data on the areas of the intima and media are expressed as the medians. The ratios of explants showing outgrowth are expressed as means and standard deviations. The unpaired t-test was used for comparison of two groups and P < 0.05 was considered significant. Results
Effect of probucol administration on intimal thickening of carotid artery (Fig. I) The medians of areas of the intimal layer in Groups I (normal diet), II (high cholesterol diet) and III (high cholesterol plus probucol diet) were 0.237, 0.475 and 0.309 mm*, respectively. The dif-
lntima
1.25,
Media
1
1 .oo
0.75
i
%
or
0
i
0
I I
0.50
t
4 0.25
t 0.00
L
I
I
HCD ND
HCD HCD+P
ND
HCD+P
Group Fig. 1. Areas of intimal and medial layers of rabbit carotid artery 2 weeks after balloon catheter injury. Each circle represents the median of areas of four portions from one rabbit. ND, Group I (normal diet), HCD, Group II (high cholesterol diet), HCD + P, Group III (high cholesterol diet containing probucol). Bars represent medians for each group. *Significant differences between the two groups (P < 0.05).
ference between the values for Groups I and II (P < 0.05) was significant. The difference between the values for Groups II and III (P < 0.05) was also significant. The medians of areas of the medial layers in Groups I, II and III were 0.886, 0.922 and 0.869 mm’, respectively, which were not significantly different. Intimal thickening observed in these experiments was mainly composed of smooth muscle cells; small amounts of monocytes or lymphocytes or granulocytes were observed as reported previously [ 121. However, significant differences in cell types among the three groups were not observed (data not shown). Probucol concentration In week 4, the mean probucol concentration in the plasma of probucol treated animals was 18.6 f 13.2 &ml and that in the aorta was 7.3 f 5.4 kg/g wet tissue. Probucol administration did not alter the plasma total cholesterol level (59 f 12, 681 f 301 and 673 f 236 mg/dl in Groups I, II and III, respectively).
146
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8’(80
60 _
- 60
- 40
20
L
0
3
5
7
9
11
13
15
--‘O 17
Day Fig. 2. Explants from rabbit aorta showing outgrowth. The numbers of explants from rabbit aorta showing outgrowth as percentages of the total numbers of explants are shown as functions of the day after explantation. Points and bars represent means and standard deviations. Open circles show results for aorta from rabbits fed 0.5% cholesterol and closed circles show these for aorta from rabbits fed 0.5% cholesterol plus 0.5% probucol. *Significant differences between the two groups (P < 0.05).
Outgrowth of explants of rabbit aorta showing outgrowth (Fig. 2) Figure 2 shows the percentages of explants of rabbit aorta showing outgrowth. The percentages for explants from rabbits given high cholesterol plus probucol diet were constantly lower than those of explants from the group given high cholesterol diet. The inhibitions of outgrowth by probucol were 42.0% on day 10 and 33.9% on day 14. Discussion The present results indicated that the intimal thickening of the carotid artery of rabbits after balloon catheter injury was inhibited by preceding administration of probucol. This effect was not due to a change in the plasma lipid level. These results are consistent with reports that probucol suppresses the development of atherosclerotic lesions in animals given high cholesterol diet without decreasing the plasma lipid level [4]. Therefore, we examined the mechanism of action
of probucol other than by decrease in the plasma lipid level. In the carotid artery, the remarkable atheromatous lesion was not observed macroscopically. There were no significant differences in the degree of lipid deposition or the number of foam cells. The difference in findings compared to those of Daugherty et al. [4] may be due to the differences in feeding periods or anatomical localization and experimental conditions. The intimal thickening after balloon catheterization can be explained by increase of SMC [ 121, due to migration of medial SMC into the intima and their proliferation in the intima. As probucol did not decrease the plasma lipid level, it possibly affected these processes directly. Therefore we examined whether it reached cells in this tissue. In our animals given high cholesterol diet and probucol, considerable probucol was detected in the arterial wall, and its plasma concentration was similar to that in probucol-treated humans [19] and WHHL rabbits [20]. The outgrowth from the explants of these probucol-containing tissues, which may reflect the migration and proliferation of the cells, was less than that from control tissue containing no probucol. Because this tissue contains lymphocytes, polymorphonuclear cells and monocyte-macrophages, the action of probucol should be clarified in relation to cell-to-cell interactions, including growth and migration factors and cytokines produced by various cells [21-231. Probucol in the specimens may reduce the proliferation of explants showing outgrowth by exerting some effect on these functions. The doubling time of SMC in passage 2 from rabbits fed a high cholesterol diet was 0.88 days and that from those on a high cholesterol diet plus probucol was 0.57 days (data not shown). However, the other effect of probucol on SMC should also be considered because SMCs play an important role in the formation of atheromatous lesions including phenotypic changes of the cells [13,24]. These present results indicate that probucol suppresses the cell migration and/or cell proliferation observed in the early stages of formation of atherosclerotic lesions, resulting in suppression of intimal thickening.
147
Acknowledgment The authors express great thanks to Dr. Bunshiro Akikusa, Second Department of Pathology, School of Medicine, Chiba University, for helpful discussions concerning the pathological findings of the carotid artery. The authors also express their gratitude to Ms. Keiko Umemiya, Tomoko Koshikawa and Mihoko Nakamura for technical assistance. This study was supported by a grant from the Japanese Ministry of Welfare to the study group led by Prof. Y. Yazaki on ‘The Molecular Biology Study on the Development of Cardiovascular Disease.’ This study was also supported by grants from the Uehara Memorial Foundation and the Funai Foundation.
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References Ross, R., The pathogenesis of atherosclerosis - an update, N. Engl. J. Med., 314 (1986) 488. Hunninghake, D.B., Bell, C. and Olson. L., Effect of probucol on plasma lipids and lipoproteins in type IIb hyperlipoproteinemia, Atherosclerosis, 37 (1980) 469. Atmeh, R.E., Stewart, J.M., Boag, D.E., Packard, C.J., Lorimer, A.R. and Shepherd, J., The hypolipidemic action of probucol: A study of its effects on high and low density lipoproteins, J. Lipid Res., 24 (1983) 588. Daugherty, A., Zweifel, B.S. and Schongeld, G. Probucol attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits, Br. J. Pharmacol., 98 (1989) 612. 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. Nat]. Acad. Sci. USA, 84 (1987) 5928. Carew, T.E., Schwenke, D.C. and Steinberg, D., Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: Evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streak and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit, Proc. Natl. Acad. Sci. USA., 84 (1987) 7725. Ku, G., Doherty, N.S., Wolos, J.A. and Jackson, R.L., Inhibition by probucol of interleukin-1 secretion and its implication in atherosclerosis, Am. J. Cardiol., 62 (1988) 778. 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.
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Bamhart, R.L., Busch, S.J. and Jackson, R.L., Concentration-dependent antioxidant activity of probucol in low density lipoproteins in vitro: probucol degradation precedes lipoprotein oxidation, J. Lipid Res., 30 (1989) 1703. Ferns, G.A.A., Raines, E.W., Sprugel, K.H., Motani, A.S., Reidy, M.A. and Ross, R., Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF, Science, 253 (1991) 1129. Linder, V. and Reidy, M.A., Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor, Proc. Natl. Acad. Sci. USA, 88 (1991) 3739. Ferns, G.A.A., Stewart-Lee, A.L. and Anggard, E.E., Arterial response to mechanical injury: balloon catheter de-endothelialization, Atherosclerosis, 92 (1992) 89. Manderson, J.A., Mosse, P.R.L., Safstrom, J.A., Young, S.B. and Campbell, G.R., Balloon catheter injury to rabbit carotid artery, Arteriosclerosis, 9 (1989) 289. Morisaki, N., Kanzaki, T., Fujiyama, Y., Oosawa, I., Shirai, K., Matsuoka, N., Saito, Y. and Yoshida, S., Metabolism of n-3 polyunsaturated fatty acids and modification of phospholipids in cultured rabbit aortic smooth muscle cells, J. Lipid Res., 26 (1985) 930. Ficher-Dzoga, K., Jones, R.M., Vesselinovitch, D. and Wissler, R.W., Ultrastructural and immunohistochemical studies of primary cultures of aortic medial cells, Exp. Mol. Pathol., 18 (1973) 162. McMurray, H.F., Parrott, D.P. and Bowyer, D.E., A standardized method of culturing aortic explants, suitable for the study of factors affecting the phenotypic modulation, migration and proliferation of aortic smooth muscle cells, Atherosclerosis, 86 (1991) 227. Satonin, D.K. and Coutant, J.E., Comparison of gas liquid and high performance chromatography chromatography for the analysis of probucol in plasma, J. Chromatogr., 380 (1986) 401. Richmond, W., Preparation and properties of cholesterol oxidase from Nocurdiu sp. and its application to the enzymatic assay of total cholesterol in serum, Clin. Chem., 19 (1973) 1350. Nakamura, T., Ueyama, Y., Funabashi, T., Yamashita, S., Kameda-Takemura, K., Kubo, M., Yamada, K. and Matsuzawa, Y., Non-macrophage-related accumulation of cholesterol during probucol treatment in familial two cases, hypercholesterolemia: reports of Atherosclerosis, 92 (1992) 193. Nagano, Y., Nakamura, T., Matsuzawa, Y., Cho, M., Ueda, Y. and Kita, T., Probucol and atherosclerosis in the Watanabe heritable hyperlipidemic rabbit - Long-term antiatherogenic effect and effect on established plaques, Atherosclerosis, 92 (1992)131. O’Brien, K., Nagano, Y., Gown, A., Kita, Y. and Chait, A., Probucol treatment affects the cellular composition but not anti-oxidized low density lipoprotein immunoreactivity of plaques from Watanabe heritable hypcrlipidemic rabbits, Arterioscler. Thromb., I I (1991) 751.
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Walker, L.N., Bowen-Pope, D.F., Ross, R. and Reidy, M.A., Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury, Proc. Natl. Acad. Sci. USA, 83 (1986) 7311. Weidinger, F.F., McLenachan, J.M., Cybulsky, MI., Fallon, J.T., Hollenberg, N.K., Cooke, J.P. and Ganz, P., Hypercholesterolemia enhances macrophage recruitment
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and dysfunction of regenerated endothelium after balloon catheter injury of the rabbits iliac artery, Circulation, 84 (1991) 755. Thyberg, J.T., Hedin, U., Sjolund, M., Palmberg, L. and Bottger, B.A., Regulation of differentiated properties and proliferation of arterial smooth muscle cells, Arterioscler. Thromb., 10 (1991) 966.
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Printed and Published in Ireland
ATHERO 04927
Inhibition of intimal thickening of the carotid artery of rabbits and of outgrowth of explants of aorta by probucol Masaki Shinomiya, Kohji Shirai, Yasushi Saito and Sho Yoshida The Second Department of Internal Medicine, School of Medicine, Chiba University Chuo-ku, Chiba Ciry, 260 fJapanJ
(Received 6 May, 1992) (Revised, received 28 July, 1992) (Accepted 3 August, 1992)
Summary
The effect of administration of probucol in preventing intimal thickening of rabbit carotid artery after balloon catheter injury and the mechanism of action of the drug were studied. Groups of 6 male New Zealand-White rabbits were given normal diet (Group I), high cholesterol diet (Group II) or high cholesterol diet plus probucol (Group III) for 4 weeks. Balloon catheter injury was made in week 2 and animals were killed in week 4. No significant differences in the total cholesterol levels in Groups II and III were found in week 4. The medians of areas of the intimal layer in cross-sections of the carotid arteries of Groups I, II and III were 0.237, 0.475 and 0.309 mm2, respectively. Thus high-cholesterol diet increased the thickness of the intimal layer and probucol reduced its effect. There were no significant differences in the areas of the medial layers in these 3 groups. For the examination of the mechanism of the effect of probucol, rabbits were given chow containing 0.5% cholesterol with and without 0.5% probucol (7 rabbits each) and then the numbers of explants from their aortas showing outgrowth were compared. The plasma total cholesterol levels of these two groups were the same. The probucol concentrations in the plasma and aorta of the former group were 18.6 f 13.2 &ml and 7.3 f 5.4 &g wet tissue, respectively. The number of explants showing outgrowth on day 14 was suppressed by 34% in the probucol-treated group. These results show that administration of probucol suppresses intimal thickening of the carotid artery after injury by a balloon catheter and that its effect may be partly explained by its suppression of smooth muscle cell migration and growth.
Key words: Balloon catheter injury; Intimal thickening; Outgrowth from explants; Probucol
Introduction Correspondence to: Dr. Yasushi Saito, The Second Department
of Internal Medicine, School of Medicine, Chiba University, Chuo-ku, Chiba City, 260 Japan. Tel.: 81-43-222-7171 ex. 2167, 2168; Fax: 81-43-222-7853.
Atheromatous lesions are identified by many criteria, such as foam cell formation, proliferations of cells and extracellular matrices, and calcium deposition. As several different processes are
144
responsible for these events [l], the prevention of these lesions might be achieved by blocking some of these processes. Probucol is used as a hypolipidemic drug [2,3] to reduce the plasma cholesterol level. In rabbits given a high cholesterol diet it is also reported to prevent atheromatous plaque formation in the arterial wall without reducing the high plasma cholesterol level significantly [4]. Probucol reduced the area of atheromatous lesion in Watanabe heritable hyperlipidemic (WHHL) rabbits without reducing plasma cholesterol level [5,6]. These findings suggest that its anti-atherogenic effect is not related to the plasma cholesterol level. Probucol has been thought to act on arterial wall cells by inhibiting the release of interleukin-1 from macrophages [7] or the oxidative moditication [8,9] on LDL induced by the interaction of LDL with endothelial cells [8,9]. These effects might be closely related to its prevention of atheromatous plaque formation. There are reports that anti-PDGF antibody [lo], anti-basic FGF antibody [ll] and other drugs [12] prevent development of intimal thickening after balloon catheter injury. However, the effect of probucol on intimal thickening of the carotid artery has not yet been reported. In the present study, we examined the effect of pretreatment with probucol on the development of intimal thickening in rabbit carotid artery. As intimal thickening is mainly caused by the migration and proliferation of smooth muscle cells, to clarify the mechanism of the action of probucol, we examined the effect of probucol treatment of rabbits on outgrowth of explants of their arterial tissue and determined probucol concentration in those tissues. Materials and Methods Animal conditions Ballooning study. Groups of 6 male New Zea-
land-white rabbits weighing 2 kg were fed for 4 weeks on 100 g/day of (I) normal diet (standard chow purchased from Oriental Co., Tokyo), (II) chow containing 0.5% cholesterol (high cholesterol diet) and (III) high cholesterol diet containing 0.5% probucol. Pure probucol was a gift from Merrell Dow Pharmaceuticals.
Balloon catheter injury. Balloon catheter injury was made in week 2 and animals were killed in week 4. The right carotid artery of the rabbits was mechanically injured by passing an inflatable embolectomy catheter (Fogarty 3F catheter, American Edwards Laboratories, Santa Ana, CA) along the length of the vessel three times [ 131. This was done under sterile conditions in animals under anesthesia with pentobarbital. Measurement of thickening of intimal layer. Two weeks after the balloon catheter injury, the rabbits were killed under anesthesia by exsanguination from the left carotid artery. The right carotid artery was removed, cut into 4 portions of equal length and stored in 10% formalin solution. Preparations were made for light microscopy and stained with Hematoxylin-Eosin and van GiesonElastica stain. The areas of intimal and medial layers in cross-sections of the artery were calculated using an image scanner (Epson model GT-lOOV, Tokyo, Japan). To determine the optimum time for determination of intimal thickening in preliminary experiments, the cross-sectional areas in weeks 0, 1, 2, 3 and 4 were compared. Intimal thickening was recognized in week 1, increased until week 3 and decreased in week 4. Subsequently, intimal thickenings in week 2 was compared. Rabbits for primary explants. For experiments on primary explants of aorta, one group of 7 rabbits was given high cholesterol diet (containing 0.5% cholesterol) and another 7 rabbits was given high cholesterol plus probucol diet (containing 0.5% cholesterol and 0.5% probucol). These rabbits were killed for examination 4 weeks later. Explants of rabbit aorta. Rabbits were killed by anesthesia with sodium pentobarbital (50 mg/kg), which satisfies the ethical concerns regarding experimental animals. Blood was removed under sterile conditions with a cannula inserted into the carotid artery. With the feeding regimen used the aorta showed no microscopic indications of atherosclerotic lesions such as fatty streaks or fibrous plaques. Explantation to examine smooth muscle cell (SMC) outgrowth was carried out as reported previously [14]. The method is based on that of Fischer-Dzoga et al. [ 151. Briefly, the tissue was cut into small pieces (2 mm x 2 mm square) and placed in T-25 flasks (Corning Co., NY) with
145
1 ml of medium containing DME (Grand Island Biological Co., Grand Island, NY) supplemented with 0.67 mg/ml of sodium bicarbonate, 0.01 mg/ml of gentamicin and 10% bovine fetal serum (Gibco Laboratories, Grand Island NY) and incubated at 37OC in a C02(5%) incubator. One hundred pieces were obtained from each rabbit. McMurray et al. showed [16] that the cutting method and time between sacrifice of animals and explant of aorta affect the proportion of explants showing outgrowth. By our method, no difference was observed between groups in the adhesion of explanted specimens. Outgrowth was examined microscopically and was judged positive when one or more cells growing out of the specimen were seen. The number of explants showing outgrowth was expressed as a percentage of the total number of explants examined and the percentages reached to 100% finally in the adhered explants. Chemical analyses A volume of 0.1 ml of serum was extracted with chloroform/methanol (2:l) and the probucol concentration of the extract was determined by high performance liquid chromatography (HPLC) using a PBondapack Cis column (13 x 300 mm, Waters Co., MA) by the procedure described by Satonin and Coutant [ 171. The mobile phase was acetonitrile/3% acetic acid (88: 12). Plasma total cholesterol was determined by the method of Richmond [18]. All reagents were of experimental grade. Statistics Data on the areas of the intima and media are expressed as the medians. The ratios of explants showing outgrowth are expressed as means and standard deviations. The unpaired t-test was used for comparison of two groups and P < 0.05 was considered significant. Results
Effect of probucol administration on intimal thickening of carotid artery (Fig. I) The medians of areas of the intimal layer in Groups I (normal diet), II (high cholesterol diet) and III (high cholesterol plus probucol diet) were 0.237, 0.475 and 0.309 mm*, respectively. The dif-
lntima
1.25,
Media
1
1 .oo
0.75
i
%
or
0
i
0
I I
0.50
t
4 0.25
t 0.00
L
I
I
HCD ND
HCD HCD+P
ND
HCD+P
Group Fig. 1. Areas of intimal and medial layers of rabbit carotid artery 2 weeks after balloon catheter injury. Each circle represents the median of areas of four portions from one rabbit. ND, Group I (normal diet), HCD, Group II (high cholesterol diet), HCD + P, Group III (high cholesterol diet containing probucol). Bars represent medians for each group. *Significant differences between the two groups (P < 0.05).
ference between the values for Groups I and II (P < 0.05) was significant. The difference between the values for Groups II and III (P < 0.05) was also significant. The medians of areas of the medial layers in Groups I, II and III were 0.886, 0.922 and 0.869 mm’, respectively, which were not significantly different. Intimal thickening observed in these experiments was mainly composed of smooth muscle cells; small amounts of monocytes or lymphocytes or granulocytes were observed as reported previously [ 121. However, significant differences in cell types among the three groups were not observed (data not shown). Probucol concentration In week 4, the mean probucol concentration in the plasma of probucol treated animals was 18.6 f 13.2 &ml and that in the aorta was 7.3 f 5.4 kg/g wet tissue. Probucol administration did not alter the plasma total cholesterol level (59 f 12, 681 f 301 and 673 f 236 mg/dl in Groups I, II and III, respectively).
146
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8’(80
60 _
- 60
- 40
20
L
0
3
5
7
9
11
13
15
--‘O 17
Day Fig. 2. Explants from rabbit aorta showing outgrowth. The numbers of explants from rabbit aorta showing outgrowth as percentages of the total numbers of explants are shown as functions of the day after explantation. Points and bars represent means and standard deviations. Open circles show results for aorta from rabbits fed 0.5% cholesterol and closed circles show these for aorta from rabbits fed 0.5% cholesterol plus 0.5% probucol. *Significant differences between the two groups (P < 0.05).
Outgrowth of explants of rabbit aorta showing outgrowth (Fig. 2) Figure 2 shows the percentages of explants of rabbit aorta showing outgrowth. The percentages for explants from rabbits given high cholesterol plus probucol diet were constantly lower than those of explants from the group given high cholesterol diet. The inhibitions of outgrowth by probucol were 42.0% on day 10 and 33.9% on day 14. Discussion The present results indicated that the intimal thickening of the carotid artery of rabbits after balloon catheter injury was inhibited by preceding administration of probucol. This effect was not due to a change in the plasma lipid level. These results are consistent with reports that probucol suppresses the development of atherosclerotic lesions in animals given high cholesterol diet without decreasing the plasma lipid level [4]. Therefore, we examined the mechanism of action
of probucol other than by decrease in the plasma lipid level. In the carotid artery, the remarkable atheromatous lesion was not observed macroscopically. There were no significant differences in the degree of lipid deposition or the number of foam cells. The difference in findings compared to those of Daugherty et al. [4] may be due to the differences in feeding periods or anatomical localization and experimental conditions. The intimal thickening after balloon catheterization can be explained by increase of SMC [ 121, due to migration of medial SMC into the intima and their proliferation in the intima. As probucol did not decrease the plasma lipid level, it possibly affected these processes directly. Therefore we examined whether it reached cells in this tissue. In our animals given high cholesterol diet and probucol, considerable probucol was detected in the arterial wall, and its plasma concentration was similar to that in probucol-treated humans [19] and WHHL rabbits [20]. The outgrowth from the explants of these probucol-containing tissues, which may reflect the migration and proliferation of the cells, was less than that from control tissue containing no probucol. Because this tissue contains lymphocytes, polymorphonuclear cells and monocyte-macrophages, the action of probucol should be clarified in relation to cell-to-cell interactions, including growth and migration factors and cytokines produced by various cells [21-231. Probucol in the specimens may reduce the proliferation of explants showing outgrowth by exerting some effect on these functions. The doubling time of SMC in passage 2 from rabbits fed a high cholesterol diet was 0.88 days and that from those on a high cholesterol diet plus probucol was 0.57 days (data not shown). However, the other effect of probucol on SMC should also be considered because SMCs play an important role in the formation of atheromatous lesions including phenotypic changes of the cells [13,24]. These present results indicate that probucol suppresses the cell migration and/or cell proliferation observed in the early stages of formation of atherosclerotic lesions, resulting in suppression of intimal thickening.
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Acknowledgment The authors express great thanks to Dr. Bunshiro Akikusa, Second Department of Pathology, School of Medicine, Chiba University, for helpful discussions concerning the pathological findings of the carotid artery. The authors also express their gratitude to Ms. Keiko Umemiya, Tomoko Koshikawa and Mihoko Nakamura for technical assistance. This study was supported by a grant from the Japanese Ministry of Welfare to the study group led by Prof. Y. Yazaki on ‘The Molecular Biology Study on the Development of Cardiovascular Disease.’ This study was also supported by grants from the Uehara Memorial Foundation and the Funai Foundation.
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