(Received for publication, November 18, 1994; and in revised form, December 19, 1994)
From the
Considerable evidence supports the involvement of acyl-CoA:cholesterol acyltransferase (ACAT) in the maintenance of intracellular cholesterol homeostasis. A number of recently developed ACAT inhibitors may have potential use as pharmacological agents to reduce the development of atherosclerosis. Recently, however, reports arose describing cytotoxic effects following administration of a specific ACAT inhibitor to experimental animals. In order to address the specific intracellular mechanisms involved with the cytotoxic effect, we examined the consequences of ACAT inhibition in cholesterol-enriched mouse peritoneal macrophages. Mouse peritoneal macrophages were cholesterol-enriched by incubation with acetylated low density lipoprotein and free cholesterol:phospholipid dispersions prior to the addition of an ACAT inhibitor, either Sandoz 58-035 or Pfizer CP-113,818. The adenine pool of the macrophages was radiolabeled prior to addition of the ACAT inhibitors, in order to monitor the release of radiolabeled adenine, a technique shown to be a sensitive method to monitor drug-induced toxicity. The ACAT inhibitors were added for up to 48 h and at concentrations up to 2 µg/ml. These conditions resulted in an approximately 2-fold increase in adenine release. The increase in cell toxicity paralleled an increase in the cellular free cholesterol content. Reducing the cellular free cholesterol content, by the addition of extracellular acceptors, decreased the cytotoxic effects of the ACAT inhibitors. Addition of an intracellular cholesterol transport inhibitor, either progesterone or U18666A, together with CP-113,818 blocked the toxic effect of CP-113,818. These results suggest that ACAT inhibition of cholesterol-enriched macrophages increases cell toxicity due to the buildup of cellular free cholesterol. Removal of free cholesterol by the addition of extracellular cholesterol acceptors or by blocking intracellular sterol transport relieves the ACAT inhibitor-induced toxicity.
Intracellular esterification of cholesterol is accomplished by
the microsomal enzyme, acyl coenzyme A:cholesterol acyltransferase
(ACAT)()(1) . This reaction is believed to play a
crucial role in the maintenance of cellular cholesterol homeostasis.
Considerable evidence supports the involvement of ACAT in the
intestinal absorption of cholesterol(1) , in the secretion of
hepatic lipoproteins(2, 3) , and in the accumulation
of cholesteryl esters in atherosclerotic lesions(4) .
Inhibition of ACAT at any of these points may be beneficial in slowing
the development of atherosclerosis.
Several ACAT inhibitors have
been developed that show beneficial effects toward reducing
atherosclerosis in experimental animals(5, 6) . The
structural characteristics of these compounds vary considerably,
although most inhibit ACAT activity with IC values less
than 1 µM(7) . While the potential benefits of
ACAT inhibitors have been widely reported, recent data suggests a
potential toxic effect of these
compounds(8, 9, 10) . These researchers noted
an acute toxic effect of a specific ACAT inhibitor PD132301-2 in
adrenal cells of both guinea pigs and beagle dogs. Vernetti et al.(10) concluded that this toxic effect may be linked to ATP
depletion of mitochondria.
Maintenance of cellular free cholesterol concentrations within small ranges appears to be critical for preservation of cellular functions (11) . Inhibition of the enzyme catalyzing cholesteryl ester formation, and therefore controlling intracellular free cholesterol concentrations, may have consequences with regard to cell viability. The aim of this study was to more closely examine the intracellular mechanisms that may be responsible for the loss of cell viability as a consequence of ACAT inhibitor treatment. We hypothesize that free cholesterol accumulation, resulting from the blocking the ACAT arm of the cholesteryl ester cycle, is responsible for the observed toxic effects following ACAT inhibitor treatment.
Cell monolayers were extracted
with isopropyl alcohol containing cholesteryl
[1-C]oleate (
50,000 dpm, 59.5 mCi/mmol)
(DuPont NEN) as an internal standard to correct for losses during
extraction. After drying under N
, the lipids were dissolved
in 50 µl of chloroform/methanol (1:1) and plated onto Silica Gel G
thin-layer chromatography plates (Whatman PE SIL G, Whatman, Ltd,
Maidstone, Kent, United Kingdom). Cholesteryl esters were separated
from other lipids using hexane/ethyl ether/acetic acid (80:20:1) as a
developing solvent and visualized by co-migration with nonradioactive
cholesteryl oleate. Areas were cut and analyzed for radioactivity by
dual isotope liquid scintillation spectroscopy (Beckman LS 3801,
Beckman Instruments, Inc., Irvine, CA).
The goal of this investigation was to establish if the intracellular accumulation of free cholesterol in macrophagederived foam cells could lead to cell toxicity. For this purpose, we used cholesterol-enriched peritoneal macrophages as a model for foam cells and induced free cholesterol accumulation by inhibiting the ACAT arm of the cholesteryl ester cycle. This approach has been used in a number of investigations and results in the cellular deposition of substantial stores of free cholesterol(26, 27, 28, 29) . Our measure of toxicity was based on the established method of monitoring the release of radiolabeled adenine from control and drug-treated cells(19) , a technique that has been demonstrated to accurately quantify cell integrity in cell lines including the cholesterol-loaded macrophage(20) . In preliminary studies, we observed that the release of radiolabeled adenine correlated with the release of lactate dehydrogenase, a well accepted marker of cell toxicity. Therefore, this assay appears to be a reliable marker for ACAT inhibitor-induced toxicity in the cholesterol-loaded macrophage.
Figure 1:
Time course of mouse peritoneal
macrophage cell toxicity following incubation with compound CP-113,818.
Mouse peritoneal macrophages were enriched with cholesterol for 24 h
with acetylated LDL, free cholesterol/phospholipid dispersion, and
fetal bovine serum as described under ``Experimental
Procedures.'' The cells were then incubated with 1 µCi of
[H]adenine for 2 h before the addition of the
ACAT inhibitor. Macrophages were incubated with CP-113,818 (2.0
µg/ml) dissolved in Me
SO or Me
SO alone as
control. The final concentration of Me
SO did not exceed
0.1%. The release of [
H]adenine from the
macrophages was monitored for up to 36 h. Values are presented as the
percent of total cellular
H cpm released to the culture
medium for control (
) and CP-113,818-treated (
)
macrophages. Values are averages from triplicate wells. *, p < 0.001 versus control at same time
point.
Fig. 2shows the results of an experiment in which monolayers
of macrophages were loaded with increasing levels of cholesteryl esters
and then exposed to the ACAT inhibitor CP-113,818 for 24 h in the
absence of any extracellular cholesterol acceptors. The different
degrees of cholesteryl ester loading were achieved by prior exposure of
the cells to acetylated LDL (100 µg of protein/ml) together with
increasing concentrations of a 2:1 (mol/mol) free
cholesterol/phospholipid dispersion. As expected(32) , this
treatment resulted in more than a 4-fold increase in cell total
cholesterol above unloaded, control macrophages, with the major
accumulation occurring in the cholesteryl ester pool (Fig. 2A). As shown in Fig. 2B, the
treatment of each group of macrophages with the ACAT inhibitor for 24 h
led to a significantly higher (p < 0.001) release of
[H]adenine than observed in parallel
cholesterol-loaded cells incubated under identical conditions but in
the absence of the inhibitor. In unloaded control macrophages the
effect of CP-113,818 (2.0 µg/ml) on cell toxicity was small but
statistically significant (p < 0.001). In cells
cholesterol-enriched by prior incubation with the highest concentration
of dispersion, the ACAT inhibitor CP-113,818 (2.0 µg/ml) increased
cell toxicity nearly 5-fold over controls. Fig. 2C illustrates that upon incubation of cholesteryl ester-enriched
macrophages with CP-113,818, these cells accumulate free cholesterol.
At all levels of added cholesterol/phospholipid dispersion tested, the
free cholesterol content of the ACAT inhibitor-treated macrophages was
significantly higher than untreated controls (p < 0.05).
Maximal [
H]adenine release and cell free
cholesterol content (time 24 h) was reached with a concentration of 250
µg of free cholesterol added via free cholesterol/phospholipid
dispersion.
Figure 2:
Effect of varying levels of macrophage
cholesterol enrichment on the accumulation of free cholesterol and the
release of adenine after treatment with ACAT inhibitors. Macrophages
were cholesterol-loaded for 24 h with fetal bovine serum, acetylated
LDL, and the specified amounts of free cholesterol/phospholipid
dispersion. Following an 18-h equilibration period in 0.2% BSA media,
some wells were analyzed for free and esterified cholesterol as
described under ``Experimental Procedures.'' The remaining
monolayers were then incubated with 1 µCi of
[H]adenine for 2 h before the addition of the
ACAT inhibitor. Macrophages were incubated in the absence (control) and
presence of CP-113,818 (2.0 µg/ml) for 24 h. A,
cholesterol content of macrophages at time 0 following cholesterol
enrichment with increasing amounts of free cholesterol via a free
cholesterol/phospholipid dispersion. The values are presented as the
average free (
) and esterified (&cjs2106;) cholesterol
(µg/well) from triplicate dishes. B, influence of
increasing cholesterol-loading on cell toxicity. The release of
[
H]adenine from the macrophages was monitored for
24 h. Values are presented as the mean ± S.D. release of
[
H]adenine (% of control) from triplicate wells. C, free cholesterol content following a 24-h incubation with
ACAT inhibitor. Values are presented as the mean ± S.D. (from
triplicate wells) for cellular free cholesterol content (µg/well)
in the absence (&cjs2106;) and presence (
) of CP-113,818 (2.0
µg/ml). a, p < 0.05 versus control.
The removal of cell cholesterol from mouse peritoneal
macrophage-derived foam cells can be accomplished by adding to the
incubation medium either native HDL (26) or reconstituted
apo-HDLphospholipid complexes (rHDL)(33) . In a
preliminary experiment, cholesterol-loaded macrophages were incubated
with CP-113,818, either alone or together with the
apoprotein-phospholipid complex or with human HDL
. In
addition, a second, extensively used ACAT inhibitor, Sandoz
58-035(34) , was added to parallel cultures alone or
together with the cholesterol acceptors. Incubation with Sandoz
58-035 (2.0 µg/ml) resulted in
[
H]adenine release comparable with that observed
with 2.0 µg/ml of CP-113,818 (176 and 205% of control,
respectively). Co-incubation of either ACAT inhibitor with HDL
or rHDL blocked this toxic response. The data for
[
H]adenine release closely paralleled the free
cholesterol content of the cells, with free cholesterol accumulating in
cells exposed to either of the ACAT inhibitors and with the deposition
of cell free cholesterol prevented by the diversion of the cholesterol
to extracellular cholesterol acceptors (data not shown). These data,
together with those of Fig. 2, indicate that ACAT
inhibitor-induced cell toxicity is the result of free cholesterol
buildup.
The ability to modulate the extent of free cholesterol
accumulation in the drug-treated cells by exposure to graded levels of
extracellular cholesterol acceptors provided us the opportunity to more
precisely examine the relationship between cell free cholesterol
content and cell toxicity as measured by labeled adenine leakage. The
data presented in Fig. 3were obtained from a study in which the
cholesteryl ester-enriched macrophages were incubated for 24 h in
medium supplemented with CP-113,818 (2.0 µg/ml) together with
increasing concentrations of rHDL complexes. In this protocol, the
cells had been previously labeled with
[H]cholesterol during the loading phase of the
experiment so that the efflux of cell cholesterol could also be
measured. Fig. 3A presents the free cholesterol mass data for
the cells after a 24-h exposure to rHDL. As the concentration of rHDL
increased, cell free cholesterol decreased until free cholesterol
contents reached 40-45 µg/well; levels that were equivalent
to untreated control cells. Fig. 3B illustrates that
the rHDL particles acted as efficient acceptors of the cell cholesterol
and stimulated cholesterol efflux in a dose-dependent manner. The
release of [
C]adenine from the treated cells was
inversely related to the fractional efflux of cell cholesterol (r
= 0.95, p < 0.001) and very
highly correlated with the actual mass of cell free cholesterol (r
= 0.92, p < 0.001, Fig. 3C).
Figure 3:
Effects of increasing apo-HDL/PC complexes
on cellular free cholesterol content, cell toxicity, and cholesterol
efflux from cholesterol-loaded macrophages. Mouse peritoneal
macrophages were cholesterol-loaded for 24 h with acetylated LDL, free
cholesterol/phospholipid dispersion, and fetal bovine serum as
described under ``Experimental Procedures.'' In wells where
[H]cholesterol efflux was measured, 4
µCi/well of [1,2-
H]cholesterol was present in
the loading medium during the period of cholesterol enrichment. In this
experiment, macrophages were labeled for 18 h with 1 µCi/well of
[
C]adenine during the equilibration period. The
appearance of [1,2-
H]cholesterol and
[
C]adenine in the media were measured after 24 h
of incubation with compound CP-113,818 (2.0 µg/well) alone or in
combination with apo-HDL/PC at amounts ranging from 0 to 200 µg of
phospholipid/ml. A, influence of increasing the media
cholesterol acceptor concentration on cell free cholesterol content.
Values are presented as the mean ± S.D. for cellular free
cholesterol (µg/well) from triplicate wells after a 24-h incubation
with CP-113,818 (2.0 µg/well) and various levels of cholesterol
acceptor. B, correlation between relative cholesterol efflux
and [
C]adenine release. Values for the
fractional efflux of [1,2-
H]cholesterol are
presented as the average of triplicate wells.
[
C]Adenine release is expressed as the mean
± S.D. (% of control) from triplicate wells. The r
value was 0.95 (p < 0.001). C, correlation between [
C]adenine
release and cellular free cholesterol content. Values for cell free
cholesterol are the average from triplicate wells after a 24-h
treatment and are the same as those in B.
[
C]Adenine release is expressed as the mean
± S.D. (% of control) from triplicate wells. The r
value was 0.92 (p <
0.001).
The correlative data in Fig. 3C indicate that the ability of extracellular cholesterol acceptors to reduce or eliminate the toxicity of ACAT inhibitors is related to their ability to prevent cell free cholesterol accumulation. However, another mechanism that could explain the protective effect of the extracellular cholesterol acceptors could be related to a possible ability to bind the ACAT inhibitors and prevent uptake into cells. Both CP-113,818 and Sandoz 58-035 are hydrophobic compounds that could associate with the cholesterol acceptors and thus have a reduced effective concentration in medium containing HDL or rHDL. Cholesterol esterification was reduced more than 97% compared with controls when cells were exposed to CP-113,818 (2.0 µg/ml) alone or together with rHDL complexes, whereas rHDL alone reduced esterification only 80%. This result indicates that the ACAT inhibitor, at the concentrations used in this study, was equally effective in blocking ACAT activity in both the absence and presence of the cholesterol acceptor.
Figure 4:
Relationships between ACAT inhibition,
cellular free cholesterol concentration, and cell toxicity with
increasing concentrations of CP-113,818 and Sandoz 58-035. Mouse
peritoneal macrophages were cholesterol-loaded for 24 h with acetylated
LDL, free cholesterol/phospholipid dispersion, and fetal bovine serum
as described under ``Experimental Procedures.'' Macrophages
were incubated with compounds CP-113,818 or Sandoz 58-035 at
concentrations up to 50 ng/ml. These concentrations shut down virtually
all cholesterol esterification activity after a 24-h incubation. Values
for [H]adenine release for CP-113,818 (
)
and Sandoz 58-035 (
), ACAT inhibition for CP-113,818
(
) and Sandoz 58-035 (
), and cellular free
cholesterol concentrations for CP-113,818 (
) and Sandoz
58-035 (
) are presented as the percent of maximum change
measured and represent means from triplicate
wells.
Figure 5:
Addition of progesterone or U18666A to
relieve ACAT inhibitor-induced cell toxicity. Mouse peritoneal
macrophages were cholesterol-loaded for 48 h with acetylated LDL, free
cholesterol/phospholipid dispersion, and fetal bovine serum as
described under ``Experimental Procedures.'' The cells were
incubated with 1 µCi of [H]adenine for 2 h
before the addition of the treatments. Macrophages were incubated for
24 h with 1 µg/ml CP-113,818 alone or in combination with 5
µg/ml progesterone or 1 µg/ml U18666A. Values for
[
H]adenine release are presented as the mean
± S.D. (% of control) from triplicate wells. a, p < 0.01 versus CP-113,818
alone.
When increasing concentrations of progesterone were added to medium containing a constant amount of CP-113,818 (1 µg/ml), we observed that the addition of progesterone, together with CP-113,818, decreased cell toxicity in a manner that was dose-dependent on the concentration of progesterone (Fig. 6). The addition of 5 µg/ml of progesterone in combination with 1 µg/ml of CP-113,818 showed no increase in toxicity compared with 5 µg/ml of progesterone alone, or control incubations with no additions.
Figure 6:
Dose
curve of the effect of progesterone on ACAT inhibitorinduced cell
toxicity. Mouse peritoneal macrophages were cholesterol-loaded for 24 h
with acetylated LDL, free cholesterol/phospholipid dispersion, and
fetal bovine serum as described under ``Experimental
Procedures.'' The cells were incubated with 1 µCi of
[H]adenine for 2 h before the addition of the
treatments. Macrophages were incubated for 24 h with up to 5 µg/ml
progesterone alone or in combination with 1 µg/ml CP-113,818.
Values for [
H]adenine release are presented as
the mean ± S.D. (% of control) from triplicate wells for
progesterone alone (
) or progesterone plus CP-113,818
(
).
Free cholesterol concentrations in macrophages treated with CP-113,818 alone or in combination with increasing concentrations of progesterone showed similar increases (data not shown).
Inhibition of cholesterol esterification may provide a mechanism by which progression of atherosclerosis is reduced(7) . However, inhibition of ACAT in intact cells, in vitro or in vivo, may result in a redistribution of cellular cholesterol by increasing the free cholesterol and decreasing the cellular cholesteryl ester contents. In this study, using cholesterol-loaded macrophages, we show that such a change in the cellular cholesterol homeostasis resulted in an increased cell toxicity.
Subacute adrenal toxicity caused by a specific inhibitor of ACAT has recently been reported(8, 9, 10) . These researchers attributed the toxic effect to a direct inhibition of mitochondrial respiration in adrenocortical cells. Short term incubation of the ACAT inhibitor with adrenocortical cells resulted in a rapid loss of cell viability. Further examination into the mechanism of cell toxicity showed that cellular ATP levels were severely compromised prior to loss of cell viability. The present cell toxicity data are consistent with these findings.
In the present study, we have shown that two
specific ACAT inhibitors, Sandoz 58-035 and CP-113,818, induce a
free cholesterol accumulation in macrophage-derived foam cells. In each
case, the free cholesterol buildup coincides with an increase in cell
toxicity. Several lines of evidence suggest that free cholesterol is
responsible for this induced toxic effect. First, increasing the
cellular content of cholesteryl esters, before ACAT inhibitor
treatment, resulted in higher free cholesterol buildup as well as
higher [H]adenine release (Fig. 2).
Second, cellular free cholesterol content and the release of
C-labeled nucleotides were highly correlated, while the
latter showed a highly negative correlation with efflux of
[
H]cholesterol from macrophages (Fig. 3).
Third, the EC
for cholesterol buildup is lower than that
for ACAT inhibition, indicating that only partial inhibition of ACAT
will produce cell free cholesterol accumulation in this closed cell
system (Fig. 4). Finally, CP-113,818-induced cell toxicity could
be blocked by the combined addition of progesterone or U18666A ( Fig. 5and Fig. 6), compounds which inhibit intracellular
sterol transport(35, 36, 38) .
The inhibition of ACAT by itself is not sufficient to produce the toxic accumulation of free cholesterol as evidenced by the observation that ACAT inhibition by progesterone does not produce toxicity equivalent to that observed with Sandoz 58-035 or CP-113,818. In addition, treatment with progesterone or U18666A, together with the ACAT inhibitor, CP-113,818, eliminates the toxicity. As shown in Fig. 7, we propose that interruption of the cholesteryl ester cycle results in the accumulation of unesterified cholesterol. Excess free cholesterol is transported from the site of hydrolysis, and its incorporation into membranes results in the eventual destabilization of the plasma membrane (Fig. 7B). We further propose that progesterone or U18666A interferes with the transport of unesterified cholesterol from intracellular sites of cholesteryl ester hydrolysis to the plasma membrane (Fig. 7C). In support of this hypothesis is the observation that the accumulation of free cholesterol in progesterone-treated cells resulted in the formation of membrane-bound vacuoles containing myelin figures(27, 28) , whereas inhibition of ACAT with pharmacological inhibitors such as Sandoz 58-035 does not induce intracellular storage of cholesterol in intracellular organelles(28) . Thus, our data would be consistent with the free cholesterol-induced toxicity resulting from a destabilization of the plasma membrane upon cholesterol enrichment and the protection produced by progesterone and U18666A being a reflection of the retention of the free cholesterol in intracellular pools.
Figure 7: Diagram of possible mechanism for ACAT inhibitor-induced cell toxicity. A, mouse peritoneal macrophages incubated for 24 h with acetylated LDL, free cholesterol/phospholipid dispersion, and fetal bovine serum have an enlarged cholesteryl ester pool compared with the free cholesterol pool. The cholesteryl ester cycle proceeds uninterrupted. B, the addition of an ACAT inhibitor blocks the conversion of free cholesterol (FC) to cholesteryl ester (CE) resulting in the intracellular buildup of free cholesterol, some of which is transferred to a membrane pool. Enrichment of this membrane pool ultimately results in a loss of cell viability. C, as above, the addition of an ACAT inhibitor causes an intracellular buildup of free cholesterol. However, co-incubation of either progesterone or U18666A, together with the ACAT inhibitor, blocks the toxic effect by inhibiting the intracellular transport of the free cholesterol to the membrane pool.
Our present observations are relevant to considerations of the development of ACAT inhibitors that function at the level of the vessel wall. Studies have shown that unstable plaques are associated with lipid accumulation and foam cells (39, 40) and suggested that a reduction in the extent of lipid deposition will result in the stabilization of the pathologic processes (for a review, see (41) ). Based on our observations, it can be predicted that if ACAT inhibitors are employed to reduce the intracellular levels of cholesteryl esters, cell death could result unless sufficient cholesterol acceptors were present to prevent free cholesterol accumulation.