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From Marc and Ruti Bell Vascular Biology Research Program of the Leon H. Charney Division of Cardiology (Department of Medicine) and the Department of Cell Biology (J.X.R., J.K., E.A.F.), New York University School of Medicine, New York, NY; and the Institute of Human Nutrition (P.O., S.L.S.) and the Department of Pediatrics (S.L.S.), Columbia University, New York, NY.
Correspondence to Edward A. Fisher, MD, PhD, TH-451, NYU School of Medicine, 550 1st Avenue, New York, NY 10016. E-mail edward.fisher@med.nyu.edu
Abstract
Objective— Studies in vitro and in vivo of macrophage foam cells have shown evidence of cytotoxicity after acyl-CoA:cholesterol acyltransferase (ACAT) inhibition. Foam cells of smooth muscle origin are also found in human and animal atherosclerotic lesions.
Methods and Results— To study whether cytotoxicity from ACAT inhibition is independent of cell type, we first established a protocol to conveniently induce aortic smooth muscle foam cell formation using cholesterol–cyclodextrin complexes (CCC). Rat aortic smooth muscle cells (ASMCs) treated for 48 hours with CCC (20 μg/mL) became foam cells by morphological (oil-red-O staining) and biochemical (1200% and 180% increase in cellular esterified and free cholesterol, respectively) criteria. ACAT activity increased 500% (P<0.01 versus untreated). Similar results were obtained in human ASMC, but ACAT activity increased to an even greater extent (3200%; P<0.01 versus untreated). Western blots indicated that CCC treatment increased human (to 380±20% of untreated, P<0.001), but not rat, ACAT protein expression. ACAT inhibition by Fujirebio compound F1394 suppressed CCC-induced foam cell formation in rat and human ASMC, but, notably, did not induce significant cytotoxicity.
Conclusion— ASMC might be more resistant to FC-induced adverse effects than are macrophages.
Aortic smooth muscle foam cell formation was induced with cholesterol–cyclodextrin complexes. Compared with current and published data in macrophages, simultaneous cholesterol-loading and ACAT inhibition led to little cytotoxicity in SMCs. Therefore, the potential for adverse consequences of ACAT inhibitors used therapeutically may be related to dose and the cell type.
Key Words: cyclodextrins ? F1394 ? lipid droplets ? smooth muscle cells ? ACAT
Introduction
The appearance of cholesteryl ester (CE)-laden foam cells in the intimal layer of the arterial wall is an early event in the development of atherosclerosis.1 Foam cells found in vivo have been demonstrated to be of either macrophage or smooth muscle cell (SMC) origin. In cells of macrophage origin, foam cell formation is thought to depend on the ability of an isoform of the enzyme acyl-CoA:cholesterol acyltransferase (ACAT), ACAT1, to convert excess free cholesterol (FC) to the lipid droplet form of CE. In tissue culture, macrophages can be easily induced to transform into foam cells by loading cholesterol from a variety of donors, including modified low-density lipoproteins (LDL) and ?-cyclodextrins.2,3 The inhibition of ACAT, by decreasing cholesteryl esterification and, presumably foam cell formation, has attracted considerable interest as a therapeutic strategy for the treatment of atherosclerosis.
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When foam cell formation in cells of macrophage origin was blocked by the ACAT inhibitor 58-035, however, significant toxicity and cell death were observed.4 This result is consistent with other results in vitro5 and in vivo5–7 that have shown that the ACAT1-dependent formation of CE in macrophages is a protective response to prevent the disruption of functions of plasma and endoplasmic reticulum (ER) membranes that result from their enrichment in FC. For example, complete deficiency of macrophage ACAT18 led to increased atherosclerotic lesion formation in LDL receptor-deficient mice, a model of human atherosclerosis. Nevertheless, partial ACAT inhibition by pharmaceutical approaches has been shown to be associated with decreased lesion size and lesional macrophage content, and without signs of systemic or vessel wall toxicity or changes in smooth muscle cell content.9,10 This indicates there might be a dose-dependent or cell type-specific cytotoxic effect of ACAT inhibition.
As alluded to, in atherosclerotic lesions there are also arterial smooth muscle foam cells. In tissue culture, SMCs can be induced to form foam cells,11–13 but the previously reported cholesterol-loading methods have been relatively slow and inconvenient.11,12,14–17 In this report, we have characterized the relative utilities of various methods of cholesterol enrichment to promote smooth muscle foam cell formation in vitro. We demonstrate the relative advantages of ?-cyclodextrins and, in conjunction with the ACAT inhibitor F1394,18 have gone on to use this method to examine the ACAT-dependence of smooth muscle foam cell formation and the potential for toxic effects of ACAT inhibition. Similar to cells of macrophage origin, smooth muscle foam cell formation appears to be dependent on ACAT1 but, in contrast, SMCs are relatively resistant to the toxicity of FC enrichment. This difference in susceptibility implies that in vivo, the toxicity of ACAT inhibition in atherosclerotic lesions may depend on the proportion of foam cells that originate from each cell type.
Methods
Chemicals
Please see http://atvb.ahajournals.org for a more detailed Methods section. The ACAT inhibitor (F-1394) was provided by Fujirebio, Inc (Japan).
Cell Culture
The conditions of growth of the rat aortic smooth muscle cells (rat ASMCs; provided by Dr Mark B. Taubman, University of Rochester Medical Center, Rochester, NY), human ASMCs (purchased from American Type Culture Collection, Manassas, Va), and human THP-1 (provided by Dr George H. Rothblat, Children’s Hospital of Philadelphia/University of Pennsylvania School of Medicine, Philadelphia, Pa) are described in the supplemental online materials.
Induction of Aortic Smooth Muscle Foam Cell Formation
ASMCs were plated on Laboratory-Tek II chamber slides (Nalge Nunc International) and were maintained in culture media until the cells reached 90% to 95% confluence. The cell monolayer was then washed with phosphate-buffered saline and incubated for 48 hours with 0, 5, 10, and 20 μg/mL of cholesterol–cycoldextrin complex (CCC) in 0.2% bovine serum albumin (BSA) fraction V in DMEM (rat ASMC) or Ham F12K culture medium (human ASMC). Alternatively, ASMCs were incubated with lipoproteins/liposomes containing cholesterol (20 μg/mL) in 0.2% BSA/DMEM for 48 hours. To inhibit foam cell formation, ASMCs were incubated for 48 hours with 20 μg/mL of CCC in 0.2% BSA/DMEM in the presence of 0, 10, 100, and 1000 nM of F1394 (in dimethyl sulfoxide, final concentration in medium was 0.1%). The monolayer was then washed with phosphate-buffered saline, fixed with 4% paraformaldehyde, and stained with Mayer hematoxylin solution and oil red O, which detects neutral lipids such as CE and triglycerides.
Cytotoxicity Assay
Cytotoxicity of ACAT inhibition was determined based on the method of Warner et al4 with modifications. Briefly, ASMCs (in 12-well plates) with or without CCC (20 μg/mL) or F1394 (1000 nM) treatment were incubated for 18 hours in 0.2% BSA. The cells were then incubated for 2 hours with [1-14C]-adenine (0.5 μCi/mL) in 0.2% BSA, followed by 10 minutes with culture medium without [1-14C]-adenine, and washed with phosphate-buffered saline. Subsequently, the cells were cultured for 24 hours in 0.2% BSA with or without CCC (20 μg/mL) or F1394 (1000 nM). Alternatively, fully differentiated human macrophage THP-1 cells were preloaded with [1-14C]-adenine as mentioned, and then incubated in 0.2% BSA/DMEM with or without CCC (20 μg/mL) or F1394 (1000 nM) for 48 hours in the presence of phorbol 12-myristate 13-acetate (100 ng/mL). The media from ASMC and THP-1 culture were collected and an aliquot was used to determine 14C released from the cells using a scintillation counter. The cell monolayer was dissolved in 0.1 N NaOH, and an aliquot was removed to determine the remaining radioactivity. The cytotoxicity was determined by % 14C release, which was calculated as follows: 14C release (%) = radioactivity in the medium (cpm) / [radioactivity remaining in the cell (cpm) + radioactivity in the medium (cpm)] x 100.
Alternatively, cytotoxicity was determined by total recovered protein mass19 after ASMCs (in a 12-well plate) were treated with or without CCC (20 μg/mL) or F1394 (30, 100, 300, and 1000 nM).
Statistics
Within an experiment, duplicate, triplicate, or quadruplicate wells were used for each condition or treatment. Data were expressed as mean±SEM. All experiments were repeated at least once. GraphPad Prism software was used to analyze differences between samples by 1-way ANOVA with the Bonferroni post-test for differences between selected pairs of samples. P<0.05 were considered significant.
Results
Induction of Aortic Smooth Muscle Foam Cell Formation by CCC
After incubation with CCC for 48 hours, rat or human ASMCs assumed the morphological appearance of foam cells with oil red O–stained lipid droplets present predominantly in the perinuclear area, but also distributed throughout the cytosol of most cells (Figure 1A and insets); in contrast, no lipid droplets were found in the untreated cells. The visual abundance of lipid droplets was grossly correlated with CCC concentration in the treatment.
Figure 1. Aortic smooth muscle foam cell formation is induced by CCC and inhibited by F1394 treatments. Rat or human ASMCs were incubated for 48 hours with indicated concentrations of CCC (A) or for 48 hours with CCC (20 μg/mL) plus indicated concentrations of F1394 in 0.2% BSA (B) in DMEM or Ham F12K culture medium. Cells were then fixed with 4% paraformaldehyde and stained with Mayer hematoxylin and oil red O. Magnification x50; inset magnification x100.
In addition to CCC, we also examined the effectiveness of other approaches previously used to deliver cholesterol to cells to stimulate smooth muscle foam cell formation. These included incubating cells with native LDL, acLDL (a modified LDL that effectively induces macrophage foam cell formation in vitro2), negatively charged liposomes (previously shown to induce macrophage foam cell formation in vitro20), positively charged liposomes, cholesterol micelles (CM), and CM–mono-olein (both of which contain the bile acid taurocholate and effectively deliver cholesterol to the human intestinal cell line, Caco-2 cells21). When rat ASMCs were incubated with these vehicles at cholesterol concentrations similar to that provided by CCC, none was comparable to CCC in promoting cholesterol accumulation and maintaining cell viability. The lipoproteins or liposomes induced little or no lipid inclusion accumulation in ASMCs, and CM or CM–mono-olein treatment lysed rat ASMCs within 6 hours, perhaps because of solubilization of the plasma membrane of rat ASMCs by the taurocholic acid component of the micelles.
CCC-Induced Aortic Smooth Muscle Foam Cell Formation Is ACAT-Dependent
As shown in Figure 1B, when rat or human ASMCs were incubated with CCC (20 μg/mL) plus ACAT inhibitor F1394, cellular lipid inclusions decreased as F1394 concentration increased. No lipid inclusions were visible by light microscopy when a 1000 nM concentration of F1394 was used. Biochemical measurements were consistent with the oil red O staining results; compared with the untreated rat ASMCs, CCC treatment for 48 hours increased CE and FC content to 1200% and 180%, respectively, of the corresponding values for the untreated cells (Table 1). When cells were treated with CCC plus F1394, however, as F1394 concentration increased, cellular CE content decreased (compared with the cells treated with CCC only), whereas FC content increased. At the highest concentration of F1394 tested (1000 nM), the CE content remained at the basal level (ie, similar to when no CCC was added), and FC content increased to 280% of the basal level. Total cholesterol content remained constant in the presence of the ACAT inhibitor.
TABLE 1. Cholesterol Content and Whole ACAT Activity in ASMC
Similarly, in human ASMCs, CCC treatment increased both CE and FC content to 300% of the untreated cells (7.6±0.5 and 27.6±2.8 μg/mg cellular protein, respectively), and F1394 (1000 nM) completely blocked CE formation while increasing FC content to 430%.
Whole-Cell ACAT Activity Increased With CCC Treatment but Decreased With F1394 Cotreatment
To definitively show that the effects of F1394 were mediated by its inhibition of ACAT, we measured ACAT activity in CCC-induced smooth muscle foam cells, before and after CCC treatment, and in the presence and absence of F1394. As summarized in Table 1, whole-cell ACAT activity increased 350% (P<0.01) in rat ASMCs after CCC (20 μg/mL) treatment for 48 hours. When increasing concentrations of F1394 were used (Table 1), the increase in ACAT activity was inhibited in a concentration-dependent manner; at the highest concentration of F1394 (1000 nM), ACAT activity declined to <6% (P<0.01) of the basal level.
As also shown in Table 1, there was even greater CCC-induced increase in the whole-cell ACAT activity in human ASMCs (to 3200% of baseline, P<0.01; Table 1), which was also inhibited in a concentration-dependent manner by F1394. The IC50 values of F1394 on ACAT activity were estimated to be 62.7 nM and 10 nM in rat and human ASMCs, respectively.
CCC Treatment Increased Human but not Rat ACAT Protein Mass
We were interested in the basis of increased ACAT activity in the CCC-treated cells. Because ACAT can be allosterically regulated by the concentration of the cholesterol substrate22 as well as by increased expression,23 we performed Western blotting using cell lysates obtained before and after CCC treatment. As shown in Figure 2, a band of 45 kDa, similar to what was reported for rat ACAT1,24 was detected in the rat cell lysates by an anti-ACAT1 antibody (originally prepared by J. Bilheimer et al, Dupont-Merck Pharmaceuticals). There was little or no change in rat ACAT protein abundance on treatment with CCC (20 μg/mL) alone (Figure 2) or CCC with F1394 (1000 nM; not shown).
Figure 2. Western blot analysis of ACAT protein in ASMCs with or without cholesterol loading. Rat or human ASMCs were incubated for 48 hours with or without CCC (20 μg/mL) in 0.2% BSA in DMEM or Ham F12K culture medium. Total cellular proteins were separated by SDS-PAGE, transferred to polyvinylidenefluoride (PVDF) membrane, and probed with rabbit anti-human ACAT antibody (see Methods). Blot shown is representative of 2 independent experiments.
In the human cell lysate, a band of 45 kDa, similar to that was reported for human ACAT1 monomer,25 was detected by the same antibody. Surprisingly, unlike the rat ACAT, on CCC treatment human ACAT mass increased to 380±20% of untreated (P<0.001). This difference in rat and human ACAT regulation on cholesterol loading was confirmed (data not shown) in separate experiments when Western blotting was performed using new cell preparations and a different antibody (anti-ACAT1DM10,26 from Drs T.Y. and C.C. Chang, Dartmouth Medical School, Hanover, NH).
The reactivity of ASMC ACAT with antibodies prepared against ACAT1 suggests that this is the form expressed by this cell type, consistent with the reports that ACAT2 is expressed only in the liver and small intestine of humans and rodents.27 Nonetheless, because F1394 inhibits both ACAT1 and ACAT2, to confirm that the species expressed in ASMCs was indeed ACAT1, reverse-transcription polymerase chain reaction28 using RNA from human ASMCs was performed, which showed only an ACAT1 amplicon (data not shown), consistent with a recent report that no ACAT2 was detected in rodent ASMCs.29
ACAT Inhibition Resulted in Minimal Cytotoxiciy
In both in vitro studies of macrophages and in vivo studies in ACAT1 knockout mice,6 toxicity related to FC accumulation has been reported. In particular, in a series of reports by Rothblat et al,4,30,31 FC loading in the presence of an ACAT inhibitor was associated with induced cytotoxicity in the form of damage to the plasma membrane and leakage of preloaded 14C-adenine.4 Consistent with their observations, we found in cholesterol-loaded human THP-1 (Table 2A), ACAT inhibition by F1394 led to increased 14C-adenine release compared with control or unloaded cells. The amount of release with F1394+CCC was 1.5-times that with control treatment, which is comparable to the 2-times increase Rothblat et al observed on ACAT inhibition of cholesterol-loaded mouse peritoneal macrophages.4
TABLE 2A. 14C Release From ASMC and THP-1 Macrophage-Like Cells
TABLE 2B. Total Recovered Protein Mass From Rat ASMC
To examine whether there was a similar induction phenomenon in ASMCs under our experimental conditions, we also measured in basal (ie, when no CCC or F1394 was added) and cholesterol-loaded cells the release of preloaded 14C-adenine. As shown in Table 2A, treating rat ASMCs with CCC (20 μg/mL) alone or CCC plus F1394 (1000 nM) did not increase 14C-adenine release compared with the basal level. This result is consistent with our finding that over a range of F1394 concentrations (0 to 1000 nM), there were no differences in total recovered protein mass (Table 2B). Taken together, it appears that rat ASMCs, compared with the macrophages, exhibit little induced cytotoxicity when ACAT is inhibited and FC accumulates.
In contrast to the rat ASMCs, 14C adenine release was higher in human ASMCs under basal conditions (Table 2A). Nonetheless, CCC alone or F1394 alone did not change 14C adenine release. CCC plus F1394 treatment, however, tended to increase 14C adenine release over basal values, but this did not reach statistical significance. Overall, then, these results indicated that there was minimal cytotoxicity of ACAT inhibition and FC accumulation in rat or human ASMCs under our experimental conditions.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
There have been a number of recent studies in vitro and in vivo that have shown that the inhibition of ACAT in cholesterol-loaded cells can result in cytotoxicity and cell death.4–6,31,32 This issue has attracted significant attention given the potential for ACAT as an anti-atherosclerosis drug target. The present study in ASMCs was undertaken to first develop a convenient cholesterol-loading protocol for this cell type. Then, we used this protocol to investigate the relevance of the previous studies in vitro,4,31,32 which were conducted exclusively in cells of macrophage origin, to another cell type capable of forming foam cells in vitro12,19 and in atherosclerotic lesions.33,34 The major results of the present studies are: (1) a convenient, relatively rapid, cyclodextrin-based SMC cholesterol-loading protocol has been achieved; (2) compared with cells of macrophage origin, simultaneous cholesterol-loading and ACAT inhibition in ASMCs led to comparatively little cytotoxicity; (3) as in human macrophages, the major ACAT form expressed in human SMCs is ACAT1; and (4) rat and human ACAT activity exhibited differential regulation at the protein level.
Lipid-laden, SMC-derived foam cells have been demonstrated in early and late atherosclerotic lesions from human and animal models.1 There have been numerous efforts to establish in vitro approaches to induce and study smooth muscle foam cell formation, but with only limited successes.11–17 Using a commercially available reagent, CCC, we successfully delivered cholesterol to rat and human ASMCs and induced foam cell formation. The esterification of cholesterol delivered by CCC and the formation of foam cells were dependent on ACAT activity. Importantly, the use of CCC was more effective than using a number of other cholesterol-delivering vehicles, including native LDL, acLDL, negatively charged liposomes, positively charged liposomes, CM, and CM–mono-olein. Furthermore, because of the commercial availability of CCC, the method is much more convenient than those using other reagents, such as cationized LDL13 and lipid inclusions isolated from lipid-laden macrophages.12
It has been long known that ACAT can be activated by substrate availability. In the present studies, we also found large increases in ACAT activity in the cholesterol-loaded cells. Interestingly, increased ACAT activity in rat ASMCs was not accompanied by an increase in enzyme mass (as measured by Western blot), whereas in human ASMCs, it was. This difference could not be explained by a species difference in the form of ACAT expressed—both rodent29 and human ASMCs (determined by reverse-transcription polymerase chain reaction; data not shown) expressed ACAT1 (as do macrophages). The increase in ACAT protein mass in human ASMCs is consistent with studies in human monocytes that ACAT mRNA was increased during differentiation and foam cell formation.23 Thus, in addition to the previously proposed allosteric activation of ACAT by substrate,22 which may explain the rat ASMC results, there may be other modes of regulating ACAT activity depending on the species of cells and, perhaps, cell type.
We were initially surprised by our finding that the accumulation of FC after ACAT inhibition did not induce cytotoxicity (as assessed by an assay previously used for this purpose4). In comparison to previous results4,5,31 and ours (Table 1) with cells of macrophage origin, this discrepancy could not be explained by less ACAT inhibition or cholesterol loading in the present study. Previous studies have implicated excess FC in either the plasma4 or the ER membrane5 as the provocateur of adverse consequences ultimately culminating in cell death. Enrichment of these depots depends on sterol trafficking pathways, which themselves are under complex control35 and which may vary among cell types. In addition, cytotoxicity and cell death pathways are also multifactorial, with multiple regulatory molecules. Thus, variations either in the delivery of FC to critical sites or in the response to the FC so delivered may ultimately determine the susceptibility of a cell to the adverse effects of cholesterol loading. The relative resistance of ASMCs to cholesterol loading-induced toxicity suggests that in advanced atherosclerotic lesions, a significant fraction of the foam cells may be of SMC origin, consistent with SMCs being a more prominent histological feature of such lesions.
In summary, we have established a new convenient cell culture model to study the formation of smooth muscle foam cells and the potential cytoxicity of cholesterol loading. Taken with previous results in vivo from us and others,9,10 in which partial inhibition of ACAT was effective in reducing atherosclerosis in animal models, the potential for adverse consequences of ACAT inhibitors used therapeutically would appear to be related to dose and cell type. Further investigation will be needed to identify the molecular basis of the variation in the susceptibility of a cell to the toxicity of FC. Nonetheless, the differential susceptibility of macrophage and SMC foam cells implies that in vivo, the toxicity of ACAT inhibition in atherosclerotic lesions may depend on the proportion of foam cells that originate from each cell type.
Acknowledgments
The study was supported by National Institutes of Health grant HL61814 (E.A.F.) and an American Heart Association Heritage Affiliate grant-in-aid (S.L.S.).
Footnotes
Current affiliation for J.K.: Tsukuba Research Institute, Banyu Pharmaceutical Co, LTD, Tsukuba, Ibaraki, Japan.
J.X.R. and J.K. contributed equally and may be cited interchangeably.
Received January 20, 2004; accepted October 5, 2004.
References
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Discussion
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