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Characterization of the Interaction of Acetylated LDL and Oxidatively Modified LDL With Human Liver Parenchymal and Kupffer Cells in Culture Jan A.A.M. Kamps, J. Kar Kruijt, Johan Kuiper, and Theo J.C. van Berkel The interaction of acetylated low density lipoprotein (Ac-LDL) and oxidatively modified low density lipoprotein (Ox-LDL) with cultured human liver parenchymal cells and human Kupffer cells was investigated to define, for humans, the presence of scavenger receptors in the liver. A direct comparison of the capacity of Kupffer and parenchyma] cells to interact with Ac-LDL and Ox-LDL indicated that the capacity of Kupffer cells per milligram of cell protein to degrade Ac-LDL and Ox-LDL is 14-fold and sixfold higher, respectively, than that of parenchymal cells. The degradation of both Ac-LDL and Ox-LDL by parenchymal cells and Kupffer cells could be inhibited by chloroquine and ammonium chloride, indicating that degradation occurs in the lysosomes. Competition studies showed that unlabeled Ox-LDL competed efficiently with the cell association and degradation of 125I-labeled Ac-LDL by human parenchyma] cells and human Kupffer cells. However, unlabeled Ac-LDL did not compete (parenchymal cells) or only partially competed (40% in Kupffer cells) with the cell association and degradation of '"1-labeled Ox-LDL. Polyinosink acid completely blocked the cell association and degradation of Ac-LDL and Ox-LDL with Kupffer cells while no significant effect on parenchymal cells was noted. It is concluded that human liver parenchymal cells contain a scavenger receptor that interacts with Ac-LDL and Ox-LDL and an additional recognition site that recognizes Ox-LDL specifically. On human Kupffer cells, as on rat Kupffer cells, in addition to the scavenger (Ac-LDL) receptor that interacts efficiently with both Ac-LDL and Ox-LDL, a specific Ox-LDL receptor is present at a relatively high concentration. The identification of various recognition sites for modified LDL on human liver cells indicates that the liver protection system against circulating modified LDL, identified earlier in rats, is also highly relevant for humans. (Arteriosclerosis and Thrombosis 1992;12:1079-1087)
KEY WORDS • oxidatively modified low density lipoproteins • acetylated low density lipoproteins • scavenger receptors • human liver parenchymal cells • human Kupffer cells
O
ne characteristic in the early development of atherosclerotic lesions is the formation of foam cells.1 The conversion of macrophages into foam cells cannot be provoked by native low density lipoprotein (LDL).2 Various modifications of LDL, such as acetylation, acetoacetylation, malondialdehyde treatment, and oxidative modification, lead to LDL uptake via scavenger receptors, which leads to foam cell formation.3 Evidence has recently been provided that various classes of scavenger receptors are involved in the uptake of modified LDL by peritoneal mouse macrophages.4-5 Also, in rat liver it has been established that different recognition sites are involved in the uptake of differently modified LDL.6 When injected into rats, both From the Division of Biopharmaceutics, Center for Bio-Pharmaceutical Sciences, Sylvius Laboratory, University of Leiden, Leiden, The Netherlands. Supported by a grant from the Dutch Heart Foundation (grant No. 87001). Address for correspondence: Prof. Dr. ThJ.C. van Berkel, Division of Biopharmaceutics, Center for Bio-Pharmaceutical Sciences, Sylvius Laboratory, University of Leiden, PO Box 9503, 2300 RA Leiden, The Netherlands. Received April 21, 1992; accepted May 29, 1992.
acetylated LDL (Ac-LDL) and oxidatively modified LDL (Ox-LDL) are rapidly cleared from the blood circulation by the liver. Separation of the liver into parenchymal cells, endothelial cells, and Kupffer cells indicated that endothelial cells were mainly responsible for the uptake of Ac-LDL, whereas Kupffer cells were responsible for the uptake of most of the Ox-LDL. In vitro studies with isolated liver cells provided further evidence that various scavenger receptors are involved in the liver uptake of modified LDL. In addition to the Ac-LDL receptor on liver endothelial cells,7-8 which interacts with both Ac-LDL and Ox-LDL, a specific recognition site for Ox-LDL that was highly concentrated on Kupffer cells was reported.6 Because several lines of evidence suggest that oxidative modification can occur in vivo1-910 and because Ox-LDL, when injected into rats, is rapidly cleared from the circulation by the liver, it was suggested that in vivo Kupffer cells will form the major protection system against the occurrence of atherogenic Ox-LDL particles in the blood. However, the relevance of these studies for humans is unclear especially because it was reported that human hepatocytes express no surface receptors for modified LDL,11 although Edge et al12 found that modified LDL
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was bound and degraded by hepatocytes at 30-50% of the level of native LDL. Kume et al13 indicated that human endothelial cells, in contrast to mouse peritoneal macrophages, do not have any additional receptors specific only for Ox-LDL. In earlier studies we found that both human parenchymal and Kupffer cells interact with LDL through the classic LDL receptor,14 although the association of LDL with Kupffer cells is more efficiently coupled to degradation than with parenchymal cells. The availability of cultured human liver parenchymal cells and human Kupffer cells enabled us to compare directly the interaction of Ac-LDL and Ox-LDL with human liver cells. Methods Materials Collagenase type I, human serum albumin (fraction V), dexamethasone, and chloroquine were purchased from Sigma Chemical Co. (St. Louis, Mo.); Nycodenz was obtained from Nycomed A/S (Oslo, Norway); and 125 I was purchased from Amersham International (Amersham, Bucks, UK). Fetal calf serum, penicillin, and streptomycin were obtained from Boehringer (Mannheim, Germany), and Williams' E culture medium and kanamycin were purchased from Flow Laboratories (Irvine, Scotland, UK). RPMI-1640 cell culture medium was from GIBCO (Paisley, Scotland, UK), and multiwell cell culture dishes were from Costar (Cambridge, Mass.). All other chemicals were of analytical grade. Isolation, Iodination, Oxidation, and Acetylation of LDL Human LDL was obtained from the blood of healthy volunteers who had fasted overnight. Isolation of LDL (1.02425I-labeled acetylated (Ac) low density Upoprotein (LDL) or 123I-labeled oxidized (Ox) LDL with human parenchymal cells. Parenchymal cells were incubated for 3 hours at 37°C with 10 /Jg/ml 125I-^Ac-LDL (panel A) or 125I-Ox-LDL (panel B) and with the indicated amounts of unlabeled LDL (m),Ac-LDL (•), Ox-LDL (A), or polyinosinic acid (T). Cell association of l25I^4c-LDL or 12}I-Ox-LDL is expressed as the radioactivity obtained in the absence of a competitor. Values for 100% cell association of 125I-Ac-LDL and 125 I-Ox-LDL with parenchymal cells are 48 and 156 ng apolipoprotein per milligram of cell protein, respectively. Data represent mean of two or three experiments. When indicated, bars represent SEM.
about 70% by unlabeled Ac-LDL or polyinosinic acid. Unlabeled Ox-LDL appears to be slightly less efficient as a competitor than Ac-LDL. Native LDL was relatively ineffective. The cell association and degradation of 125I-Ox-LDL by Kupffer cells is efficiently inhibited by Ox-LDL itself and by polyinosinic acid. Ac-LDL is clearly less efficient as a competitor than Ox-LDL. The cell association and degradation of 125I-Ox-LDL is only slightly inhibited by native LDL. Discussion In the present work we analyzed the interaction of 125 I-Ac-LDL and 125I-Ox-LDL with human liver parenchymal cells and human Kupffer cells. Recent studies in rats indicate that Kupffer cells appear to be the main site in the liver for the uptake of Ox-LDL.6 Because Ox-LDL is considered to be the pathological form of modified LDL, it was suggested that Kupffer cells may form a major protection system against the action of Ox-LDL.6 Furthermore, in vitro studies with isolated liver cell types (parenchymal, endothelial, and Kupffer cells) provided evidence that in addition to the Ac-LDL receptor, which is concentrated on liver endothelial cells,8 Kupffer cells contain a scavenger receptor that specifically interacts with Ox-LDL.6 So far there are no data concerning the role of human liver parenchymal cells and human Kupffer cells in the metabolism of Ac-LDL and Ox-LDL nor on the possible involvement of various scavenger receptors in the uptake of modified LDL. The availability of human parenchymal cells and Kupffer cells enabled us to address these questions for the human situation. Human liver endothelial cells could not be isolated from human liver, probably because of a loss of viable liver endothelial cells, which has been observed to occur when cold ischemic liver material is reperfused with warm oxygenated buffer.30
calcium-independent, specific recognition sites that recognize Ac-LDL and/or Ox-LDL. Competition experiments indicate that human liver parenchymal cells contain 1) a specific Ac-LDL recognition site, which is partly competed for by Ox-LDL and 2) a specific Ox-LDL recognition site, which is not competed for by Ac-LDL and is hardly by polyinosinic acid. Human Kupffer cells express 1) an Ac-LDL recognition site, which is also blocked by Ox-LDL and polyinosinic acid and 2) an Ox-LDL recognition site, which interacts only partially with Ac-LDL and is completely blocked by polyinosinic acid. These results are comparable with the results that were found for the interaction of Ac-LDL and Ox-LDL with rat liver parenchymal cells and Kupffer cells,6 indicating that in addition to a scavenger receptor, which interacts with both Ac-LDL and OxLDL, a specific Ox-LDL receptor exists on liver cells. It is possible that this receptor is similar to the class III binding sites as defined on basis of the specificity of reconstituted hepatic scavenger receptors.31-32 The specific recognition of Ox-LDL by Kupffer cells cannot be explained by aggregation phenomena because we used a relatively low apolipoprotein concentration (200 fig/tnl) for oxidation. Moreover, we subjected Ox-LDL, directly before the incubations, to filtration over a 22-/xm filter; also, complete blockade of the interaction of Ox-LDL with Kupffer cells by polyinosinic acid points to the involvement of scavenger receptors and not bulk-phase endocytosis. To investigate whether lysosomes are involved in the degradation of Ac-LDL and Ox-LDL, chloroquine and ammonium chloride were used. These two unrelated compounds inhibit lysosomal proteolysis by increasing the lysosomal pH.33 Based on their inhibitory action, it appeared that the uptake of both AcLDL and Ox-LDL by parenchymal cells and Kupffer cells was coupled to rysosomal degradation.
The data in this study demonstrate that both human parenchymal cells and Kupffer cells in culture possess
There is a dispute about the role of human parenchymal cells in the uptake of modified LDL. Edge et al12
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Interaction of Modified LDL With Human liver Cells
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FIGURE 5. Line plots comparing the ability ofunlabeled lipoproteins or potyinosinic acid to compete for the cell association and degradation of I25I-labeled acetylated (Ac) low density lipoprotein (LDL) or I25I-labeled oxidized (Ox) LDL by human Kupffer cells. Kupffer cells were incubated for 3 hours at 37°C with 10 uglml 125I-Ac-LDL ("panels A and B) or 125I-Ox-LDL (panels C and D) and with the indicated amounts of unlabeled LDL (•), Ac-LDL (•), Ox-LDL (A), or polyinosinic acid ( • ) . Cell association (panels A and C) and degradation (panels B and D) of125l-Ac-LDL or I25I-Ox-LDL are expressed as the radioactivity and l25I-Ox-LDL with Kupffer cells are obtained in the absence of a competitor. Values for 100% cell association ofml-Ac-LDL 498 and 978 ng apolipoprotein per milligram of cell protein, respectively, and for degradation are 273 and 432 ng apolipoprotein per milligram of cell protein, respectively. Data represent mean of two experiments.
demonstrated that Ac-LDL was bound and degraded at 30-50% of the level of that of native LDL. Babaev et al11 reported that binding, internalization, and degradation of malondialdehyde-treated LDL by cultured human hepatocytes was caused by contamination of the primary cultures by endothelial and Kupffer cells. We observed a specific recognition site for modified LDL in human parenchymal cells, which cannot be due to contamination of the parenchymal cells with nonparenchymal cells, as polyinosinic acid does not compete for the interaction of Ox-LDL with parenchymal cells while polyinosinic acid is a very effective competitor for the interaction of Ac-LDL and Ox-LDL with Kupffer cells. Also, with rat liver cells it has been shown that the interaction of Ac-LDL, Ox-LDL, and Ox-/3-VLDL with
endothelial and Kupffer cells is effectively inhibited by polyinosinic acid, whereas with parenchymal cells this interaction is not blocked.6-34 ' Human liver endothelial cells could not be isolated from the human liver, thus, no information about the importance of these cell types for the relative interaction with Ac-LDL and Ox-LDL could be obtained. From in vivo and in vitro studies with rats it has been shown that rat liver endothelial cells account for 52% of the total uptake of Ac-LDL and for 36% of the total uptake of Ox-LDL.6 Recent studies in which human liver tissue blocks were perfused in vitro with l,l'-dioctadecyl-333'»3'-tetramethyl indocarbocyanine perchlorate-labeled Ac-LDL provide evidence that in the human liver also, Ac-LDL is mainly taken up by liver endothelial cells.33
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The capacity of human Kupffer cells for cell association and degradation of Ac-LDL and Ox-LDL is considerably higher than that of parenchymal cells (respectively, 4.8- and 4.9-fold per milligram of cell protein) for which, in addition to a scavenger receptor, which interacts with both Ac-LDL and Ox-LDL, a specific Ox-LDL receptor is responsible. It appears that the recognition sites for Ac-LDL and Ox-LDL on human parenchymal and human Kupffer cells exert similar properties as those on rat liver cells. Earlier, we demonstrated that human Kupffer cells also have a higher capacity than do parenchymal cells to catabolize native LDL.14 Together, these data indicate that Kupffer cells may contribute significantly to the regulation of the level of (modified) LDL in the human situation and thus in the protection against the development of atherosclerosis. Several lines of evidence, such as the occurrence of autoantibodies against oxidized LDL in human sera3* and the prevention of the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits by probucol,37-38 indicate that Ox-LDL is the pathophysiologicalry occurring form of modified LDL. The presence of a specific Ox-LDL recognition site on human liver parenchymal cells and especially on human Kupffer cells may form a major protection in humans against the pathological action of Ox-LDL in the blood compartment. References 1. Steinberg D: Metabolism of lipoproteins and their role in the pathogenesis of atherosclerosis. Atheroscla Rev 1988;18:l-23 2. Brown MS, Basu SK, Falck JR, Ho YK, Goldstein JL: The scavenger cell pathway for lipoprotein degradation: Specificity of the binding site that mediates the uptake of negatively-charged LDL by macrophages. J Supramol Struct 1980;13:67-81 3. Brown MS, Goldstein JL: Lipoprotein metabolism in the macrophage: Implication for cholesterol deposition in atherosclerosis. Annu Rev Biochan 1983^2:223-261 4. Sparrow CP, Parthasarathy S, Steinberg D: A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein. / Biol Chan 1989;264:2599-2604 5. Arai H, Kita T, Yokode M, Narumiya S, Kawai C: Multiple receptors for modified low density lipoproteins in mouse peritoneal macrophages: Different uptake mechanisms for acetylated and oxidized low density lipoproteins. Biochan Biophys Res Commun 1989-,159:1375-1382 6. Van Berkel ThJC, De Rijke YB, Kruijt JK: Different fate in vivo of cnridativery modified low density lipoprotein and acetylated low density lipoprotein in rats: Recognition by various scavenger receptors on Kupffer and endothelial liver cells. / Biol Chan 1991; 266:2282-2289 7. Van Berkel ThJC, Nagelkerke JF, Harkes L, Kruijt JK: Processing of acetylated human low-density lipoprotein by parenchymal and non-parenchymal liver cells; Involvement of calmodulin? Biochan J 1982208:493-503 8. Nagelkerke JF, Barto KP, Van Berkel ThJC: In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells. J Biol Chan 1983^58:12221-12227 9. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL: Beyond cholesterol: Modifications of low density lipoprotein that increase its atherogenicity. N Engl J Med 1989^20:915-923 10. Wiklund O, Mattsson L, Bjornheden T, Camejo G, Bondjers G: Uptake and degradation of low density lipoproteins in atherosclerotic rabbit aorta: Role of local LDL modification. / Upid Res 199132:55-62 11. Babaev VR, Kosykh VA, Tsibulsky VP, Ivanov VO, Repin VS, Smirnov VN: Binding and uptake of native and modified lowdensity lipoproteins by human hepatocytes in primary culture. Hepatology 1989;10-.56-60 12. Edge SB, Hoeg JM, Triche T, Schneider PD, Brewer HB J r Cultured human hepatocytes: Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor. / Biol Chan 1986;261:3800-3806
September 1992 13. Kume N, Arai H, Kawai C, Kita T: Receptors for modified lowdensity lipoproteins on human endothelial cells: Different recognition for acetylated low-density lipoprotein and oxidized lowdensity lipoprotein. Biochim Biophys Acta 1991;1091:63-67 14. Kampt JAAM, Kruijt JK, Kuiper J, Van Berkel ThJC: Uptake and degradation of human low-density lipoprotein by human liver parenchymal and Kupffer cells in culture. Biochan J 1991^76:135-140 15. Redgrave TG, Roberts DCK, West CE: Separation of plasma lipoproteins by density gradient ultracentrifugation. Anal Biochan 1975;65:42-49 16. Basu SK, Goldstein JL, Anderson RGW, Brown MS: Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Nad Acad Sci U S A 1976;73:3178-3182 17. Steinbrecher UP, Witztum JL, Parthasarathy S, Steinberg D: Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL: Correlation with changes in receptor mediated catabolism. Arteriosclerosis 1987;7:135-143 18. McFarlane AS: Efficient trace-labeling of proteins with iodine. Nature 1958;182:53 19. Bilheimer DW, Eisenberg S, Levy RI: The metabolism of very tow density lipoproteins: I. Preliminary in vitro and in vivo observations. Biochim Biophys Acta 1972;260:212-221 20. Princen HMG, Huijsmans CMG, Kuipers F, Vonk RJ, Kempen HJM: Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7a-bydroxylase. J Clin Invest 1986;78:1064-1071 21. Kwekkeboom J, Princen HMG, Van Voorthuizen EM, Merjer P, Kempen HJM: Comparison of taurocholate accumulation in cultured hepatocytes of pig, man and rat. Biochan Biophys Res Commun 1989;162:619-625 22. Forte TM, Nordhausen RW, Princen HMG: Structural properties of lipoproteins isolated from human hepatocyte cultures, (abstract) Anaiosclerosis 1989;9:693a 23. Kooistra T, Bosma PJ, Tons HAM, Van den Berg AP, Meijer P, Princen HMG: Plasminogen activator inhibitor 1: Biosynthesis and mRNA level are increased by insulin in cultured human hepatocytes. Thmmb Haanost 1989;62:723-728 24. Krenning EP, De Jong M, Vos RA, Bernard HF, Docter R, Menneman G: Transport and metabolism of thyroid hormones in human hepatocytes. Endocrinology 1989;122:T-36 25. Scbouten D, Kleinherenbrink-Stins MF, Brouwer A, Knook DL, Kamps JAAM, Kuiper J, Van Berkel ThJC: Characterization in vitro of interaction of human apolipoprotein E-free high density lipoprotein with human hepatocytes. Arteriosclerosis 1990;10:1127-1135 26. Kamps JAAM, Kuiper J, Kruijt JK, Van Berkel ThJC: Complete down-regulation of low-density lipoprotein receptor activity in human liver parenchymal cells by /S-very-tow-density lipoprotein. FEBS Lett 1991^87:34-38 27. Van Berkel ThJC, Kruijt JK, Van Gent T, Van Tol A: Saturable high affinity uptake and degradation of rat plasma lipoproteins by isolated parenchymal and non-parenchymal cells from rat liver. Biochim Biophys Acta 1981;665:22-33 28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chan 1951;193: 265-275 29. Ho YK, Brown MS, Bilheimer DW, Goldstein JL: Regulation of low density lipoprotein receptor activity in freshly isolated human lymphocytes. / Clin Invest 1976^8:1465-1474 30. Lemasters JL, Caldwell-Kenkel JC, Currin RT, Yanaka Y, Marzi I, Thurman RG: Endothelial cell killing and activation of Kupffer cells following reperfusion of rat liver stored in Euro-Collins solution, in Wisse E, Knook DL, Decker K (eds): Cells of the Hepatic Sinusoid. 1989, vol 2, pp 277-280 31. Ottnad E, Via DP, Sinn H, Friedrich E, Ziegler R, Dresel HA: Binding characteristics of reduced hepatic receptors for acetylated low-density lipoprotein and maleylated bovine serum albumin. Biochan J 1990-^65:689-698 32. Ottnad E, Via DP, Frflbis J, Sinn H, Friedrich E, Ziegler R, Dresel HA: Differentiation of binding sites on reconstituted hepatic scavenger receptors using oxidized tow-density lipoprotein. Biochan J 1992281:745-751 33. Ohkuma S, Poole B: Fluorescence probe measurements of the intrarysosomal pH in living cells and the perturbation of pH by various agents. Proc NatlAcad Sci U S A 1978;75:3327-3331 34. De Rijke YB, Hessels EMAJ, Van Berkel ThJC: Recognition sites on rat liver cells for aridatively modified /J-very low density lipoproteins. Arterioscla Thmmb 1992;12:41-49 35. Kleinherenbrink-Stins MF, Van de Boom JH, Schouten D, Roholl PJM, Van der Heyde MN, Brouwer A, Van Berkel ThJC, Knook
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Kamps et al DL: Visualization of the interaction of native and modified lipoproteins with parenchymal, endothelial and Kupffer cells from human liver. Hepalology 1991;14:79-90 36. Palinski W, Rosenfeld ME, YlS-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL: Low density lipoprotein undergoes oxidative modification in vivo. Proc NatlAcad Sd U S A 1989-,86:1372-1376 37. Kita T, Nagano Y, Yokode M, Ishii K, Kume N, Ooshima A, Yoshida H, Kawai C: Piobucol prevents the progression of athero-
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sclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc NatlAcad SdU SA 1987;84:5928-5931 38. Carew TE, Swenke DC, 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 streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc NatlAcad Sd U SA 1987;84:7725-7729
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Characterization of the interaction of acetylated LDL and oxidatively modified LDL with human liver parenchymal and Kupffer cells in culture. J A Kamps, J K Kruijt, J Kuiper and T J van Berkel Arterioscler Thromb Vasc Biol. 1992;12:1079-1087 doi: 10.1161/01.ATV.12.9.1079 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1992 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636
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Characterization of the Interaction of Acetylated LDL and Oxidatively Modified LDL With Human Liver Parenchymal and Kupffer Cells in Culture Jan A.A.M. Kamps, J. Kar Kruijt, Johan Kuiper, and Theo J.C. van Berkel The interaction of acetylated low density lipoprotein (Ac-LDL) and oxidatively modified low density lipoprotein (Ox-LDL) with cultured human liver parenchymal cells and human Kupffer cells was investigated to define, for humans, the presence of scavenger receptors in the liver. A direct comparison of the capacity of Kupffer and parenchyma] cells to interact with Ac-LDL and Ox-LDL indicated that the capacity of Kupffer cells per milligram of cell protein to degrade Ac-LDL and Ox-LDL is 14-fold and sixfold higher, respectively, than that of parenchymal cells. The degradation of both Ac-LDL and Ox-LDL by parenchymal cells and Kupffer cells could be inhibited by chloroquine and ammonium chloride, indicating that degradation occurs in the lysosomes. Competition studies showed that unlabeled Ox-LDL competed efficiently with the cell association and degradation of 125I-labeled Ac-LDL by human parenchyma] cells and human Kupffer cells. However, unlabeled Ac-LDL did not compete (parenchymal cells) or only partially competed (40% in Kupffer cells) with the cell association and degradation of '"1-labeled Ox-LDL. Polyinosink acid completely blocked the cell association and degradation of Ac-LDL and Ox-LDL with Kupffer cells while no significant effect on parenchymal cells was noted. It is concluded that human liver parenchymal cells contain a scavenger receptor that interacts with Ac-LDL and Ox-LDL and an additional recognition site that recognizes Ox-LDL specifically. On human Kupffer cells, as on rat Kupffer cells, in addition to the scavenger (Ac-LDL) receptor that interacts efficiently with both Ac-LDL and Ox-LDL, a specific Ox-LDL receptor is present at a relatively high concentration. The identification of various recognition sites for modified LDL on human liver cells indicates that the liver protection system against circulating modified LDL, identified earlier in rats, is also highly relevant for humans. (Arteriosclerosis and Thrombosis 1992;12:1079-1087)
KEY WORDS • oxidatively modified low density lipoproteins • acetylated low density lipoproteins • scavenger receptors • human liver parenchymal cells • human Kupffer cells
O
ne characteristic in the early development of atherosclerotic lesions is the formation of foam cells.1 The conversion of macrophages into foam cells cannot be provoked by native low density lipoprotein (LDL).2 Various modifications of LDL, such as acetylation, acetoacetylation, malondialdehyde treatment, and oxidative modification, lead to LDL uptake via scavenger receptors, which leads to foam cell formation.3 Evidence has recently been provided that various classes of scavenger receptors are involved in the uptake of modified LDL by peritoneal mouse macrophages.4-5 Also, in rat liver it has been established that different recognition sites are involved in the uptake of differently modified LDL.6 When injected into rats, both From the Division of Biopharmaceutics, Center for Bio-Pharmaceutical Sciences, Sylvius Laboratory, University of Leiden, Leiden, The Netherlands. Supported by a grant from the Dutch Heart Foundation (grant No. 87001). Address for correspondence: Prof. Dr. ThJ.C. van Berkel, Division of Biopharmaceutics, Center for Bio-Pharmaceutical Sciences, Sylvius Laboratory, University of Leiden, PO Box 9503, 2300 RA Leiden, The Netherlands. Received April 21, 1992; accepted May 29, 1992.
acetylated LDL (Ac-LDL) and oxidatively modified LDL (Ox-LDL) are rapidly cleared from the blood circulation by the liver. Separation of the liver into parenchymal cells, endothelial cells, and Kupffer cells indicated that endothelial cells were mainly responsible for the uptake of Ac-LDL, whereas Kupffer cells were responsible for the uptake of most of the Ox-LDL. In vitro studies with isolated liver cells provided further evidence that various scavenger receptors are involved in the liver uptake of modified LDL. In addition to the Ac-LDL receptor on liver endothelial cells,7-8 which interacts with both Ac-LDL and Ox-LDL, a specific recognition site for Ox-LDL that was highly concentrated on Kupffer cells was reported.6 Because several lines of evidence suggest that oxidative modification can occur in vivo1-910 and because Ox-LDL, when injected into rats, is rapidly cleared from the circulation by the liver, it was suggested that in vivo Kupffer cells will form the major protection system against the occurrence of atherogenic Ox-LDL particles in the blood. However, the relevance of these studies for humans is unclear especially because it was reported that human hepatocytes express no surface receptors for modified LDL,11 although Edge et al12 found that modified LDL
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was bound and degraded by hepatocytes at 30-50% of the level of native LDL. Kume et al13 indicated that human endothelial cells, in contrast to mouse peritoneal macrophages, do not have any additional receptors specific only for Ox-LDL. In earlier studies we found that both human parenchymal and Kupffer cells interact with LDL through the classic LDL receptor,14 although the association of LDL with Kupffer cells is more efficiently coupled to degradation than with parenchymal cells. The availability of cultured human liver parenchymal cells and human Kupffer cells enabled us to compare directly the interaction of Ac-LDL and Ox-LDL with human liver cells. Methods Materials Collagenase type I, human serum albumin (fraction V), dexamethasone, and chloroquine were purchased from Sigma Chemical Co. (St. Louis, Mo.); Nycodenz was obtained from Nycomed A/S (Oslo, Norway); and 125 I was purchased from Amersham International (Amersham, Bucks, UK). Fetal calf serum, penicillin, and streptomycin were obtained from Boehringer (Mannheim, Germany), and Williams' E culture medium and kanamycin were purchased from Flow Laboratories (Irvine, Scotland, UK). RPMI-1640 cell culture medium was from GIBCO (Paisley, Scotland, UK), and multiwell cell culture dishes were from Costar (Cambridge, Mass.). All other chemicals were of analytical grade. Isolation, Iodination, Oxidation, and Acetylation of LDL Human LDL was obtained from the blood of healthy volunteers who had fasted overnight. Isolation of LDL (1.02425I-labeled acetylated (Ac) low density Upoprotein (LDL) or 123I-labeled oxidized (Ox) LDL with human parenchymal cells. Parenchymal cells were incubated for 3 hours at 37°C with 10 /Jg/ml 125I-^Ac-LDL (panel A) or 125I-Ox-LDL (panel B) and with the indicated amounts of unlabeled LDL (m),Ac-LDL (•), Ox-LDL (A), or polyinosinic acid (T). Cell association of l25I^4c-LDL or 12}I-Ox-LDL is expressed as the radioactivity obtained in the absence of a competitor. Values for 100% cell association of 125I-Ac-LDL and 125 I-Ox-LDL with parenchymal cells are 48 and 156 ng apolipoprotein per milligram of cell protein, respectively. Data represent mean of two or three experiments. When indicated, bars represent SEM.
about 70% by unlabeled Ac-LDL or polyinosinic acid. Unlabeled Ox-LDL appears to be slightly less efficient as a competitor than Ac-LDL. Native LDL was relatively ineffective. The cell association and degradation of 125I-Ox-LDL by Kupffer cells is efficiently inhibited by Ox-LDL itself and by polyinosinic acid. Ac-LDL is clearly less efficient as a competitor than Ox-LDL. The cell association and degradation of 125I-Ox-LDL is only slightly inhibited by native LDL. Discussion In the present work we analyzed the interaction of 125 I-Ac-LDL and 125I-Ox-LDL with human liver parenchymal cells and human Kupffer cells. Recent studies in rats indicate that Kupffer cells appear to be the main site in the liver for the uptake of Ox-LDL.6 Because Ox-LDL is considered to be the pathological form of modified LDL, it was suggested that Kupffer cells may form a major protection system against the action of Ox-LDL.6 Furthermore, in vitro studies with isolated liver cell types (parenchymal, endothelial, and Kupffer cells) provided evidence that in addition to the Ac-LDL receptor, which is concentrated on liver endothelial cells,8 Kupffer cells contain a scavenger receptor that specifically interacts with Ox-LDL.6 So far there are no data concerning the role of human liver parenchymal cells and human Kupffer cells in the metabolism of Ac-LDL and Ox-LDL nor on the possible involvement of various scavenger receptors in the uptake of modified LDL. The availability of human parenchymal cells and Kupffer cells enabled us to address these questions for the human situation. Human liver endothelial cells could not be isolated from human liver, probably because of a loss of viable liver endothelial cells, which has been observed to occur when cold ischemic liver material is reperfused with warm oxygenated buffer.30
calcium-independent, specific recognition sites that recognize Ac-LDL and/or Ox-LDL. Competition experiments indicate that human liver parenchymal cells contain 1) a specific Ac-LDL recognition site, which is partly competed for by Ox-LDL and 2) a specific Ox-LDL recognition site, which is not competed for by Ac-LDL and is hardly by polyinosinic acid. Human Kupffer cells express 1) an Ac-LDL recognition site, which is also blocked by Ox-LDL and polyinosinic acid and 2) an Ox-LDL recognition site, which interacts only partially with Ac-LDL and is completely blocked by polyinosinic acid. These results are comparable with the results that were found for the interaction of Ac-LDL and Ox-LDL with rat liver parenchymal cells and Kupffer cells,6 indicating that in addition to a scavenger receptor, which interacts with both Ac-LDL and OxLDL, a specific Ox-LDL receptor exists on liver cells. It is possible that this receptor is similar to the class III binding sites as defined on basis of the specificity of reconstituted hepatic scavenger receptors.31-32 The specific recognition of Ox-LDL by Kupffer cells cannot be explained by aggregation phenomena because we used a relatively low apolipoprotein concentration (200 fig/tnl) for oxidation. Moreover, we subjected Ox-LDL, directly before the incubations, to filtration over a 22-/xm filter; also, complete blockade of the interaction of Ox-LDL with Kupffer cells by polyinosinic acid points to the involvement of scavenger receptors and not bulk-phase endocytosis. To investigate whether lysosomes are involved in the degradation of Ac-LDL and Ox-LDL, chloroquine and ammonium chloride were used. These two unrelated compounds inhibit lysosomal proteolysis by increasing the lysosomal pH.33 Based on their inhibitory action, it appeared that the uptake of both AcLDL and Ox-LDL by parenchymal cells and Kupffer cells was coupled to rysosomal degradation.
The data in this study demonstrate that both human parenchymal cells and Kupffer cells in culture possess
There is a dispute about the role of human parenchymal cells in the uptake of modified LDL. Edge et al12
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Interaction of Modified LDL With Human liver Cells
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FIGURE 5. Line plots comparing the ability ofunlabeled lipoproteins or potyinosinic acid to compete for the cell association and degradation of I25I-labeled acetylated (Ac) low density lipoprotein (LDL) or I25I-labeled oxidized (Ox) LDL by human Kupffer cells. Kupffer cells were incubated for 3 hours at 37°C with 10 uglml 125I-Ac-LDL ("panels A and B) or 125I-Ox-LDL (panels C and D) and with the indicated amounts of unlabeled LDL (•), Ac-LDL (•), Ox-LDL (A), or polyinosinic acid ( • ) . Cell association (panels A and C) and degradation (panels B and D) of125l-Ac-LDL or I25I-Ox-LDL are expressed as the radioactivity and l25I-Ox-LDL with Kupffer cells are obtained in the absence of a competitor. Values for 100% cell association ofml-Ac-LDL 498 and 978 ng apolipoprotein per milligram of cell protein, respectively, and for degradation are 273 and 432 ng apolipoprotein per milligram of cell protein, respectively. Data represent mean of two experiments.
demonstrated that Ac-LDL was bound and degraded at 30-50% of the level of that of native LDL. Babaev et al11 reported that binding, internalization, and degradation of malondialdehyde-treated LDL by cultured human hepatocytes was caused by contamination of the primary cultures by endothelial and Kupffer cells. We observed a specific recognition site for modified LDL in human parenchymal cells, which cannot be due to contamination of the parenchymal cells with nonparenchymal cells, as polyinosinic acid does not compete for the interaction of Ox-LDL with parenchymal cells while polyinosinic acid is a very effective competitor for the interaction of Ac-LDL and Ox-LDL with Kupffer cells. Also, with rat liver cells it has been shown that the interaction of Ac-LDL, Ox-LDL, and Ox-/3-VLDL with
endothelial and Kupffer cells is effectively inhibited by polyinosinic acid, whereas with parenchymal cells this interaction is not blocked.6-34 ' Human liver endothelial cells could not be isolated from the human liver, thus, no information about the importance of these cell types for the relative interaction with Ac-LDL and Ox-LDL could be obtained. From in vivo and in vitro studies with rats it has been shown that rat liver endothelial cells account for 52% of the total uptake of Ac-LDL and for 36% of the total uptake of Ox-LDL.6 Recent studies in which human liver tissue blocks were perfused in vitro with l,l'-dioctadecyl-333'»3'-tetramethyl indocarbocyanine perchlorate-labeled Ac-LDL provide evidence that in the human liver also, Ac-LDL is mainly taken up by liver endothelial cells.33
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Arteriosclerosis and Thrombosis
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The capacity of human Kupffer cells for cell association and degradation of Ac-LDL and Ox-LDL is considerably higher than that of parenchymal cells (respectively, 4.8- and 4.9-fold per milligram of cell protein) for which, in addition to a scavenger receptor, which interacts with both Ac-LDL and Ox-LDL, a specific Ox-LDL receptor is responsible. It appears that the recognition sites for Ac-LDL and Ox-LDL on human parenchymal and human Kupffer cells exert similar properties as those on rat liver cells. Earlier, we demonstrated that human Kupffer cells also have a higher capacity than do parenchymal cells to catabolize native LDL.14 Together, these data indicate that Kupffer cells may contribute significantly to the regulation of the level of (modified) LDL in the human situation and thus in the protection against the development of atherosclerosis. Several lines of evidence, such as the occurrence of autoantibodies against oxidized LDL in human sera3* and the prevention of the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits by probucol,37-38 indicate that Ox-LDL is the pathophysiologicalry occurring form of modified LDL. The presence of a specific Ox-LDL recognition site on human liver parenchymal cells and especially on human Kupffer cells may form a major protection in humans against the pathological action of Ox-LDL in the blood compartment. References 1. Steinberg D: Metabolism of lipoproteins and their role in the pathogenesis of atherosclerosis. Atheroscla Rev 1988;18:l-23 2. Brown MS, Basu SK, Falck JR, Ho YK, Goldstein JL: The scavenger cell pathway for lipoprotein degradation: Specificity of the binding site that mediates the uptake of negatively-charged LDL by macrophages. J Supramol Struct 1980;13:67-81 3. Brown MS, Goldstein JL: Lipoprotein metabolism in the macrophage: Implication for cholesterol deposition in atherosclerosis. Annu Rev Biochan 1983^2:223-261 4. Sparrow CP, Parthasarathy S, Steinberg D: A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein. / Biol Chan 1989;264:2599-2604 5. Arai H, Kita T, Yokode M, Narumiya S, Kawai C: Multiple receptors for modified low density lipoproteins in mouse peritoneal macrophages: Different uptake mechanisms for acetylated and oxidized low density lipoproteins. Biochan Biophys Res Commun 1989-,159:1375-1382 6. Van Berkel ThJC, De Rijke YB, Kruijt JK: Different fate in vivo of cnridativery modified low density lipoprotein and acetylated low density lipoprotein in rats: Recognition by various scavenger receptors on Kupffer and endothelial liver cells. / Biol Chan 1991; 266:2282-2289 7. Van Berkel ThJC, Nagelkerke JF, Harkes L, Kruijt JK: Processing of acetylated human low-density lipoprotein by parenchymal and non-parenchymal liver cells; Involvement of calmodulin? Biochan J 1982208:493-503 8. Nagelkerke JF, Barto KP, Van Berkel ThJC: In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells. J Biol Chan 1983^58:12221-12227 9. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL: Beyond cholesterol: Modifications of low density lipoprotein that increase its atherogenicity. N Engl J Med 1989^20:915-923 10. Wiklund O, Mattsson L, Bjornheden T, Camejo G, Bondjers G: Uptake and degradation of low density lipoproteins in atherosclerotic rabbit aorta: Role of local LDL modification. / Upid Res 199132:55-62 11. Babaev VR, Kosykh VA, Tsibulsky VP, Ivanov VO, Repin VS, Smirnov VN: Binding and uptake of native and modified lowdensity lipoproteins by human hepatocytes in primary culture. Hepatology 1989;10-.56-60 12. Edge SB, Hoeg JM, Triche T, Schneider PD, Brewer HB J r Cultured human hepatocytes: Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor. / Biol Chan 1986;261:3800-3806
September 1992 13. Kume N, Arai H, Kawai C, Kita T: Receptors for modified lowdensity lipoproteins on human endothelial cells: Different recognition for acetylated low-density lipoprotein and oxidized lowdensity lipoprotein. Biochim Biophys Acta 1991;1091:63-67 14. Kampt JAAM, Kruijt JK, Kuiper J, Van Berkel ThJC: Uptake and degradation of human low-density lipoprotein by human liver parenchymal and Kupffer cells in culture. Biochan J 1991^76:135-140 15. Redgrave TG, Roberts DCK, West CE: Separation of plasma lipoproteins by density gradient ultracentrifugation. Anal Biochan 1975;65:42-49 16. Basu SK, Goldstein JL, Anderson RGW, Brown MS: Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Nad Acad Sci U S A 1976;73:3178-3182 17. Steinbrecher UP, Witztum JL, Parthasarathy S, Steinberg D: Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL: Correlation with changes in receptor mediated catabolism. Arteriosclerosis 1987;7:135-143 18. McFarlane AS: Efficient trace-labeling of proteins with iodine. Nature 1958;182:53 19. Bilheimer DW, Eisenberg S, Levy RI: The metabolism of very tow density lipoproteins: I. Preliminary in vitro and in vivo observations. Biochim Biophys Acta 1972;260:212-221 20. Princen HMG, Huijsmans CMG, Kuipers F, Vonk RJ, Kempen HJM: Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7a-bydroxylase. J Clin Invest 1986;78:1064-1071 21. Kwekkeboom J, Princen HMG, Van Voorthuizen EM, Merjer P, Kempen HJM: Comparison of taurocholate accumulation in cultured hepatocytes of pig, man and rat. Biochan Biophys Res Commun 1989;162:619-625 22. Forte TM, Nordhausen RW, Princen HMG: Structural properties of lipoproteins isolated from human hepatocyte cultures, (abstract) Anaiosclerosis 1989;9:693a 23. Kooistra T, Bosma PJ, Tons HAM, Van den Berg AP, Meijer P, Princen HMG: Plasminogen activator inhibitor 1: Biosynthesis and mRNA level are increased by insulin in cultured human hepatocytes. Thmmb Haanost 1989;62:723-728 24. Krenning EP, De Jong M, Vos RA, Bernard HF, Docter R, Menneman G: Transport and metabolism of thyroid hormones in human hepatocytes. Endocrinology 1989;122:T-36 25. Scbouten D, Kleinherenbrink-Stins MF, Brouwer A, Knook DL, Kamps JAAM, Kuiper J, Van Berkel ThJC: Characterization in vitro of interaction of human apolipoprotein E-free high density lipoprotein with human hepatocytes. Arteriosclerosis 1990;10:1127-1135 26. Kamps JAAM, Kuiper J, Kruijt JK, Van Berkel ThJC: Complete down-regulation of low-density lipoprotein receptor activity in human liver parenchymal cells by /S-very-tow-density lipoprotein. FEBS Lett 1991^87:34-38 27. Van Berkel ThJC, Kruijt JK, Van Gent T, Van Tol A: Saturable high affinity uptake and degradation of rat plasma lipoproteins by isolated parenchymal and non-parenchymal cells from rat liver. Biochim Biophys Acta 1981;665:22-33 28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chan 1951;193: 265-275 29. Ho YK, Brown MS, Bilheimer DW, Goldstein JL: Regulation of low density lipoprotein receptor activity in freshly isolated human lymphocytes. / Clin Invest 1976^8:1465-1474 30. Lemasters JL, Caldwell-Kenkel JC, Currin RT, Yanaka Y, Marzi I, Thurman RG: Endothelial cell killing and activation of Kupffer cells following reperfusion of rat liver stored in Euro-Collins solution, in Wisse E, Knook DL, Decker K (eds): Cells of the Hepatic Sinusoid. 1989, vol 2, pp 277-280 31. Ottnad E, Via DP, Sinn H, Friedrich E, Ziegler R, Dresel HA: Binding characteristics of reduced hepatic receptors for acetylated low-density lipoprotein and maleylated bovine serum albumin. Biochan J 1990-^65:689-698 32. Ottnad E, Via DP, Frflbis J, Sinn H, Friedrich E, Ziegler R, Dresel HA: Differentiation of binding sites on reconstituted hepatic scavenger receptors using oxidized tow-density lipoprotein. Biochan J 1992281:745-751 33. Ohkuma S, Poole B: Fluorescence probe measurements of the intrarysosomal pH in living cells and the perturbation of pH by various agents. Proc NatlAcad Sci U S A 1978;75:3327-3331 34. De Rijke YB, Hessels EMAJ, Van Berkel ThJC: Recognition sites on rat liver cells for aridatively modified /J-very low density lipoproteins. Arterioscla Thmmb 1992;12:41-49 35. Kleinherenbrink-Stins MF, Van de Boom JH, Schouten D, Roholl PJM, Van der Heyde MN, Brouwer A, Van Berkel ThJC, Knook
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Kamps et al DL: Visualization of the interaction of native and modified lipoproteins with parenchymal, endothelial and Kupffer cells from human liver. Hepalology 1991;14:79-90 36. Palinski W, Rosenfeld ME, YlS-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL: Low density lipoprotein undergoes oxidative modification in vivo. Proc NatlAcad Sd U S A 1989-,86:1372-1376 37. Kita T, Nagano Y, Yokode M, Ishii K, Kume N, Ooshima A, Yoshida H, Kawai C: Piobucol prevents the progression of athero-
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sclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc NatlAcad SdU SA 1987;84:5928-5931 38. Carew TE, Swenke DC, 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 streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc NatlAcad Sd U SA 1987;84:7725-7729
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Characterization of the interaction of acetylated LDL and oxidatively modified LDL with human liver parenchymal and Kupffer cells in culture. J A Kamps, J K Kruijt, J Kuiper and T J van Berkel Arterioscler Thromb Vasc Biol. 1992;12:1079-1087 doi: 10.1161/01.ATV.12.9.1079 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1992 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636
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