Influence of the acyl-CoA:cholesterol O-acyltransferase inhibitor, CL 277082, on cholesteryl ester accumulation in rabbit macrophage-rich granulomas and hepatic tissue

Kelley, Jim; Suenram, C. Alan; Rozek, Marius M.; Schaffer, Sheldon A.; Schwartz, Colin J.

doi:10.1016/0005-2760(88)90012-4

Cited by 18 publications

(7 citation statements)

References 24 publications

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“…1314 Melinamide 28 and a furobenzochromone described by Gammill and colleagues 29 were reported to blunt a diet-induced hypercholesterolemia by 46% and 88%, respectively. Kelley et al 30 have reported that after 6 weeks of treatment with CL277082, plasma triglycerides were elevated eightfold. In contrast to CL277082, plasma triglycerides were unaffected by CI-976.…”

Section: Discussionmentioning

confidence: 99%

Comparison of CI-976, an ACAT inhibitor, and selected lipid-lowering agents for antiatherosclerotic activity in iliac-femoral and thoracic aortic lesions. A biochemical, morphological, and morphometric evaluation.

Bocan¹,

Mueller²,

Uhlendorf³

et al. 1991

Arterioscler Thromb

151

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Due to the potential importance of acyl-coenzyme A:cholesterol 0-acyltransferase (ACAT) in the generation of lipid-filled monocytes-macrophages, the ACAT inhibitor CI-976 (2,2-dimethyl-AT-(2,4,6-trimethoxyphenyl)dodecanamide) was evaluated relative to selected lipidlowering agents for their effect on atherosclerotic lesion regression and progression. Atherosclerotic lesions comparable in composition to human fatty streaks were induced by chronic endothelial denudation in the iliac-femoral artery of hypercholesterolemic New Zealand White rabbits before intervention, while naturally occurring fatty streaks developed in the thoracic aorta. CI-976 administered in a hypercholesterolemic diet at a dose that did not lower plasma cholesterol prevented the accumulation of monocytes-macrophages within the preestablished iliac-femoral lesion and reduced the foam cell area by 27-29% relative to the initiation of intervention. CI-976 also blunted the development of thoracic aortic fatty streak-like lesions and decreased the cholesteryl ester enrichment by 46%. CI-976 had no effect on plasma triglycerides and, more importantly, had no effect or decreased liver, iliac-femoral, and thoracic aortic free cholesterol content Dietary intervention alone increased monocytemacrophage involvement in the iliac-femoral lesion despite reductions in plasma, liver, and thoracic aortic cholesterol content Conventional lipid-lowering therapy such as cholestyramine or cholestyramine/niacin required substantial decreases in plasma cholesterol levels to achieve comparable vascular changes. We conclude that inhibition of ACAT within the arterial wall by the potent and specific ACAT inhibitor CI-976, even in the absence of plasma cholesterol lowering, can result in the inhibition of atherosclerotic lesion progression and can enhance regression. (Arteriosclerosis and Thrombosis 1991;ll:1830-1843) A cyl-coenzyme A: cholesterol O-acyltransferase / \ (EC 2.3.1.26; ACAT), the primary enzyme JL \~ responsible for the esterification of cholesterol in all mammalian cells, has been implicated as a key enzyme involved in cholesterol absorption, very low density lipoprotein secretion, and the formation of lipid-filled macTophages and smooth muscle cells (SMCs).1 In addition, monocyte-macrophage foam cells have been shown to be integral components of human and animal atherosclerosis. In humans, both Received March 19, 1991; revision accepted August 19, 1991. fatty streaks and fibrous plaques are characterized by the presence of lipid-filled macrophages; however, in fibrous plaques the appearance of abundant extracellular lipid and lipid-filled SMCs is also noted. 2 Several investigators, using monoclonal antibodies to monocytes-macrophages, have shown that monocytes-macrophages in animals are involved in the initiation and development of the fatty streak and the conversion to the fibrous plaque. "10 Since ACAT is responsible for the esterification and storage of cholesterol in the macrophage, one might hypothesize that inhibition of arterial wall ACAT may ...

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Section: Discussionmentioning

confidence: 99%

Comparison of CI-976, an ACAT inhibitor, and selected lipid-lowering agents for antiatherosclerotic activity in iliac-femoral and thoracic aortic lesions. A biochemical, morphological, and morphometric evaluation.

Bocan¹,

Mueller²,

Uhlendorf³

et al. 1991

Arterioscler Thromb

151

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“…Additionally, cholesterol content was increased about three times, from 0. 24 The enhanced cholesterol-esterifying activity demonstrated by hyperlipemic swine monocytes was studied further, as this reaction, catalyzed by ACAT, is prominently displayed by macrophages and atherosclerotic tissue as well. A time course of ACAT activity over 90 minutes in normal and hyperlipemic swine monocytes is shown in Figure 1, in which cholesterol esterification to [ 14 C]oleate was evaluated.…”

Section: Resultsmentioning

confidence: 99%

“…To determine the sensitivity of the monocytic ACAT activity to chemical inhibition, hyperlipemic swine monocytes were incubated in the presence of the ACAT inhibitor CL277-082, 24 and ACAT was evaluated by following the incorporation of [ 14 C]oleate into cholesteryl esters (Table 3). Inhibition of monocytic ACAT was achievable and dose related.…”

Section: Resultsmentioning

confidence: 99%

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Evidence for an altered lipid metabolic state in circulating blood monocytes under conditions of hyperlipemia in swine and its implications in arterial lipid metabolism.

Bell

Gerrity

1992

Arterioscler Thromb

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Circulating blood monocytes were isolated from normal and hypercholesterolemic swine, and the monocyte lipid compositions and lipid biosynthesis profiles were assessed. The data indicate that monocytes freshly isolated from hyperlipemic swine have increased phospholipid and cholesterol contents and have increased biosynthetic capability for synthesizing phospholipids, triglycerides, and cholesteryl esters, but not cholesterol. The profile of the stimulated lipid synthesis capability is similar to that of the swine aortic intima undergoing atherogenic change. These studies indicate that circulating blood monocytes in hyperlipemic swine, which are known to give rise to intimal foam cells in the early fatty streak lesion, can contribute to altered vessel lipid metabolism without a requirement for in situ modification by wall factors. 11 Although some studies have been performed with arterial (monocyte-derived macrophage) foam cells, 12 many of the macrophage studies to date have used macrophages elicited from peritoneum and lung or a macrophage cell line. 5101314 In our studies, we have focused on lipid metabolism in the freshly isolated blood monocyte, as this is the cellular entity that invades the vessel wall, and we previously reported that monocytes from hypercholesterolemic rabbits have a stimulated lipid synthesis profile that differs significantly from that of normolipemic blood monocytes and that resembles the lipid synthesis profile of atherosclerotic vessels. 15 In the present studies, we have successfully isolated monocytes from the blood of normal and hypercholesterolemic swine ]oleate, which resembles the lipid metabolic profile of cholesterol-enriched or lesioned aortic intima. The data suggest that monocytes invading the vessel wall from hypercholesterolemic plasma are capable of contributing to altered wall lipid metabolism without an obligatory requirement for in situ modification. Methods Animals and DietsPairs of male Yorkshire swine (6 weeks of age, 15-20 kg at initiation) were fed either normal hog chow or chow supplemented with 1.5% cholesterol and 19.5% lard 16 for periods of 15-20 weeks. Serum cholesterol levels ranged from 70 to 110 mg/dl in normal swine and between 400 and 600 mg/dl in the hyperlipemic swine. Details of euthanasia and tissue collection have been previously described.

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Differences in the potency of ACAT inhibitors in two assay systems

Harte

Jackson

Suckling

et al. 1993

Biochemical Society Transactions

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Acyl CoA:cholesterol acyltransferase (ACAT) inhibitors have been shown to prevent cholesterol absorption in the intestine [l], cholesterol storage and apo B secretion from the liver [2], and cholesterol accumulation in the aortic artery [3,4]. Compounds have been established as ACAT inhibitors by their ability to inhibit incorporation of labelled fatty acyl CoA into cholesteryl esters i n microsomal preparations. This assay involves an unknown amount of cholesterol and modulators of the enzyme may be present [5,6]. Therefore in order to assess the action of ACAT inhibitors i n a more controlled environment, a reconstitution assay was set up.Firstly, the enzyme was solubilised using detergent and then it was reconstituted into liposomes providing a defined lipid environment. The method was based on that of Cadigan and Chang for human skin fibroblasts and Chinese hamster ovary cells 151. The assay showed protein and time dependence. The kinetics of compounds in the two assays were compared.Liver microsomes were prepared from male Sprague-Dawley rats which had been fed 2% cholesterol to stimulate ACAT activity. They were stored freeze-dried and stored at -2OOC and resuspended in 0.05M KP04/0.05M NaF buffer (pH 7.4) as required. Liposomes were made by the cholestyramine method [7] and contained cholesterol and phosphatidylcholine at a molar ratio of 0.3. Solubilisation was carried out using 3% DOC and solubilised protein was then diluted 1 in 240 into liposomes to effectively dilute out the detergent which would otherwise inhibit ACAT activity.Assays contained 3 . 3~9 reconstituted protein, 5pg fatty acid-free BSA, 1mM DTT in 50mM Tris, 1mM EDTA buffer (pH 7.7) in a final volume of 0.1 ml. Compounds were added in dimethylsulphoxide (DMSO) and after a 5 minute preincubation, the reaction was started by the addition of 1 nmoie [14C]oleoyl CoA and incubated at 37OC for 10 minutes. Reactions were stopped using 1 ml of a propan-2-olfheptane mix (4:l) containing [3H]cholesteryl oleate (approximately 20,000 dpm/ml) as a recovery marker. Lipids were extracted with 0.4ml heptane, 0.6ml water and 0.4ml of the top layer was removed and reaction products were separated using silica gel columns [8]. Activities were expressed as a percentage of the DMSO control. Microsomal assays were carried out as described previously [9].The results show that octimibate is active as an inhibitor of ACAT activity in both assay systems ( Fig. l(a)). However, CI 976 ( Fig. l(b)) does not inhibit in the reconstituted system and DuP 128 shows an intermediate response (Fig. l(c)). Table 1 shows differences in iC50s. (M) and a reconstituted ( R ) assay. The ratio of the two IC50s is also indicated ( R / M ) . octimibate 45 40 1 CI 976 0 . 1 3 14 1 0 8 DuP 128 0 . 0 0 3 5 0.11 3 1 Table 2. Effect of adding an incubation mixture for a reconstitution assay containing CI 976 to a microsomal a s s a y . T h e c o n c e n t r a t i o n o f C I 9 7 6 i n t h e reconstitution assay ( R ) and the final concentration i n t h e m i c r o s o m a l a s s a y (M)...

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Influence of the acyl-CoA:cholesterol O-acyltransferase inhibitor, CL 277082, on cholesteryl ester accumulation in rabbit macrophage-rich granulomas and hepatic tissue

Cited by 18 publications

References 24 publications

Comparison of CI-976, an ACAT inhibitor, and selected lipid-lowering agents for antiatherosclerotic activity in iliac-femoral and thoracic aortic lesions. A biochemical, morphological, and morphometric evaluation.

Comparison of CI-976, an ACAT inhibitor, and selected lipid-lowering agents for antiatherosclerotic activity in iliac-femoral and thoracic aortic lesions. A biochemical, morphological, and morphometric evaluation.

Evidence for an altered lipid metabolic state in circulating blood monocytes under conditions of hyperlipemia in swine and its implications in arterial lipid metabolism.

Differences in the potency of ACAT inhibitors in two assay systems

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