Differential Effects of Lovastatin on the Trafficking of Endogenous and Lipoprotein-Derived Cholesterol in Human Monocyte–Derived Macrophages

From Institut für Arterioskleroseforschung an der Universität Münster, Münster, Germany.

Correspondence to Paul Cullen, Institut für Arterioskleroseforschung an der Universität Münster, Domagkstrasse 3, 48149 Münster, Germany. E-mail cullen{at}uni-muenster.de

	Abstract

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Abstract—Lovastatin has been shown to reduce cholesterol esterification in cholesterol-loaded human macrophages. Surprisingly, in nonloaded macrophages, lovastatin produces the opposite effect, lowering free cholesterol and increasing cholesteryl ester levels, as measured by high-performance liquid chromatography. In cholesterol-loaded cells, lovastatin reduced the cholesteryl esters of unsaturated but not those of saturated fatty acids. In nonloaded cells, by contrast, the cholesteryl esters of unsaturated fatty acids tended to increase after lovastatin treatment. Total (free plus esterified) cellular cholesterol content in nonloaded cells fell by 18% with 12-µmol/L lovastatin treatment but did not change in cholesterol-loaded cells. Lovastatin had no effect on the binding or uptake of acetylated low density lipoprotein, acyl coenzyme A:cholesterol acyltransferase (ACAT) activity, the secretion of [³H]cholesterol into the medium, or lysosomal hydrolysis of cholesteryl esters. Apolipoprotein (apo) E mRNA levels increased but apoE secretion into the medium decreased with lovastatin treatment in both cholesterol-loaded and nonloaded cells. Cholesterol of exogenous origin has been shown to pass via the cell membrane before its esterification by ACAT. We postulate that this is not the case for endogenous cholesterol, which may have direct access to ACAT. Our findings therefore suggest that lovastatin hinders the delivery of intracellular cholesterol to the plasma membrane, resulting in increased free cholesterol and lower levels of cholesteryl ester in cholesterol-loaded cells. In nonloaded cells, virtually all cholesterol is of endogenous origin and is normally translocated to the cell membrane. Lovastatin prevents this process, thus shunting newly synthesized cholesterol toward esterification and leading to an increase in the concentration of cholesteryl esters, even in the face of a drop in total and free cholesterol levels. Intracellular apoE may play a role in this process.

Key Words: lovastatin • cholesterol esterification • apoE • human macrophages

	Introduction

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Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (statins) are very commonly used for treatment of hypercholesterolemia, and they comprise one of the most commonly prescribed classes of drug in the United States and many other developed countries. Their main action is to lower LDL cholesterol via an upregulation of hepatic LDL receptors.¹ In controlled clinical trials, statins have been shown to decrease the incidence of coronary heart disease (CHD) and to reduce CHD mortality.^{2 3} Furthermore, angiographic studies have shown that statins may halt, and perhaps even reverse, atherosclerotic changes in the coronary arteries.^{4 5 6 7 8} Recently, it has been reported that radical lowering of LDL with statins also prevents restenosis after percutaneous transluminal coronary angioplasty.⁹

These effects have generally been ascribed to the lowering of circulating LDL. However, it is known that statins have a number of other important pharmacological effects other than reducing LDL. These direct effects may influence cell types that are important components of the atherosclerotic lesion. Statins inhibit smooth muscle cell proliferation and hence neointima formation.^{10 11} In higher concentrations, they have also been shown to induce apoptosis in smooth muscle and other cells.^{12 13} In cholesterol-loaded human macrophages, Kempen et al¹⁴ have shown that statins inhibit cholesteryl ester formation by a mechanism that does not involve direct inhibition of acyl coenzyme A:cholesterol acyltransferase (ACAT).

We recently developed a high-performance liquid chromatography (HPLC) method that allows measurement of individual cholesteryl ester species in human monocyte–derived macrophages.¹⁵ We therefore investigated in detail the effects of statins on cellular cholesterol and cholesteryl ester levels. Radiolabeled cholesteryl esters were used to investigate the intracellular metabolism of cholesterol.

Macrophages in the arterial wall produce substantial amounts of apolipoprotein (apo) E. This apoE is thought to play an important role in cholesterol efflux, even in the absence of extracellular cholesterol acceptors,¹⁶ and to exert a strong atheroprotective effect.¹⁷ The regulation of apoE transcription and secretion in macrophages is intimately connected with intracellular cholesterol metabolism.¹⁷ We therefore also investigated the effects of statins on the transcription and secretion of apoE in human monocyte–derived macrophages in vitro. Finally, to exclude the possibility that differences seen may have arisen owing to a lovastatin-induced alteration in expression of macrophage scavenger receptors, we measured the effects of the drug on the binding of radiolabeled acetylated (Ac) LDL to the cells.

Our results show that statins lower total cholesterol levels in non–cholesterol-loaded macrophages and prevent cholesterol ester accumulation in cholesterol-loaded macrophages. These effects may contribute to the antiatherogenic action of this drug class.

	Methods

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Cell Culture and Drug Treatment
Monocytes were isolated from healthy volunteer donors by elutriation and countercurrent centrifugation as previously described.¹⁸ All donors gave informed, written consent, and none was receiving medications of any kind. The procedure for monocyte isolation was approved by the Hospital Ethics Committee. Purity of isolated monocytes was >95% as revealed by fluorescence-activated cell sorting analysis. Monocytes were maintained in RPMI 1640 medium (Gibco) supplemented with 20% pooled human serum for 14 days to allow differentiation into macrophages. Thereafter, the cells were washed 3 times with serum-free RPMI 1640 and incubated for 24 hours in the same medium containing 0.2% fatty acid–free BSA and lovastatin. The cells were then exposed to fresh medium containing lovastatin and, where indicated, AcLDL (80 to 100 µg protein/mL) for 24 hours. In some cases, fresh medium was added for an additional 24 hours. Mevalonate (100 µmol/L) was present throughout the incubations where indicated. Lovastatin was a kind gift of Dr K. Bestehorn, MSD Sharpe & Dohme, Munich, Germany. Cell viability was assessed by calculating the amount of cell protein in each dish or culture flask and by means of the trypan blue exclusion test.

Lipoprotein Isolation and Labeling
LDL was obtained from human plasma from healthy volunteer donors by sequential ultracentrifugation (d=1.019 to 1.063 g/mL). AcLDL was obtained by repeated additions of acetic anhydride to LDL as previously described by Basu et al.¹⁹ For labeling, AcLDL was incubated with 200 µCi/mL [³H]cholesteryl linoleate or 25 µCi/mL cholesteryl [¹⁴C]linoleate (NEN-Du Pont) in the presence of lipoprotein-deficient serum for 6 hours at 37°C.²⁰ The reaction mixture was then adjusted to d=1.063 g/mL and ultracentrifuged for 2.5 hours at 4°C in a TL100 rotor (Beckman Instruments) in 1.5-mL polypropylene tubes. Top fractions were pooled and thoroughly dialyzed in 0.9% NaCl. The product was analyzed by agarose gel electrophoresis to confirm the mobility shift of AcLDL versus normal LDL. Protein content was measured by the method of Lowry et al.²¹

Quantitative Reverse Transcription (RT)–Polymerase Chain Reaction (PCR)
Macrophages were incubated in the presence or absence of 80 µg/mL AcLDL for 24 hours and washed twice with sterile PBS. RNA was obtained by guanidinium-phenol-chloroform extraction.²² Quantitative RT-PCR was performed as described by Grassi et al²³ with slight modifications. In brief, an internal RNA standard differing from wild-type apoE mRNA only by a 20-base insertion in the middle was prepared by sequential PCRs and transcription by T7-RNA polymerase. Its concentration was evaluated by densitometric analysis by comparison with known amounts of standard RNA. Increasing amounts of internal standard were added to 0.2-µg aliquots of cell RNA before the RT step. After denaturation for 3 minutes at 94°C, 30 cycles of PCR were performed at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds, followed by a final 5-minute extension at 72°C. The primers used for this step were as follows: forward primer, 5'-aag gac gtc ctt ccc cag gag c-3'; reverse primer, 5'-ctt cat ggt ctc gtc cat cag c-3'. The bands corresponding to wild-type RNA (308 bases) and internal standard (328 bases) were analyzed by densitometric scanning. The internal standard to wild type RNA band intensity ratio was plotted against the number of molecules of internal standard that had been added. The concentration of wild-type apoE mRNA was read from the point on the regression line at which the ratio of internal standard to wild-type RNA was equal to 1. In preliminary studies, this assay showed a coefficient of variation of 13%.

Measurements of ApoE Secretion
Macrophages were incubated in the presence or absence of 80 µg AcLDL for 24 hours, washed, and cultivated for another 24 hours in serum-free RPMI 1640 medium. This medium was collected, centrifuged to remove cell debris, and analyzed for apoE secretion by sandwich ELISA as previously described.²⁴

Binding of ¹²⁵I-AcLDL to the Cell
AcLDL was iodinated according to the Iodo-bead (Pierce Chemical Co) method as described.²⁵ Final specific activities ranged between 230 and 320 cpm/ng protein. Macrophages were preincubated for 24 hours in the presence or absence of 5 µmol/L lovastatin. The cells were chilled to 4°C for 30 minutes and then incubated with increasing concentrations of ¹²⁵I-AcLDL for 2 hours at 4°C with or without lovastatin. Binding experiments were otherwise performed as previously described.²⁶

Cellular Cholesterol Measurement
Macrophages were loaded with cholesterol by incubation with 80 µg/mL AcLDL in serum-free RPMI 1640 for 24 hours. Control macrophages were incubated in RPMI 1640 only for the same period of time. Cells were washed 3 times with serum-free medium and equilibrated in the same medium for another 24 hours. After extensive washing with PBS, cells were harvested in distilled water and analyzed for cholesterol accumulation by HPLC, as previously described.¹⁵

Rate of Intracellular Cholesteryl Ester Formation
The rate of cholesteryl ester formation within the cells was assessed by measuring the rate of incorporation of [¹⁴C]oleate into cholesteryl esters. After preincubation (with or without lovastatin), the cells were incubated with 0.5 µCi [¹⁴C]oleic acid (specific activity, 700 mCi/mmol) per milliliter of medium in the presence or absence of AcLDL (100 µg/mL) for the indicated time. Oleic acid was presented to the cells in a BSA–sodium oleate complex as described.^{25 27} At the end of the assay, the cells were washed, harvested, and homogenized. [¹⁴C]Oleic acid incorporation into cholesteryl esters was measured after lipid extraction and separation of the neutral lipids on high-performance thin-layer chromatographic (TLC) plates (Merck) in petroleum ether/diethylether/acetic acid, 90:10:1 vol/vol/vol. The cholesteryl ester spots were scraped off and counted in a liquid scintillation counter.

Uptake and Metabolism of Radiolabeled AcLDL by Cells
To further investigate the uptake and metabolism of radiolabeled AcLDL, macrophages were incubated for 24 hours with 100 µg/mL AcLDL labeled with either [³H]cholesteryl linoleate (ie, labeled in the cholesterol moiety; specific activity, 60 to 100 Ci/mmol) or cholesteryl-[¹⁴C]linoleate (ie, labeled in the linoleate moiety; specific activity, 45 to 60 mCi/mmol), washed 3 times with serum-free medium, and equilibrated in the same medium overnight. Cells and media samples were collected after each incubation step and analyzed for cholesterol or cholesteryl ester content by TLC. Samples were delipidated with chloroform/methanol, 1:2 vol/vol, dried, and redissolved in 20 µL chloroform. Two microliters was applied to Kieselgel 60 TLC plates (Merck) which were then eluted with hexane/heptane/diethylether/acetic acid, 63:18.5:18.5:1 vol/vol/vol/vol. For experiments involving separation of cholesteryl oleate and cholesteryl linoleate, heptane/chloroform 60:40 vol/vol was used.²⁸ The spots corresponding to free and esterified cholesterol were scraped off the plates and analyzed by scintillation counting (LKB).

Statistics
An explorative data analysis was performed by using the statistical package for the social sciences (SPSS-X).²⁹ Relationships between variables and concentration were calculated by Pearson regression analysis after logarithmic transformation of the concentration values and by ANOVA. In addition, values at individual concentrations and the values in nonloaded and cholesterol-loaded cells were compared by using t tests. All P values are two-tailed. P values 0.05 were taken to be significant.

	Results

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Intracellular Levels of Total Cholesterol, Free Cholesterol, and Cholesteryl Esters
In initial concentration-ranging experiments, application of lovastatin >12 µmol/L was found to be toxic to non–cholesterol-loaded cells. For this reason and for better comparison with the original work on this topic,¹⁴ a maximum lovastatin concentration of 12 µmol/L was used in all experiments described below. In nonloaded cells, lovastatin produced a significant decrease in total cholesterol levels (-18% at 12 µmol/L lovastatin, P<0.05, Table 1). This decrease in total cholesterol was due to a significant drop in free cholesterol, which reached a maximum of 30% at 12 µmol/L lovastatin (Figure 1). By contrast, cholesteryl esters increased 2.2-fold in the presence of 12 µmol/L lovastatin (Figure 1).

View larger version (16K): [in this window] [in a new window]   Figure 1. Levels of free cholesterol (FC) and cholesteryl esters (CEs) as determined by HPLC in control 14-day-old human monocyte–derived macrophages (nonloaded) and in cells that were loaded with cholesterol by incubation with 80 µg/mL AcLDL (+AcLDL) for 24 hours. All cells were incubated with lovastatin at the concentrations shown during the loading phase and during the preceding 24 hours. n=8, all values are mean±SEM; *P<0.05, **P<0.01 for individual values compared with cells that were not treated with lovastatin. Values of loaded cells were significantly greater than those of nonloaded cells at all lovastatin concentrations (P=0.001 to 0.033). Regression analysis showed significant concentration responses for FC+AcLDL, FC nonloaded, and CE+AcLDL (P<0.05).

Cholesterol loading of untreated cells led to an increase in both intracellular free cholesterol (+30%, P<0.05) and cholesteryl esters (11-fold). In cholesterol-loaded cells, total cholesterol content was not altered by treatment with lovastatin (Table 1). However, in contrast to the effect of the drug on non–cholesterol loaded cells, treatment of cholesterol-loaded with lovastatin increased free cholesterol by 30% while decreasing the cholesteryl ester content by 50% at a dose of 12 µmol/L. All effects of lovastatin on intracellular cholesterol and cholesteryl ester were abolished by administration of 100 µmol/L mevalonate (data not shown).

The use of a novel HPLC method described by our laboratory¹⁵ allowed quantification of the major cholesteryl ester species in the cells. In the loaded cells, the cholesteryl esters of the unsaturated fatty acids linoleic and oleic acid, which accounted for some 85% of total esters, decreased on treatment with lovastatin (Figure 2). The reduction in arachidonate levels failed to achieve statistical significance. The levels of cholesteryl esters of the saturated fatty acids palmitic and stearic, by contrast, were not reduced, even with 12 µmol/L lovastatin. In the non–cholesterol-loaded cells, levels of unsaturated cholesteryl esters (arachidonate, linoleate, and oleate) were much lower than those in the loaded cells but increased significantly with the highest dose of lovastatin used (12 µmol/L). No cholesteryl stearate could be detected in the non–cholesterol-loaded cells (Figure 2).

View larger version (24K): [in this window] [in a new window]   Figure 2. Levels of individual cholesteryl ester species as determined by HPLC in control 14-day-old human monocyte–derived macrophages (non-loaded, black bars) and in macrophages that were loaded with cholesterol by incubation with 80 µg/mL AcLDL for 24 hours (+AcLDL, white bars). All cells were incubated with lovastatin at the concentrations shown during the loading phase and during the preceding 24 hours. n=10, all values are mean±SEM; *P<0.05 compared with cells that were not treated with lovastatin. For all esters, values of loaded cells were significantly greater than those of nonloaded cells at all lovastatin concentrations (P<0.001, P<0.05 for palmitate) except for palmitate at 0.1 µmol/L lovastatin (NS). Regression analysis showed a significant concentration response for cholesteryl linoleate and cholesteryl oleate in loaded cells (P<0.05).

Binding Studies
Specific binding of ¹²⁵I-AcLDL to the scavenger receptor was determined at 4°C by using concentrations between 0 and 50 µg/mL medium (protein concentration), and binding reached saturation at a concentration of 20 µg ¹²⁵I-AcLDL/mL medium. The amount of maximally bound AcLDL ranged between 50 and 58 ng/mg cell protein in both treated and untreated cells. No significant difference could be detected between the slope of the binding curve of the lovastatin-treated cells and that of the control cells, indicating a similar affinity of the scavenger receptor for AcLDL in both cell populations (data not shown).

Radioisotope Studies of Cholesterol Metabolism
To investigate the mechanisms by which lovastatin alters intracellular cholesterol metabolism, macrophages were incubated for 24 hours with AcLDL labeled with [³H]cholesteryl linoleate in the presence or absence of lovastatin. Treatment of the cells with lovastatin did not significantly affect the uptake of cholesterol and cholesteryl ester from AcLDL (Table 2). After a subsequent 24-hour incubation in serum-free medium (secretion phase), the level of [³H]cholesterol and [³H]cholesteryl ester did not differ significantly between untreated and lovastatin-treated cells (Table 2).

Cholesteryl linoleate is the most abundant ester of AcLDL, whereas the most abundant ester in cells is cholesteryl oleate. For this reason, the response of these 2 cholesteryl ester species to loading with [³H]cholesteryl linoleate–labeled AcLDL was investigated. Treatment with 5 µmol/L lovastatin was associated with increased uptake of [³H]cholesteryl linoleate during the loading phase (Figure 3). However, during a subsequent 24-hour incubation in serum-free medium (secretion phase), the level of intracellular [³H]cholesteryl linoleate increased in untreated cells but not in cells treated with lovastatin. This finding is consistent with a lovastatin-induced inhibition of cholesterol esterification (Figure 3). After its uptake into the cells, [³H]cholesteryl linoleate undergoes hydrolysis, and the [³H]cholesterol is reesterified by ACAT, primarily to [³H]cholesteryl oleate. Treatment with lovastatin reduced the level of [³H]cholesteryl oleate during both the loading (-40%) and secretion (-30%) phases, consistent with a reduced intracellular cholesterol re-esterification. However, [³H]cholesteryl oleate increased to a similar extent during the secretion phase in both the untreated (+30%) and treated (+50%) cells, indicating that lovastatin does not directly affect the rate of intracellular cholesterol esterification but rather reduces the supply of substrate for this process (Figure 3). The secretion of [³H]cholesterol into the culture medium was not affected by addition of lovastatin (not shown).

View larger version (18K): [in this window] [in a new window]   Figure 3. Intracellular [3H]cholesteryl linoleate and oleate levels in 14-day-old human untreated macrophages (control) and macrophages that were incubated with 5 µmol/L lovastatin throughout the experiment (lovastatin). All cells were incubated with 100 µg/mL [3H]cholesterol linoleate–labeled AcLDL for 24 hours (loading) and then cultured for 24 hours in serum-free medium (secretion). Lipids were separated by TLC and measured by scintillation counting as described in Methods. Mean±SEM; values shown are from a representative experiment performed in triplicate. *P<0.01, **P<0.001 lovastatin treated versus untreated; §P<0.001 untreated, secretion versus loading; and #P<0.001 lovastatin treated, secretion versus loading.

To further examine the effects of lovastatin on hydrolysis of lipoprotein-derived cholesteryl esters, the cells were incubated with AcLDL labeled with cholesteryl-[¹⁴C]linoleate (ie, labeling of the linoleate moiety). Treatment with lovastatin appeared to increase the amount of intracellular [¹⁴C]linoleic acid by 20% (loading phase) or 30% (secretion phase), although this change did not achieve statistical significance (Figure 4). Incoporation of [¹⁴C-]linoleic acid into cholesteryl linoleate and triglycerides, however, was reduced by lovastatin (Figure 4). Because ACAT activity in lovastatin-treated cells appears to be normal (Figure 3), and since lysosomal hydrolysis of cholesteryl esters is also at least normal (Figure 4) in lovastatin-treated cells, our findings indicate that reduced esterification in cholesterol-loaded, lovastatin-treated cells is due to a reduced supply of substrate.

View larger version (18K): [in this window] [in a new window]   Figure 4. Intracellular [14C]linoleic acid, [14C]linoleate-triglyceride, and cholesteryl-[14C]linoleate levels in 14-day-old human untreated macrophages (control) and macrophages that were incubated with 5 µmol/L lovastatin throughout the experiment (lovastatin). All cells were incubated with 100 µg/mL cholesteryl-[14C]linoleate–labeled AcLDL for 24 hours (loading) and then cultured for 24 hours in serum-free medium (secretion). Lipids were separated by TLC and measured by scintillation counting as described in Methods. Mean±SEM; values shown are from a representative experiment performed in triplicate. *P<0.05 treated versus control; **P<0.01 treated versus control.

To further investigate these effects, cholesterol-loaded and nonloaded cells were incubated in the presence of [¹⁴C]oleate complexed with albumin and treated with lovastatin. As previously demonstrated by others,¹⁴ lovastatin reduced oleate incorporation into the cholesteryl ester fraction (data not shown).

Effect of Lovastatin on ApoE mRNA Levels and Secretion
ApoE is involved in cholesterol metabolism and efflux in macrophages.¹⁷ We therefore measured apoE mRNA levels at the end of the cholesterol-loading phase and apoE protein secretion into serum-free medium during the following 24 hours. In nonloaded macrophages, lovastatin significantly increased apoE mRNA levels, by 22% at 1.2 µmol/L and by 43% at 12 µmol/L concentrations. In untreated macrophages, cholesterol loading produced a 55% increase in apoE mRNA levels (P<0.05). Treatment with lovastatin produced a further increase in apoE mRNA, with a maximum of 75% at 12 µmol/L compared with untreated, cholesterol-loaded macrophages (Figure 5). By contrast, secretion of apoE in both loaded and nonloaded cells decreased after treatment with lovastatin (Figure 6). Secretion of apoE in cholesterol-loaded cells was greater than that in nonloaded cells at all concentrations of lovastatin tested except for the maximum concentration of 12 µmol/L. The effects of lovastatin on apoE mRNA levels and protein secretion were reversed by simultaneous incubation with 100 µmol/L mevalonate (data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 5. Levels of apoE mRNA as determined by quantitative RT-PCR in control 14-day-old human monocyte–derived macrophages (nonloaded) and in macrophages that had been loaded with cholesterol by incubation with 80 µg/mL AcLDL for 24 hours (+AcLDL). All cells were incubated with lovastatin at the concentrations shown during the loading phase and during the preceding 24 hours. n=11, all values are mean±SEM; *P<0.05 compared with cells that were not treated with lovastatin. Values of loaded cells were significantly greater than those of nonloaded cells at all lovastatin concentrations (P=0.008 to 0.05). Regression analysis showed significant concentration responses for mRNA levels in both loaded and nonloaded cells (P<0.05).

View larger version (13K): [in this window] [in a new window]   Figure 6. Secretion of apoE into serum-free medium of 14-day-old macrophages. After the first incubation with 80 µg/mL AcLDL (+ AcLDL) or without AcLDL (nonloaded) in the presence of lovastatin as indicated, cells were washed and incubated for another 24 hours in serum-free medium with or without lovastatin as indicated. n=10, all values are mean±SEM; *P<0.05 compared with cells that were not treated with lovastatin. ApoE secretion in loaded cells was not significantly greater than that in nonloaded cells at any lovastatin concentration. Regression analysis showed significant concentration responses for apoE protein secretion in loaded cells only (P<0.05).

	Discussion

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The results of our studies confirm those of Kempen et al,¹⁴ who were the first to show that statins inhibit cholesteryl ester formation by an ACAT-independent mechanism in human macrophages. However, our findings extend those results in several important respects. We have shown that the effect of statins on cholesterol esterification is critically dependent on cholesterol loading of the cells. Whereas in loaded cells, the effect of lovastatin is as described by Kempen et al,¹⁴ ie, an increase in free cholesterol and a decrease in cholesteryl esters, in nonloaded cells lovastatin had exactly the opposite effect, by decreasing free cholesterol and increasing cholesteryl esters (Figure 1). This decrease in the cholesteryl ester content of loaded cells was confined to esters of unsaturated fatty acids.

The concentration range of lovastatin used in our studies encompasses the plasma level found after oral administration of standard concentrations in human subjects.^{30 31} Because similar results to ours have also been obtained for other statins,^{14 32 33} the effects described here probably apply to most if not all members of this drug class. Moreover, because the effects of lovastatin on cholesterol mass and cholesteryl ester storage and on apoE transcription and secretion were reversed by mevalonate, our findings are almost certainly due to specific inhibition of HMG-CoA reductase and not to a nonspecific or toxic action of the drug. This indicates that even in AcLDL-loaded macrophages, HMG-CoA reductase continues to show residual activity, as has been demonstrated previously.¹⁴

How does lovastatin produce such apparently contradictory effects in loaded and nonloaded macrophages? These findings can be most parsimoniously accounted for by a lovastatin-induced modulation of intracellular cholesterol trafficking (Figure 7). Current theories suggest that at least 2 pools of free cholesterol exist within the macrophage, 1 derived from endogenous synthesis (C_C for cholesterol of cellular origin) and a second derived from lysosomal hydrolysis of cholesteryl esters contained in lipoproteins such as AcLDL (C_L for cholesterol of lipoprotein origin). C_L and C_C are rapidly transported to the cell membrane, it is thought, by 2 distinct pathways.³⁴ In the non–cholesterol-loaded macrophage, virtually all of the cholesterol within the cell is C_C. Usually, most C_C is transported to the cell membrane and only a very small amount is esterified by ACAT (Figure 7A), probably in an endoplasmic reticulum–related compartment.³⁵ In the cholesterol-loaded cell, the situation is quite different. Here, the bulk of cholesterol is C_L, not only because it is supplied in large amounts by endocytosis but also because large amounts of C_L downregulate HMG-CoA reductase, thus reducing synthesis of C_C³⁶ (Figure 7C). The amount of C_L in AcLDL-loaded cells greatly exceeds the storage capacity of cell membranes, so that the bulk is moved into the esterification pathway. However, before undergoing esterification, C_L must first cycle via the plasma membrane³⁵ (Figure 7C). On the basis of our results, we propose that C_C, by contrast, is able to gain direct access to ACAT without first passing via the cell membrane. We further propose that in macrophages, lovastatin blocks the translocation of cholesterol from both intracellular pools to the cell membrane. In nonloaded cells, treatment with lovastatin reduces cholesterol synthesis and thus reduces the levels of free C_C (Figure 1) and total C_C (Table 2). However, the translocation of C_C to the cell membrane is also blocked, increasing the delivery of C_C to ACAT, to which it has direct access. This leads in turn to the observed increase in cholesteryl ester (C_CE, Figure 7B). In loaded cells, lovastatin blocks translocation of C_L to the cell membrane, hence reducing delivery of C_L to ACAT and explaining the observed rise in free C_L and fall in C_LE (Figure 1). Because under conditions of cholesterol loading net endogenous cholesterol synthesis is low, lovastatin does not measurably change the total amount of cholesterol in loaded cells (Table 2 and Figure 7D). This differential handling of C_L and C_C may also be in some way related to the differential effects of statins on the incorporation of saturated and unsaturated fatty acids into cholesteryl esters.

View larger version (30K): [in this window] [in a new window]   Figure 7. Postulated statin-induced block in transport of intracellular cholesterol to the cell membrane, with effects on intracellular cholesterol metabolism. A, In nonloaded cells, most cholesterol is of endogenous origin (CC) and is synthesized de novo from acetyl CoA in the smooth endoplasmic reticulum (SER). Under normal circumstances, most CC is transported to the cell membrane and only a small amount is esterified. B, CC appears to have direct access to ACAT (ie, without traversing the cell membrane). Lovastatin hinders transport of CC to the cell membrane, shunting CC into the esterification pathway. Thus, the level of intracellular cholesteryl ester increases despite a statin-induced fall in total cellular cholesterol content. C, Cholesteryl ester of exogenous origin (CLE) is taken up within acetylated LDL (AcLDL) and undergoes lysosomal hydrolysis with release of free cholesterol (CL). CL must cycle through the cell membrane before its esterification by ACAT and storage within a cholesteryl ester (CE) droplet. D, Lovastatin blocks not only de novo cholesterol synthesis but also transport of free cholesterol to the cell membrane. Hence, in cholesterol-loaded cells, where most cholesterol is CL, lovastatin induces an increase in free cholesterol and a drop in cholesteryl esters.

This hypothesis of a block in delivery of intracellular cholesterol to the cell membrane is compatible with the results of our radioisotope experiments. Other steps in cholesterol transport within the cells appear not to have been perturbed by lovastatin. The binding of AcLDL to the scavenger receptor in our cells was normal, as previously described in this and other systems.^{14 32} Uptake of radiolabeled cholesterol via AcLDL appeared to be normal under treatment with lovastatin (Table 1 and Figure 3), in contrast to results obtained by Bernini et al³² in murine macrophages. Our data (Figure 3) and those of Kempen et al¹⁴ indicate that ACAT is not inhibited by statins. Yet the amount of cholesteryl oleate formed within the cells was reduced on lovastatin treatment (Figure 3), consistent with a reduced delivery of substrate. Might there also be a problem with delivery of the fatty acid moiety for cholesteryl ester formation? Our data indicate that this is not the case. In cells incubated with AcLDL labeled with cholesteryl-[¹⁴C]linoleate, the level of free [¹⁴C]linoleic acid was not reduced by lovastatin during either the loading or the secretion phase (Figure 4). This indicates normal functioning of lysosomal acid lipase. In view of the marked decrease in intracellular cholesteryl esters that is seen after lovastatin treatment in cholesterol-loaded cells, the increase in cholesteryl esters in non–cholesterol-loaded cells cannot be attributed to a statin-induced reduction in the activity of neutral cholesteryl ester hydrolase. Kempen et al¹⁴ concluded that statins induce a defect in the delivery of cholesterol to ACAT, by trapping it within phospholipid-containing pools. Our findings support this conclusion for exogenous but not for endogenous cholesterol, which undergoes increased esterification on treatment with lovastatin.

How might the postulated lovastatin-induced block in the delivery of intracellular cholesterol to the plasma membrane arise? Several lines of evidence suggest the involvement of proteins in the lipoprotein-induced esterification of cholesterol in macrophages.^{37 38 39} To function correctly, these proteins may require coupling to isoprene units, which are products of the mevalonate pathway.⁴⁰ Bernini et al showed that the statin-induced reduction of cholesterol esterification in macrophages was reversed by addition of geranylgeraniol, but not by addition of farnesol, indicating that a geranylgeranylated protein is important in this process.^{32 33}

In nonloaded and to a greater extent in cholesterol-loaded cells, lovastatin increased the apoE mRNA level (Figure 6). ApoE transcription is thought to be at least partially regulated by the level of intracellular cholesterol or a derivative thereof. This concept is supported by the observation that apoE mRNA levels are greater in loaded that in nonloaded cells at all lovastatin concentrations and by the increase in apoE mRNA in loaded cells in parallel with the increase in free cholesterol in them. More puzzling is the increase in apoE mRNA in nonloaded cells in the face of falling free cholesterol levels. This may indicate that the determining factor regulating apoE transcription/stability is not the bulk of free cholesterol but a pool of free cholesterol that is not quantified when total cholesterol is measured. Work from our laboratory on the effect on the common apoE polymorphism on apoE transcription and secretion also showed changes in apoE mRNA levels unrelated to changes in intracellular free cholesterol levels.⁴¹ In contrast to the lovastatin-induced increase in apoE mRNA, apoE protein secretion fell in both loaded and nonloaded cells in response to the drug (Figure 7). Previous work by our group⁴¹ and others^{42 43} has shown that not all translated apoE is secreted and that a portion may undergo degradation in a lysosomal compartment. ApoE is known to mediate cholesterol efflux from macrophages^{16 44} ; however, little is known of its role in intracellular lipid trafficking. Tabas et al⁴⁵ have shown that the apoE content of lipoprotein particles affects their intracellular targeting. The effect of lovastatin treatment on macrophages in culture is to increase the amount of apoE protein not destined for secretion. It is possible that this nonsecreted apoE may play a role in the alteration in cholesterol trafficking induced by lovastatin.

The studies presented here and those of Kempen et al¹⁴ and Bernini et al^{32 33} illustrate the usefulness of statins in the study of intracellular cholesterol trafficking and underline how much remains to be understood about this process. Several studies have shown that statins may arrest or even reverse coronary atherosclerosis.^{4 5 6 7 8} The statin-induced reduction in cholesteryl ester storage in loaded cells and total cholesterol content in nonloaded cells may contribute to this effect and produce therapeutic benefit over and above that derived from effects on circulating lipoproteins.

	Acknowledgments

 
The authors would like to express their gratitude to Karin Tegelkamp for her excellent technical assistance and to Dr Helmut Schulte and Michael Stennecken for their invaluable advice in performing statistical analysis.

Received June 13, 1997; accepted March 16, 1998.

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