- Email: [email protected]
Effect of probucol on macrophages, leading to regression of xanthomas and atheromatous vascular lesions
Effect of Probucol on Macrophages, Leading to Regression of Xanthomas and Atheromatous Vascular Lesions Akira Yamarnoto, MD, PhD, Hitoshi Hara, MD, PhD, Shigeko Takaichi, PhD, Jun-lchiro Wakasugi, PhD, and Munehiro Tomikawa, PhD p
To explain the strong effect of probucol on xanthomas, the drug's effect on lipid storage in macrophages in the presence of denatured low-density lipoprotein (LDL) was studied. Two macrophage cell lines, UE-12 and THP-1, were used. Those cells stored lipids and became foam cells when they were incubated with acetylated LDL (acetyl-LDL). When probucol was added into the medium either in ethanolic solution or in the form bound to LDL, the storage of cholesterol and other lipide and the development of macrophages into foam cells were greatly suppressed. Two functions of probucol should he considered: (1) It inhibited the uptake of acetyl-LDL by macrophages; and (2) it enhanced the release of cholesterol from these cells. Cells were first incubated with probucol. After the cells were washed with fresh medium, the radiolaheled acetyl-LDL was added to the medium and the degradation of acetyl-LDL was measured. Increasing the concentration of probucol led to a decrease in degradation of acetyl-LDL by macrophages. Probucol also suppressed the uptake of albumin. Macrophages were incubated with acetyl-LDL, washed once, then incubated with or without probucol and high-density lipoprotein (HDL). Addition of HDL caused a rapid decrease in cholesterol content in the cells, and this phenomenon was enhanced by probucol for both kinds of cells. The secretion of apoUpoprotein E was also stimulated by the addition of probucol. These 2 sets of experimental results suggest that probucol prevents lipid storage in macrophages by both suppressing the uptake and stimulating the release of cholesterol and other lipide into or from the macrophages. The prevention of oxidative modification of LDL and the direct effect on macrophages appear to work synergistically to prevent the excess accumulation of lipids in peripheral tissues and to accelerate the metabolism of cholesterol through the liver, leading to regression of atherosclerotic vascular lesions. (Am J Cardiol 1988;62:31B-36B) From the Department of Etiology and Pathophysiology, National Cardiovascular Center Research Institute, Osaka, Japan (and Central Research Laboratories, Dai-ichi Pharmaceutical Company, Tokyo, Japan). Address for reprints: Akira Yamamoto, MD, PhD, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka, 565, Japan.
robucol has unique characteristics among the many antilipidemic drugs. First, it is the only drug that is effective in treating homozygous familial hypercholesterolemia (FH). 1,2 Both cholestyramine and compactin are nearly ineffective in treating homozygous FH, possibly because of the induction of 3-hydroxy-3methylglutaryl coenzyme a (HMG-CoA) reductase in the liver.3,4 Second, probucol causes a rapid and marked regression of cutaneous and tendon xanthomas, which serve as models of atherosclerotic vascular lesions.t.2,5 Regression of xanthomas can be estimated by measuring the size of the Achilles tendon. In heterozygous FH patients, probucol alone--and in homozygous FH, combined use of plasmapheresis and probucol--caused a rapid decrease in thickness of the Achilles tendon. 5 Such a potent effect on xanthomas correlates well with the clinical epidemiologic data of Miettinen et al, 6,7 who demonstrated that probucol is effective in the primary prevention of ischemic heart disease. Furthermore, there is a correlation between the reduction of high-density lipoprotein (HDL) levels, which occurs during probucol treatment, and the regression of Achilles tendon xanthoma thickness. 5 M E C H A N I S M OF ACTION IN X A N T H O M A REGRESSION
Several hypotheses have been offered to explain how probucol causes xanthoma regression despite a reduction of HDL cholesterol. One explanation is that the decrease in HDL cholesterol caused by probucol reflects the suppression of cholesterol synthesis, thus aiding cholesterol homeostasis. Tawara et al 8 demonstrated a suppression of cholesterol synthesis in mouse liver. A second explanation is that probucol affects lipoprotein metabolism. Although probucol lowers HDL cholesterol levels, the decreases in apolipoprotein A-I and HDL phospholipids are much less than the decrease in cholesterol levels. Hence, the ratio of phospholipid to cholesterol and the ratio of apolipoprotein A-I to cholesterol are significantly increased by probucol. 5 Analysis of the size of HDL particles using high-performance liquid chromatography done by Matsuzawa et al 9 confirmed that the HDL particles of FH patients become much smaller after probucol treatment. The smaller HDL particles may be biologically more active and, therefore, beneficial to the reverse cholesterol transport from peripheral tissue to the liver. Furthermore, both lecithin-cholesterol acyltransferase and cholesteryl ester transfer activities were not reduced in patients receiving treatment with probucol. THE AMERICAN JOURNAL OF CARDIOLOGY JULY25, 1988
31B
A SYMPOSIUM: HYPERCHOLESTEROLEMIA
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Parthasarathy et al 1° and Chisolm nl both reported that probucol protects LDL from oxidative modification, which could explain probucol's effect on xanthoma regression. Our laboratory 12 demonstrated probucol's direct action on peripheral tissues, which could also explain the drug's ability to shrink xanthomas. To further investigate how probucol works to reduce xanthomas, we studied the effect of probucol on lipid storage and metabolism in macrophages in the presence of denatured LDL. EFFECT OF PROBUCOL ON MACROPHAGES
Two macrophage cell lines, UE-12 and THP-1, were used. The UE-12 cells were derived from human histiocytic lymphoma cells, or cell line U-93713, through a mutation caused by metanesulfonic acid ethyl ester, or Nmethyl-N-nitro-N-nitrosoguanidine. 14 The cells were maintained in RPMI 1640 containing 10% fetal calf serum in a culture bottle placed in a humidified incubator with 5% carbon dioxide at 37°C. The THP-1 cell line was established by Tsuchiya and co-workers 15,16 from a patient with an acute monocytic leukemia. This cell line retained an ability to synthesize and secrete lipoprotein lipase and apolipoprotein(apo)E when stimulated by a porbol ester. 16 Macrophage-like cells derived from this line are much more like blood-monocyte macrophages than are UE-12 cells with regard to the development of lipoprotein receptors.t 7 Five to 30 #1 of acetylated LDL (acetyl-LDL) solution at a concentration of 8 mg of protein/ml were added to 3 ml of the culture medium, which contained 10% fetal calf serum at a concentration of 2 mg/ml. Either an ethanolic solution of probucol (20 mg/ml) or a solution of LDL obtained from patients who had been given probucol was added to the medium. For routine assay, probucol was added to the medium to give a final concentration of 100 to 200 #g/ml. After 8 to 48 hours of incubation, the medium was removed, and macrophages were stained with either toluidine blue or oil red O for light microsco32B
..
FIGURE I. Elecb'on microscopic observation of macrophages derived from THP-I cells. Left, typical macrophages incubated in ordinary medium. Center, typical foam cells after incubation in medium containing acetyl-low-density lipoprotein (LDL) at a conconb'ation of 24 ~ protein/3 ml medium. Right, typical macrophages incubated in medium containing acetyl-LDL in the presence of probucni (207 ~g/ml).
THE AMERICANJOURNALOF CARDIOLOGY VOLUME62
py, or were fixed with glutaraldehyde for electron microscopic observation. Determination of free and total cholesterol was made by the microscale enzymatic assay system described by Heider and Boyett, 18 or by using high-performance liquid chromatography. Incubation of UE-12 cells with acetyl-LDL caused lipid storage in macrophages, observable as empty vacuoles or lipid droplets. Foam cells, which contained a number of empty vacuoles, were discriminated from nonfoam cells, the vesicles of which contained inclusion bodies with lamellar structure. Macrophages usually have intracytoplasmic inclusions with lamellar structures. Intracellular contents of cholesterol, primarily in esterified form, were increased by increasing the concentration of acetyl-LDL in the medium. Probucol, added either in ethanolic solution or bound to LDL isolated from probucol-treated patients, greatly suppressed the appearance of foam cells for both UE-12 cells 12 and THP-1 cells (Fig. 1). Ordinary LDL receptors similar to those in blood monocytes are found in THP-1 cells. When they were transformed into macrophages by 12-O-tetradecanoylphorbol-13-acetate (TPA), LDL receptors disappeared in 1 or 2 days and acetyl-LDL receptors appeared on the cell surface. 17 To observe the extent of the development of foam cells more precisely, we counted cells under electron microscopy and classified them into 4 categories: (-): cells without lipid droplets; (+): cells in which <20 tiny droplets were seen only in the peripheral zone of cytoplasm; (++): cells in which many small droplets were scattered all over the cytoplasm; and ( + + + ) : cells that were enlarged with the presence of large vacuoles made by the fusion of small lipid droplets. Table I lists the results of the counts for THP-1 cells. Compared with cells incubated with acetyl-LDL without probucol, there was a smaller number of cells moderately ( + + ) or heavily ( + + + ) loaded with lipids, and a greater
TABLE I Effect of Probucol on Foam Cell Formation from THP-1 Macrophages: Classification of Lipid-Laden Cells into Three Categories According to the L~ent of Lipid Storage Control AcetyI-LDL(-)
(%)
Non-foam cells Class (-) 0* Foam cells Class (+) 79 Class (++) 20 Class (+++) 0
Prob ;col (-) Acet;-LDL (+)
(%)
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Probucol(+) AcetyI-LDL(+)
(%)
t =L
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O
0
0
0
92 8 0
10 82 7.7
20 68 11.8
82 19 0
79 21 0
o
total chol. 10
* Data from 2 experiments are shown under each column head; each value is the average of 3 determinations (three dishes); 30 pl of ethanolic solution of probucol (20.7 mg/ml) were added to 3 ml of medium. Values represent the percentage of cells belonging to each class with respect to the total number of cells. Class ( - ) = without fat droplets; Class (+) = less than 20 tiny droplets in the peripheral zone of the cytoplasm; class (++) = many small lipid droplets scattered all over the cytoplasm; Class (+++) = cells are greatly enlarged with large vacuoles in the cytoplasm.
free chol.
T
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2;0
3;0
Drobucol. ~g/ml
number of cells that were slightly (+) loaded with lipids, in the probucol-treated group. The results are essentially the same as those obtained for UE-12 cells, t2 although THP-1 cells are originally provided with a larger number of intracytoplasmic granules and smaller vacuoles than are UE- 12 cells. When we measured the cholesterol content of the macrophages, we found that the accumulation of cholesterol was increasingly suppressed at greater degrees with increased amounts of probucol (Fig. 2). The change in cholesterol content occurred in cholesteryl ester. The change in free cholesterol fraction was not significant. Thus both histologic and biochemical observation clearly show that probucol prevents excess lipid storage in macrophages. U P T A K E A N D D E G R A D A T I O N OF M O D I F I E D LOW-DENSITY LIPOPROTEIN
The uptake and degradation of denatured LDL are important functions of macrophages and provide a scavenger pathway for LDL metabolism. However, an excessive accumulation of lipids causes a loss of macrophage function, eventually leading to the death and breakdown of the foam cells. This process is especially important in the pathogenesis of atherosclerotic vascular lesions because it is difficult to remove the cell debris, including cholesterol, that had once been dispersed in the extracellular space. Therefore, to prevent atherosclerosis, it is necessary to prevent macrophages from accumulating an excess of lipids. H O W PROBUCOL P R E V E N T S FOAM-CELL FORMATION
To discover the mechanism by which probucol prevents foam-cell formation, we studied the influence of probucol on the uptake and degradation of modified LDL. THP-1 cells were first incubated with probucol at varying concentrations, from 0 to 200 gg/ml for 16 hours. After the cells were washed with fresh medium, radiolabeled acetyl-LDL and radiolabeled albumin were added
Effect of 10robucol on accumulation of cholesterol FIGURE 2. Cha.ges in contents of the total and free cholesterol (chol.) in macropbages (UE-12 cells) incubated with acetyl-low-density lipoprotein (LDL) in the presence of varying concentrations of probucol. UE-12 cells were incubated in RPMI 1 6 4 0 containing 1 0 % fetal calf serum for 4 8 hours. Acofyi-LDL: 75 pg protein/ml medium; probucoi: 0 - 3 0 0 pg/ ml. ~ appropriate quantity of the 2 0 mg/ml ethanolic solution was added to 3 ml of medium.)
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FIGURE 3. Degradation of :zSi-labeied acofyl-low-density lipoprotein (LDL) by THP-1 cells preincabated with probucei at varying concentrations. THP-1 cells were used for the experiments 5 days after treatment with 12-O-tetradecanoylpherbol-13-acetate (TPA). Cells were incubated in RPMI medium added with probucal at a concentration of 0 to 200 pg/ml for 16 hours. After the cells were washed with Hanks sokdion, t2Sl-labeied acofyi-LDL was added at a concentration of 5 p8 protein/ml with or without cold acofyI-LDL (100 iAg/ml). After incabation for 5 hours, S2Slin the trichleroncetic aeid-solubfe liraction was determined.
THE AMERICAN JOURNAL OF CARDIOLOGY JULY25, 1988
3 3 II
A SYMPOSIUM: HYPERCHOLESTEROLEMIA
esterified
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to the medium. The degradation of acetyl-LDL and radiolabeled albumin were measured after 5 hours of incubation. Increasing concentrations of probucol at incubation resulted in a gradual decrease in degradation of acetylLDL by macrophages (Fig. 3). When added just before the experiment, probucol did not affect the uptake of acetyl-LDL. Therefore it can be presumed that the direct action of probucol on macrophages caused the decrease in lipid storage. The uptake of radioiodine-labeled albumin by macrophages was also reduced by pretreatment with probucol. We therefore assume that probucol suppresses pinocytic activity, or fluid absorption in general. To observe whether an oxidant stimulates the membrane or fluid absorption activities of macrophages, we examined the effect of alloxan on lipid storage in THP-1 cells in the presence of acetyl-LDL. The extent of lipid storage in macrophages was judged by counting the cells with vacuoles under electron microscopy or those stained with oil red O under light microscopy. Alloxan stimulated the storage of lipids through the uptake of acetyl-LDL (Fig. 4). This stimulatory effect was suppressed by the addition of probucol into the medium. RELEASE
Macrophages release cholesterol from the cell surface into the medium in the presence of HDL. 19 Another explanation of the mechanism of action of probucol is that it enhances cholesterol release. To investigate this possibility, we incubated macrophages with acetyl-LDL for 24 hours, washed them once, then incubated them in a 348
HDL
THE AMERICANJOURNALOF CARDIOLOGY VOLUME62
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FIGURE 4. Effects of alloxon on foam cell formaUon from macrophage. After stimulation with 12-O-tetradecanoylphorbol-13-acetate, THP-1 cells were incubated in R P M I - 1 6 4 0 medium containing acetyl-low-density lipoprotein at a concentration of 2 4 . g protein/3 ml medium in the presence of alloxan (0, 20, and 5 0 ,g/ml). In I series of experiments probucol was added at a concentration of 10 #g/ml and the number of foam cells moderately or heavily loaded with Iipids was counted after 2 4 hours of incubation under electron microscopy.
CHOLESTEROL
-8
None
HDL & Probucol
FIGURE 5. The effect of probucol on release of cholesterol horn macrophages into the medium in the presence of highdensity iipopretein (HDL). UE-12 cells and THP-1 derived macrophages were incubated with acetyl-low-density lipoprorein (LDL) at a concentration of 2 4 pg protein/3 ml medium for 4 8 hours. After being washed with Hanks solution, cells were incubated in R P M I - 1 6 4 0 containing HDL at a concentration of 5 0 pg cholesterol/ml with or without the addition of probucol (100 #g/ml). After incubation for 2 4 hours, ceils were homogenized and the total and free cholesterol were determined by microenzymatic assay.
medium containing HDL with or without probucol. Addition of HDL in the medium caused a rapid decrease in cholesterol content in the macrophages (Fig. 5). This phenomenon was slightly enhanced by probucol for both UE-12 and THP-1 cells. We also incubated UE- 12 with LDL labeled with 14Ccholesteryl oleate for 42 hours. After the cells were washed, the release of taC-cholesterol into the medium was measured in the absence or presence of probucol. Increases in the concentrations of probucol added to the medium corresponded to increases in the release of ]4Ccholesterol (Fig. 6). THP-1 cells synthesize and secrete apoE after stimulation by TPA. ApoE binds to HDL and makes HDL particles available to carry cholesterol. We observed the effect of probucol on the secretion of apoE by measuring the radioactivity of 35S-methionine precipitated with an anti-apoE antibody from the centrifugally separated lipo-
- - Culfur e 4 2 hr ~ -- RPMI/640
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protein fraction. Cellular proteins were labeled with 35Smethionine, and the apoprotein precipitated with the specific antibody was subjected to electrophoresis. Pretreatment of the THP-1 cells with probucol resulted in a slight increase in apoE levels in the lipoprotein fraction of the medium. However, neither the amount of apoE itself nor the amount of messenger RNA in THP- 1 cells was affected by treatment with probucol. Therefore it is possible that the probucol accelerated the release of cholesterol from the cell surface without increasing the synthesis and secretion of apoE. Our experiments failed to demonstrate that probucol suppresses acyl CoA cholesterol acyltransferase activity. When UE-12 cells were incubated with ]4C-oleate, there was a decrease in the incorporation of oleate into cholesteryl ester fraction. However, the radioactivity in free fatty acid fraction was also decreased, suggesting the decrease in permeability for free fatty acids (Fig. 7). CONCLUSION
o rr
Control Probucol Probucol 50Hg/m£ 200Mg/m£ ( P < O . 0 5 vs c o n t r o l )
FIGURE 6. Effect of probucol on the hydrolysis of cholesteryi esters in UE-12 cells. UE-12 cells were incubated (4 X 10 s ceils per dish) in 2 ml RPMI-1640 containing 10% fetal calf serum with low-deneity iipoprotein (LDL) labeled with 14Ccholesterol oleafe (75 .l/dish) and cold LDL (400/~g protein/ dish). After incubation for 42 hours, cells were washed and a new med'un (RPMI) with or wilhout probucol (50 or 200 , g / ml) was added. After furlher incubation for 24 hours without LDL, the radioactivity of 14C-cholesterol in the medium was determined.
FIGURE 7. Effect of probucoi on the incorporaUon of z4C-oloate into free fatty arid fraclion, cholesteryl esters and triglycerides in UE-12 ceils. UE-12 cells (1 × 10 s cells per dishl were incubated with low-density lipoprotein (400 t*g protein/dish), z4C-oloate-albumin (30 "i/dish in 0.5% bovine serum albumin-RPMI 1640 1 mi). After incubaUon for 24 hours, cells were homogenized and the radioactivity in each lipid fraction was determined.
These experimental results indicate that probucol prevents foam-cell formation in macrophages by both suppressing the uptake and stimulating the release of cholesterol and other lipids into or from the macrophages. Previous results of Parthasarathy et al, I° Chisolm l] and Ku et al, 2° together with the direct effect of macrophages found by us, suggest that probucol (1) suppresses the permeability of LDL, (2) prevents the oxidative modification of LDL, (3) suppresses the uptake of modified LDL by macrophages, and (4) suppresses the secretion of smooth muscle cell proliferation factor. Such direct actions on plasma lipoproteins and macrophages could work together to prevent the excess accumulation of lipids in peripheral tissues and to accelerate the metabolism of cholesterol through the liver, leading to
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THE AMERICANJOURNALOF CARDIOLOGY JULY25,1988 3 5 B
A SYMPOSIUM: HYPERCHOLESTEROLEMIA
regression of atherosclerotic vascular lesions. However, further research must be done to learn more about the mechanism of uptake or of modified LDL by macrophages, especially with regard to the role of superoxide produced by macrophages. 21 Our recent experiments show that probucol did not suppress the uptake of oxidized LDL. More knowledge is needed about the nature of macrophage receptors.
REFERENCES 1. Baker SG, Joffe BI, Mendelsohn D, Seftel HC. Treatment of homozygous familial hypercholesterolemia with probucol. S Afr Med J 1982,'62:7-11. Z. Yamamoto A, Matsuzawa Y, Kishino B, Hayashi R, Hirobe K, Kikkawa T. Effects of probucol on homozygous cases of familial hypercholesterolemia. Atherosclerosis 1983;48.'157-166. 3. Moutafis CD, Simmons LA, Myant NB, Adams PW, Wynn V. The effect of cholestyramine on thefecal excretion of bile acids and neutral steroids infamilial hypercholesterolemia. Atherosclerosis 1977;26:329-334. 4. Yamamoto A, Sudo H, Endo A. Therapeutic effects of ML-236B in primary hypercholesterolemia. Atherosclerosis 1980;35.'259-266. 5. Yamamoto A, Matsuzawa Y, Yokoyama S, Funahashi T, Yamamura T, Kichino B. Effects of probucol on xanthoma regression in familial hypercholesterolemia. Am J Cardiol 1986;57:29H-35H. 6. Miettinen TA, Huttenen JK, Strandberg T, Naukkarinen V, Mattila S, Kumlin T. LoweredHDL cholesterol and incidence of ischaemic heart disease [letter]. Lancet 1981;2;478. 7. Miettinen TA, Huttunen JK, Naukkarinen V, Strandberg T, Vanhanen H. Long-term use of probucol in the muhifactorial primary prevention of vascular disease. Am J Cardiol 1986;57:49H-54H. 8. Tawara K, Tomikawa M, Abiko Y. Mode of action ofprobucol in reducing serum cholesterol in mice. J Jpn Atheroscler Soc 1982-1983;10.'1119-1124 [abstr in English]. 9. Matsuzawa Y, Kawamoto S, Nakamura T, Tarui S, Okazaki M, Hara 1.
Mechanism of action of probucol in familial hypercholesterolemia. Proceedings of the Japanese Atherosclerosis Society, Nagoya, 1985 [abstr in English]. 10. Parthasarathy S, Young SG, Witztum JL, Pittman RC, Steinberg D. Probucol inhibits oxidative modification of low-density lipoprotein. J Clin Invest 1986;77:641-644. 11. Chisolm G, Morel DW. Lipoprotein oxidation and cytotoxicity." effect of probucol on streptozotocin-treated rats. Am J Cardiol 1988,'62:20B-26B. 12. Yamamoto A, Takaichi S, Hara H, Nishikawa O, YokoyamaS, Yamamura T, Yamaguchi T. Probucolprevents lipid storage in macrophages. Atherasclerosis 1986,,62:209-217. 13. Larrick JW, Fisher DG, Anderson SJ, Koren HS. Characterization of a human macrophage-like cell line stimulated in vitro--a model of macrophage functions. J Immunol 1980;125:6-12. 14. Kiyotaki C, Kuritani T, Yoshizaki K. Treatment ofU.937 cells with mutagens and establishment of adherent clones. Proceedings of the Japanese Society of Immunology, December, 1985. 15. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K. Establishment and characterization of a human acute monocytic leukemia cell line (THP-I). Int J Cancer 1980;26:171-176. 16. Tajima S, Hayashi R, Tsuchiya S, Miyake Y, Yamamoto A. Cells of a human monocytic leukemic cell line (THP-1) synthesize and secrete apolipoprotein E and lipoprotein lipase. Biochem Biophys Res Communol 1985;126.'526531. 17. Hara H, Tanishita H, Yokoyama S, Tajima S, Yamamoto A. Induction of acetylated low density lipoprotein receptor and suppression of low density lipoprotein receptor on the cells of human monocytic leukemia cell line ( THP- 1 cell). Biochem Biophys Res Communol 1987;146.'802-808. 18. Heider JG, Boyett RL. The picomole determination o f f tee and total cholesterol in cells in culture. J Lipid Res 1978;19.'514-518. 19. Basu SK, Ho YK, Brown MS, Bilheimer D, Anderson RGW, Goldstein JL. Biochemical and genetic studies of the apoprotein E secreted by mouseperitoneal macrophages and human monocytes. J Biol Chem 1982;257:2788-2795. 2 0 . Ku G, Doherty NS, Wolos JA, Jackson RL. Inhibition by probucol of interleukin I secretion and its implication in atherosclerosis. Am J Cardiol 1988,62:78B-82B. 2 1 . Hiramatsu K, Rosen H, Heinecke JW, Wolfbauer G, Chait A. Superoxide initiates oxidation of low density lipoprotein by human monOcytes. Arteriosclerosis 1987;7:55-60.
Discussion Question." Could you say something about the possible toxicity of probucol to the cultured cells? Your concentrations of 200 to 300 #g/ml are extremely high. Was the probucol all in solution, and did you see any signs of toxicity to the cells? Dr. Yamamoto: We did not find any toxicity in our UE-12 and THP-1 cells. An important problem in this kind of study is delivering probucol to the cells, since it is very insoluble. One way is to use some kind of micelles that we could target into macrophages. Fortunately, our UE-12 cells have both LDL receptors and acetylated LDL receptors, enabling us to deliver the probucol by using LDL isolated from probucol-treated patients. In the latter case, the concentration of probucol was much smaller than in the experiments in which probucol was directly added in ethanolic solution.
36B
THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 62
Question." Did you test the HDL composition before and after you put it in the medium? Dr. Yamamoto: No, I didn't. In an in vivo study, the phospholipid-to-cholesterol ratio was increased in those particles after they had been put in the medium. Question." You've shown us the effect of probucol on the release of cholesterol from macrophages in the medium containing HDL. In that experiment, was the HDL incubated directly with the probucol, or was it isolated from patients treated with probucol? Dr. Yamamoto." We wanted to use HDL from probucol-treated patients. However, the concentration of HDL was very low, especially in homozygous patients, and it was difficult to prepare HDL in sufficient quantity. So in that experiment we added probucol directly into the medium containing HDL from normal subjects.
To explain the strong effect of probucol on xanthomas, the drug's effect on lipid storage in macrophages in the presence of denatured low-density lipoprotein (LDL) was studied. Two macrophage cell lines, UE-12 and THP-1, were used. Those cells stored lipids and became foam cells when they were incubated with acetylated LDL (acetyl-LDL). When probucol was added into the medium either in ethanolic solution or in the form bound to LDL, the storage of cholesterol and other lipide and the development of macrophages into foam cells were greatly suppressed. Two functions of probucol should he considered: (1) It inhibited the uptake of acetyl-LDL by macrophages; and (2) it enhanced the release of cholesterol from these cells. Cells were first incubated with probucol. After the cells were washed with fresh medium, the radiolaheled acetyl-LDL was added to the medium and the degradation of acetyl-LDL was measured. Increasing the concentration of probucol led to a decrease in degradation of acetyl-LDL by macrophages. Probucol also suppressed the uptake of albumin. Macrophages were incubated with acetyl-LDL, washed once, then incubated with or without probucol and high-density lipoprotein (HDL). Addition of HDL caused a rapid decrease in cholesterol content in the cells, and this phenomenon was enhanced by probucol for both kinds of cells. The secretion of apoUpoprotein E was also stimulated by the addition of probucol. These 2 sets of experimental results suggest that probucol prevents lipid storage in macrophages by both suppressing the uptake and stimulating the release of cholesterol and other lipide into or from the macrophages. The prevention of oxidative modification of LDL and the direct effect on macrophages appear to work synergistically to prevent the excess accumulation of lipids in peripheral tissues and to accelerate the metabolism of cholesterol through the liver, leading to regression of atherosclerotic vascular lesions. (Am J Cardiol 1988;62:31B-36B) From the Department of Etiology and Pathophysiology, National Cardiovascular Center Research Institute, Osaka, Japan (and Central Research Laboratories, Dai-ichi Pharmaceutical Company, Tokyo, Japan). Address for reprints: Akira Yamamoto, MD, PhD, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka, 565, Japan.
robucol has unique characteristics among the many antilipidemic drugs. First, it is the only drug that is effective in treating homozygous familial hypercholesterolemia (FH). 1,2 Both cholestyramine and compactin are nearly ineffective in treating homozygous FH, possibly because of the induction of 3-hydroxy-3methylglutaryl coenzyme a (HMG-CoA) reductase in the liver.3,4 Second, probucol causes a rapid and marked regression of cutaneous and tendon xanthomas, which serve as models of atherosclerotic vascular lesions.t.2,5 Regression of xanthomas can be estimated by measuring the size of the Achilles tendon. In heterozygous FH patients, probucol alone--and in homozygous FH, combined use of plasmapheresis and probucol--caused a rapid decrease in thickness of the Achilles tendon. 5 Such a potent effect on xanthomas correlates well with the clinical epidemiologic data of Miettinen et al, 6,7 who demonstrated that probucol is effective in the primary prevention of ischemic heart disease. Furthermore, there is a correlation between the reduction of high-density lipoprotein (HDL) levels, which occurs during probucol treatment, and the regression of Achilles tendon xanthoma thickness. 5 M E C H A N I S M OF ACTION IN X A N T H O M A REGRESSION
Several hypotheses have been offered to explain how probucol causes xanthoma regression despite a reduction of HDL cholesterol. One explanation is that the decrease in HDL cholesterol caused by probucol reflects the suppression of cholesterol synthesis, thus aiding cholesterol homeostasis. Tawara et al 8 demonstrated a suppression of cholesterol synthesis in mouse liver. A second explanation is that probucol affects lipoprotein metabolism. Although probucol lowers HDL cholesterol levels, the decreases in apolipoprotein A-I and HDL phospholipids are much less than the decrease in cholesterol levels. Hence, the ratio of phospholipid to cholesterol and the ratio of apolipoprotein A-I to cholesterol are significantly increased by probucol. 5 Analysis of the size of HDL particles using high-performance liquid chromatography done by Matsuzawa et al 9 confirmed that the HDL particles of FH patients become much smaller after probucol treatment. The smaller HDL particles may be biologically more active and, therefore, beneficial to the reverse cholesterol transport from peripheral tissue to the liver. Furthermore, both lecithin-cholesterol acyltransferase and cholesteryl ester transfer activities were not reduced in patients receiving treatment with probucol. THE AMERICAN JOURNAL OF CARDIOLOGY JULY25, 1988
31B
A SYMPOSIUM: HYPERCHOLESTEROLEMIA
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.
5"
' ~ ' A C E T Y L L D L (+1 ~P R O B U C O L (+)
Parthasarathy et al 1° and Chisolm nl both reported that probucol protects LDL from oxidative modification, which could explain probucol's effect on xanthoma regression. Our laboratory 12 demonstrated probucol's direct action on peripheral tissues, which could also explain the drug's ability to shrink xanthomas. To further investigate how probucol works to reduce xanthomas, we studied the effect of probucol on lipid storage and metabolism in macrophages in the presence of denatured LDL. EFFECT OF PROBUCOL ON MACROPHAGES
Two macrophage cell lines, UE-12 and THP-1, were used. The UE-12 cells were derived from human histiocytic lymphoma cells, or cell line U-93713, through a mutation caused by metanesulfonic acid ethyl ester, or Nmethyl-N-nitro-N-nitrosoguanidine. 14 The cells were maintained in RPMI 1640 containing 10% fetal calf serum in a culture bottle placed in a humidified incubator with 5% carbon dioxide at 37°C. The THP-1 cell line was established by Tsuchiya and co-workers 15,16 from a patient with an acute monocytic leukemia. This cell line retained an ability to synthesize and secrete lipoprotein lipase and apolipoprotein(apo)E when stimulated by a porbol ester. 16 Macrophage-like cells derived from this line are much more like blood-monocyte macrophages than are UE-12 cells with regard to the development of lipoprotein receptors.t 7 Five to 30 #1 of acetylated LDL (acetyl-LDL) solution at a concentration of 8 mg of protein/ml were added to 3 ml of the culture medium, which contained 10% fetal calf serum at a concentration of 2 mg/ml. Either an ethanolic solution of probucol (20 mg/ml) or a solution of LDL obtained from patients who had been given probucol was added to the medium. For routine assay, probucol was added to the medium to give a final concentration of 100 to 200 #g/ml. After 8 to 48 hours of incubation, the medium was removed, and macrophages were stained with either toluidine blue or oil red O for light microsco32B
..
FIGURE I. Elecb'on microscopic observation of macrophages derived from THP-I cells. Left, typical macrophages incubated in ordinary medium. Center, typical foam cells after incubation in medium containing acetyl-low-density lipoprotein (LDL) at a conconb'ation of 24 ~ protein/3 ml medium. Right, typical macrophages incubated in medium containing acetyl-LDL in the presence of probucni (207 ~g/ml).
THE AMERICANJOURNALOF CARDIOLOGY VOLUME62
py, or were fixed with glutaraldehyde for electron microscopic observation. Determination of free and total cholesterol was made by the microscale enzymatic assay system described by Heider and Boyett, 18 or by using high-performance liquid chromatography. Incubation of UE-12 cells with acetyl-LDL caused lipid storage in macrophages, observable as empty vacuoles or lipid droplets. Foam cells, which contained a number of empty vacuoles, were discriminated from nonfoam cells, the vesicles of which contained inclusion bodies with lamellar structure. Macrophages usually have intracytoplasmic inclusions with lamellar structures. Intracellular contents of cholesterol, primarily in esterified form, were increased by increasing the concentration of acetyl-LDL in the medium. Probucol, added either in ethanolic solution or bound to LDL isolated from probucol-treated patients, greatly suppressed the appearance of foam cells for both UE-12 cells 12 and THP-1 cells (Fig. 1). Ordinary LDL receptors similar to those in blood monocytes are found in THP-1 cells. When they were transformed into macrophages by 12-O-tetradecanoylphorbol-13-acetate (TPA), LDL receptors disappeared in 1 or 2 days and acetyl-LDL receptors appeared on the cell surface. 17 To observe the extent of the development of foam cells more precisely, we counted cells under electron microscopy and classified them into 4 categories: (-): cells without lipid droplets; (+): cells in which <20 tiny droplets were seen only in the peripheral zone of cytoplasm; (++): cells in which many small droplets were scattered all over the cytoplasm; and ( + + + ) : cells that were enlarged with the presence of large vacuoles made by the fusion of small lipid droplets. Table I lists the results of the counts for THP-1 cells. Compared with cells incubated with acetyl-LDL without probucol, there was a smaller number of cells moderately ( + + ) or heavily ( + + + ) loaded with lipids, and a greater
TABLE I Effect of Probucol on Foam Cell Formation from THP-1 Macrophages: Classification of Lipid-Laden Cells into Three Categories According to the L~ent of Lipid Storage Control AcetyI-LDL(-)
(%)
Non-foam cells Class (-) 0* Foam cells Class (+) 79 Class (++) 20 Class (+++) 0
Prob ;col (-) Acet;-LDL (+)
(%)
e~
Probucol(+) AcetyI-LDL(+)
(%)
t =L
O*
O
0
0
0
92 8 0
10 82 7.7
20 68 11.8
82 19 0
79 21 0
o
total chol. 10
* Data from 2 experiments are shown under each column head; each value is the average of 3 determinations (three dishes); 30 pl of ethanolic solution of probucol (20.7 mg/ml) were added to 3 ml of medium. Values represent the percentage of cells belonging to each class with respect to the total number of cells. Class ( - ) = without fat droplets; Class (+) = less than 20 tiny droplets in the peripheral zone of the cytoplasm; class (++) = many small lipid droplets scattered all over the cytoplasm; Class (+++) = cells are greatly enlarged with large vacuoles in the cytoplasm.
free chol.
T
o
"
2;0
3;0
Drobucol. ~g/ml
number of cells that were slightly (+) loaded with lipids, in the probucol-treated group. The results are essentially the same as those obtained for UE-12 cells, t2 although THP-1 cells are originally provided with a larger number of intracytoplasmic granules and smaller vacuoles than are UE- 12 cells. When we measured the cholesterol content of the macrophages, we found that the accumulation of cholesterol was increasingly suppressed at greater degrees with increased amounts of probucol (Fig. 2). The change in cholesterol content occurred in cholesteryl ester. The change in free cholesterol fraction was not significant. Thus both histologic and biochemical observation clearly show that probucol prevents excess lipid storage in macrophages. U P T A K E A N D D E G R A D A T I O N OF M O D I F I E D LOW-DENSITY LIPOPROTEIN
The uptake and degradation of denatured LDL are important functions of macrophages and provide a scavenger pathway for LDL metabolism. However, an excessive accumulation of lipids causes a loss of macrophage function, eventually leading to the death and breakdown of the foam cells. This process is especially important in the pathogenesis of atherosclerotic vascular lesions because it is difficult to remove the cell debris, including cholesterol, that had once been dispersed in the extracellular space. Therefore, to prevent atherosclerosis, it is necessary to prevent macrophages from accumulating an excess of lipids. H O W PROBUCOL P R E V E N T S FOAM-CELL FORMATION
To discover the mechanism by which probucol prevents foam-cell formation, we studied the influence of probucol on the uptake and degradation of modified LDL. THP-1 cells were first incubated with probucol at varying concentrations, from 0 to 200 gg/ml for 16 hours. After the cells were washed with fresh medium, radiolabeled acetyl-LDL and radiolabeled albumin were added
Effect of 10robucol on accumulation of cholesterol FIGURE 2. Cha.ges in contents of the total and free cholesterol (chol.) in macropbages (UE-12 cells) incubated with acetyl-low-density lipoprotein (LDL) in the presence of varying concentrations of probucol. UE-12 cells were incubated in RPMI 1 6 4 0 containing 1 0 % fetal calf serum for 4 8 hours. Acofyi-LDL: 75 pg protein/ml medium; probucoi: 0 - 3 0 0 pg/ ml. ~ appropriate quantity of the 2 0 mg/ml ethanolic solution was added to 3 ml of medium.)
E
1251-ocetyl LDL 5jJglmt
L~
6~
unlabeled acetyt LDL (-)
s00 U
O I
"o "O O k,.
"o
unlabeled acetyl LDL. 100pg/ml o~ o
¢) "O 0
5'0
probucol,
~~
260
l.Jg/m I
FIGURE 3. Degradation of :zSi-labeied acofyl-low-density lipoprotein (LDL) by THP-1 cells preincabated with probucei at varying concentrations. THP-1 cells were used for the experiments 5 days after treatment with 12-O-tetradecanoylpherbol-13-acetate (TPA). Cells were incubated in RPMI medium added with probucal at a concentration of 0 to 200 pg/ml for 16 hours. After the cells were washed with Hanks sokdion, t2Sl-labeied acofyi-LDL was added at a concentration of 5 p8 protein/ml with or without cold acofyI-LDL (100 iAg/ml). After incabation for 5 hours, S2Slin the trichleroncetic aeid-solubfe liraction was determined.
THE AMERICAN JOURNAL OF CARDIOLOGY JULY25, 1988
3 3 II
A SYMPOSIUM: HYPERCHOLESTEROLEMIA
esterified
0
100
t-
I o
2
probucol (-)
free
20
EU-12
o
E
U
o
50
1c
:3.
(J o
~
E o o
O h,_
,--.,.~0 " " "
~ ~
probucoI ' 100 pglmI Ill
.
0
I
1
25
50
atloxan,
to the medium. The degradation of acetyl-LDL and radiolabeled albumin were measured after 5 hours of incubation. Increasing concentrations of probucol at incubation resulted in a gradual decrease in degradation of acetylLDL by macrophages (Fig. 3). When added just before the experiment, probucol did not affect the uptake of acetyl-LDL. Therefore it can be presumed that the direct action of probucol on macrophages caused the decrease in lipid storage. The uptake of radioiodine-labeled albumin by macrophages was also reduced by pretreatment with probucol. We therefore assume that probucol suppresses pinocytic activity, or fluid absorption in general. To observe whether an oxidant stimulates the membrane or fluid absorption activities of macrophages, we examined the effect of alloxan on lipid storage in THP-1 cells in the presence of acetyl-LDL. The extent of lipid storage in macrophages was judged by counting the cells with vacuoles under electron microscopy or those stained with oil red O under light microscopy. Alloxan stimulated the storage of lipids through the uptake of acetyl-LDL (Fig. 4). This stimulatory effect was suppressed by the addition of probucol into the medium. RELEASE
Macrophages release cholesterol from the cell surface into the medium in the presence of HDL. 19 Another explanation of the mechanism of action of probucol is that it enhances cholesterol release. To investigate this possibility, we incubated macrophages with acetyl-LDL for 24 hours, washed them once, then incubated them in a 348
HDL
THE AMERICANJOURNALOF CARDIOLOGY VOLUME62
THP-I "-I
o m,-
None
HDL
t I
HDL & Probucol
t(J
pglmI
FIGURE 4. Effects of alloxon on foam cell formaUon from macrophage. After stimulation with 12-O-tetradecanoylphorbol-13-acetate, THP-1 cells were incubated in R P M I - 1 6 4 0 medium containing acetyl-low-density lipoprotein at a concentration of 2 4 . g protein/3 ml medium in the presence of alloxan (0, 20, and 5 0 ,g/ml). In I series of experiments probucol was added at a concentration of 10 #g/ml and the number of foam cells moderately or heavily loaded with Iipids was counted after 2 4 hours of incubation under electron microscopy.
CHOLESTEROL
-8
None
HDL & Probucol
FIGURE 5. The effect of probucol on release of cholesterol horn macrophages into the medium in the presence of highdensity iipopretein (HDL). UE-12 cells and THP-1 derived macrophages were incubated with acetyl-low-density lipoprorein (LDL) at a concentration of 2 4 pg protein/3 ml medium for 4 8 hours. After being washed with Hanks solution, cells were incubated in R P M I - 1 6 4 0 containing HDL at a concentration of 5 0 pg cholesterol/ml with or without the addition of probucol (100 #g/ml). After incubation for 2 4 hours, ceils were homogenized and the total and free cholesterol were determined by microenzymatic assay.
medium containing HDL with or without probucol. Addition of HDL in the medium caused a rapid decrease in cholesterol content in the macrophages (Fig. 5). This phenomenon was slightly enhanced by probucol for both UE-12 and THP-1 cells. We also incubated UE- 12 with LDL labeled with 14Ccholesteryl oleate for 42 hours. After the cells were washed, the release of taC-cholesterol into the medium was measured in the absence or presence of probucol. Increases in the concentrations of probucol added to the medium corresponded to increases in the release of ]4Ccholesterol (Fig. 6). THP-1 cells synthesize and secrete apoE after stimulation by TPA. ApoE binds to HDL and makes HDL particles available to carry cholesterol. We observed the effect of probucol on the secretion of apoE by measuring the radioactivity of 35S-methionine precipitated with an anti-apoE antibody from the centrifugally separated lipo-
- - Culfur e 4 2 hr ~ -- RPMI/640
wash
w,th or w,thout p¢obucol 2 4 hrs
•
F r e e cholesterol
[]
Cholesteryl ester T o t a l cholesterol
E E
300"
c_
200
d~ ;
I00-
protein fraction. Cellular proteins were labeled with 35Smethionine, and the apoprotein precipitated with the specific antibody was subjected to electrophoresis. Pretreatment of the THP-1 cells with probucol resulted in a slight increase in apoE levels in the lipoprotein fraction of the medium. However, neither the amount of apoE itself nor the amount of messenger RNA in THP- 1 cells was affected by treatment with probucol. Therefore it is possible that the probucol accelerated the release of cholesterol from the cell surface without increasing the synthesis and secretion of apoE. Our experiments failed to demonstrate that probucol suppresses acyl CoA cholesterol acyltransferase activity. When UE-12 cells were incubated with ]4C-oleate, there was a decrease in the incorporation of oleate into cholesteryl ester fraction. However, the radioactivity in free fatty acid fraction was also decreased, suggesting the decrease in permeability for free fatty acids (Fig. 7). CONCLUSION
o rr
Control Probucol Probucol 50Hg/m£ 200Mg/m£ ( P < O . 0 5 vs c o n t r o l )
FIGURE 6. Effect of probucol on the hydrolysis of cholesteryi esters in UE-12 cells. UE-12 cells were incubated (4 X 10 s ceils per dish) in 2 ml RPMI-1640 containing 10% fetal calf serum with low-deneity iipoprotein (LDL) labeled with 14Ccholesterol oleafe (75 .l/dish) and cold LDL (400/~g protein/ dish). After incubation for 42 hours, cells were washed and a new med'un (RPMI) with or wilhout probucol (50 or 200 , g / ml) was added. After furlher incubation for 24 hours without LDL, the radioactivity of 14C-cholesterol in the medium was determined.
FIGURE 7. Effect of probucoi on the incorporaUon of z4C-oloate into free fatty arid fraclion, cholesteryl esters and triglycerides in UE-12 ceils. UE-12 cells (1 × 10 s cells per dishl were incubated with low-density lipoprotein (400 t*g protein/dish), z4C-oloate-albumin (30 "i/dish in 0.5% bovine serum albumin-RPMI 1640 1 mi). After incubaUon for 24 hours, cells were homogenized and the radioactivity in each lipid fraction was determined.
These experimental results indicate that probucol prevents foam-cell formation in macrophages by both suppressing the uptake and stimulating the release of cholesterol and other lipids into or from the macrophages. Previous results of Parthasarathy et al, I° Chisolm l] and Ku et al, 2° together with the direct effect of macrophages found by us, suggest that probucol (1) suppresses the permeability of LDL, (2) prevents the oxidative modification of LDL, (3) suppresses the uptake of modified LDL by macrophages, and (4) suppresses the secretion of smooth muscle cell proliferation factor. Such direct actions on plasma lipoproteins and macrophages could work together to prevent the excess accumulation of lipids in peripheral tissues and to accelerate the metabolism of cholesterol through the liver, leading to
•
Control
[]
probucol 50/zg/m#.
[]
probucol 2 0 0
XlO 3
XIO 4
3
15
XIO • 15
~.~_ 10
E
0
Cholesteryl ester
-.-.-. "'"
~-ree f a t t y acid
0
Triglyceride
P
THE AMERICANJOURNALOF CARDIOLOGY JULY25,1988 3 5 B
A SYMPOSIUM: HYPERCHOLESTEROLEMIA
regression of atherosclerotic vascular lesions. However, further research must be done to learn more about the mechanism of uptake or of modified LDL by macrophages, especially with regard to the role of superoxide produced by macrophages. 21 Our recent experiments show that probucol did not suppress the uptake of oxidized LDL. More knowledge is needed about the nature of macrophage receptors.
REFERENCES 1. Baker SG, Joffe BI, Mendelsohn D, Seftel HC. Treatment of homozygous familial hypercholesterolemia with probucol. S Afr Med J 1982,'62:7-11. Z. Yamamoto A, Matsuzawa Y, Kishino B, Hayashi R, Hirobe K, Kikkawa T. Effects of probucol on homozygous cases of familial hypercholesterolemia. Atherosclerosis 1983;48.'157-166. 3. Moutafis CD, Simmons LA, Myant NB, Adams PW, Wynn V. The effect of cholestyramine on thefecal excretion of bile acids and neutral steroids infamilial hypercholesterolemia. Atherosclerosis 1977;26:329-334. 4. Yamamoto A, Sudo H, Endo A. Therapeutic effects of ML-236B in primary hypercholesterolemia. Atherosclerosis 1980;35.'259-266. 5. Yamamoto A, Matsuzawa Y, Yokoyama S, Funahashi T, Yamamura T, Kichino B. Effects of probucol on xanthoma regression in familial hypercholesterolemia. Am J Cardiol 1986;57:29H-35H. 6. Miettinen TA, Huttenen JK, Strandberg T, Naukkarinen V, Mattila S, Kumlin T. LoweredHDL cholesterol and incidence of ischaemic heart disease [letter]. Lancet 1981;2;478. 7. Miettinen TA, Huttunen JK, Naukkarinen V, Strandberg T, Vanhanen H. Long-term use of probucol in the muhifactorial primary prevention of vascular disease. Am J Cardiol 1986;57:49H-54H. 8. Tawara K, Tomikawa M, Abiko Y. Mode of action ofprobucol in reducing serum cholesterol in mice. J Jpn Atheroscler Soc 1982-1983;10.'1119-1124 [abstr in English]. 9. Matsuzawa Y, Kawamoto S, Nakamura T, Tarui S, Okazaki M, Hara 1.
Mechanism of action of probucol in familial hypercholesterolemia. Proceedings of the Japanese Atherosclerosis Society, Nagoya, 1985 [abstr in English]. 10. Parthasarathy S, Young SG, Witztum JL, Pittman RC, Steinberg D. Probucol inhibits oxidative modification of low-density lipoprotein. J Clin Invest 1986;77:641-644. 11. Chisolm G, Morel DW. Lipoprotein oxidation and cytotoxicity." effect of probucol on streptozotocin-treated rats. Am J Cardiol 1988,'62:20B-26B. 12. Yamamoto A, Takaichi S, Hara H, Nishikawa O, YokoyamaS, Yamamura T, Yamaguchi T. Probucolprevents lipid storage in macrophages. Atherasclerosis 1986,,62:209-217. 13. Larrick JW, Fisher DG, Anderson SJ, Koren HS. Characterization of a human macrophage-like cell line stimulated in vitro--a model of macrophage functions. J Immunol 1980;125:6-12. 14. Kiyotaki C, Kuritani T, Yoshizaki K. Treatment ofU.937 cells with mutagens and establishment of adherent clones. Proceedings of the Japanese Society of Immunology, December, 1985. 15. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K. Establishment and characterization of a human acute monocytic leukemia cell line (THP-I). Int J Cancer 1980;26:171-176. 16. Tajima S, Hayashi R, Tsuchiya S, Miyake Y, Yamamoto A. Cells of a human monocytic leukemic cell line (THP-1) synthesize and secrete apolipoprotein E and lipoprotein lipase. Biochem Biophys Res Communol 1985;126.'526531. 17. Hara H, Tanishita H, Yokoyama S, Tajima S, Yamamoto A. Induction of acetylated low density lipoprotein receptor and suppression of low density lipoprotein receptor on the cells of human monocytic leukemia cell line ( THP- 1 cell). Biochem Biophys Res Communol 1987;146.'802-808. 18. Heider JG, Boyett RL. The picomole determination o f f tee and total cholesterol in cells in culture. J Lipid Res 1978;19.'514-518. 19. Basu SK, Ho YK, Brown MS, Bilheimer D, Anderson RGW, Goldstein JL. Biochemical and genetic studies of the apoprotein E secreted by mouseperitoneal macrophages and human monocytes. J Biol Chem 1982;257:2788-2795. 2 0 . Ku G, Doherty NS, Wolos JA, Jackson RL. Inhibition by probucol of interleukin I secretion and its implication in atherosclerosis. Am J Cardiol 1988,62:78B-82B. 2 1 . Hiramatsu K, Rosen H, Heinecke JW, Wolfbauer G, Chait A. Superoxide initiates oxidation of low density lipoprotein by human monOcytes. Arteriosclerosis 1987;7:55-60.
Discussion Question." Could you say something about the possible toxicity of probucol to the cultured cells? Your concentrations of 200 to 300 #g/ml are extremely high. Was the probucol all in solution, and did you see any signs of toxicity to the cells? Dr. Yamamoto: We did not find any toxicity in our UE-12 and THP-1 cells. An important problem in this kind of study is delivering probucol to the cells, since it is very insoluble. One way is to use some kind of micelles that we could target into macrophages. Fortunately, our UE-12 cells have both LDL receptors and acetylated LDL receptors, enabling us to deliver the probucol by using LDL isolated from probucol-treated patients. In the latter case, the concentration of probucol was much smaller than in the experiments in which probucol was directly added in ethanolic solution.
36B
THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 62
Question." Did you test the HDL composition before and after you put it in the medium? Dr. Yamamoto: No, I didn't. In an in vivo study, the phospholipid-to-cholesterol ratio was increased in those particles after they had been put in the medium. Question." You've shown us the effect of probucol on the release of cholesterol from macrophages in the medium containing HDL. In that experiment, was the HDL incubated directly with the probucol, or was it isolated from patients treated with probucol? Dr. Yamamoto." We wanted to use HDL from probucol-treated patients. However, the concentration of HDL was very low, especially in homozygous patients, and it was difficult to prepare HDL in sufficient quantity. So in that experiment we added probucol directly into the medium containing HDL from normal subjects.