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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1891-1898.)
© 1999 American Heart Association, Inc.

Vascular Biology

Crystallization of Free Cholesterol in Model Macrophage Foam Cells

G. Kellner-Weibel; P. G. Yancey; W. G. Jerome; T. Walser; R. P. Mason; M. C. Phillips; G. H. Rothblat

From the Department of Biochemistry (G.K.-W., M.C.P., G.H.R.), MCP Hahnemann University, Philadelphia, Pa; the Department of Pathology (P.G.Y., W.G.J., T.W.), Wake Forest University School of Medicine, The Bowman Gray Campus, Winston-Salem, NC; and the Laboratory for Membrane Structure Studies (R.P.M.), MCP Hahnemann University, Pittsburgh, Pa.

Correspondence to G.H. Rothblat, Department of Biochemistry, MCP Hahnemann University, 2900 Queen Ln, Philadelphia, PA 19129.

	Abstract

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Abstract—The present study examined free cholesterol (FC) crystallization in macrophage foam cells. Model foam cells (J774 or mouse peritoneal macrophages [MPMs]) were incubated with acetylated low density lipoprotein and FC/phospholipid dispersions for 48 hours, resulting in the deposition of large stores of cytoplasmic cholesteryl esters (CEs). The model foam cells were then incubated for up to 5 days with an acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor (CP-113,818) in the absence of an extracellular FC acceptor to allow intracellular accumulation of FC. FC crystals of various shapes and sizes formed in the MPMs but not in the J774 macrophages. Examination of the MPM monolayers by microscopy indicated that the crystals were externalized rapidly after formation and thereafter continued to increase in size. Incubating J774 macrophages with 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP) in addition to CP-113,818 caused FC crystal formation as a consequence of CPT-cAMP stimulation of CE hydrolysis and inhibition of cell growth. In addition, 2 separate cholesterol phases (liquid-crystalline and cholesterol monohydrate) in the plane of the membrane bilayer were detected after 31 hours of ACAT inhibition by the use of small-angle x-ray diffraction of J774 macrophage foam cells treated with CPT-cAMP. Other compounds reported to inhibit ACAT, namely progesterone (20 µg/mL) and N-acetyl-D-sphingosine (c₂-ceramide, 10 µg/mL), induced cellular toxicity in J774 macrophage foam cells and FC crystallization when coincubated with CPT-cAMP. Addition of the extracellular FC acceptors apolipoproteins (apo) E and A-I (50 µg/mL) reduced FC crystal formation. In MPMs, lower cell density and frequent changes of medium were conducive to crystal formation. This may be due to "dilution" of apoE secreted by the MPMs and is consistent with our observation that the addition of exogenous apoE or apoA-I inhibits FC crystal formation in J774 macrophage foam cells cotreated with CP-113,818 plus CPT-cAMP. These data demonstrate that FC crystals can form from the hydrolysis of cytoplasmic stores of CEs in model foam cells. FC crystal formation can be modulated by the addition of extracellular FC acceptors or by affecting the cellular rate of CE hydrolysis. This process may contribute to the formation of FC crystals in atherosclerotic plaques.

Key Words: macrophages • foam cells • cholesterol crystals • atherosclerosis

	Introduction

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Extracellular lipid deposits are responsible for a large portion of atherosclerotic plaque volume.¹ Several studies have demonstrated that the lipid component of the lesion is directly related to the incidence of plaque rupture and thrombosis.^{2 3 4} Thus, lesions that have a large lipid component are the most unstable. Lipid in an atheroma consists mainly of cholesterol (both free, FC, and esterified, EC) and phospholipid, with small amounts of triacylglycerol and fatty acid.¹ FC in a plaque either can be associated with phospholipid and EC, or it can be present as crystalline cholesterol.^{1 5 6} Crystalline cholesterol is a prominent feature of lesions in both humans and animal models.^{7 8 9 10} Unlike membrane cholesterol, which can exchange from the plaque with lipoprotein acceptors in the plasma, crystalline cholesterol within the lesion appears to be practically inert.¹

Although the origin of these FC crystals is unknown, it has been proposed that they form either from intracellular lipid accumulated by foam cells^{11 12} or from extracellular lipid trapped in the matrix of the lesion.⁸ In the first case, macrophages or smooth muscle cells would accumulate lipid through the uptake of native or oxidized lipoproteins and through the phagocytosis of lipid expelled from neighboring dead foam cells. Once a critical mass is reached, an intracellular nucleating event could occur, leading to cholesterol crystallization. Alternatively, extracellular lipids deposited by lipoproteins or dying foam cells could crystallize in the milieu of the lesion. Recently, we demonstrated the in vitro formation of FC crystals from lipid-enriched mouse macrophages.¹¹ In this system, acyl-coenzyme A:cholesterol acyltransferase (ACAT) is inhibited, so that cytoplasmic CE hydrolysis leads to FC accumulation. These intracellular FC stores are further enhanced by exclusion of an extracellular acceptor. Once sufficient FC concentrations are reached, crystallization occurs. In the present study, we have extended these studies to characterize the crystals and examine events responsible for their formation.

	Methods

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Materials
FBS, BSA (essentially fatty acid free), gentamicin, unesterified (ie, free) cholesterol (FC), cholesteryl methyl ether, progesterone, N-acetyl-D-sphingosine (c₂-ceramide), and 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP) were purchased from Sigma Chemical Co. Organic solvents were products of Fisher Scientific. Tissue-culture flasks and plates were obtained through Corning or Falcon. [1,2-³H]Cholesterol and [³H]adenine were purchased from New England Nuclear. Tissue-culture media were obtained from Gibco. 1-Palmitoyl-2-oleoyl phosphatidylcholine (PC) was purchased from Avanti Polar Lipids. FC and PC dispersions were made by the method of Arbogast et al.¹³ Human LDL (1.019 g/mL<d>1.063 g/mL) was fractionated by sequential ultracentrifugation and acetylated according to Basu et al.¹⁴ Human plasma apolipoproteins were obtained by ultracentrifugation and purified by anion-exchange chromatography.¹⁵ Before use, the purified apolipoproteins were solubilized in 6 mol/L guanidine HCl and dialyzed against Tris buffer (0.01 mol/L Tris, 1.0 mmol/L EDTA, and 0.15 mol/L NaCl). An ACAT inhibitor, Pfizer CP-113,818, was a generous gift from Dr Mark Bamberger, Pfizer Pharmaceuticals (Groton, Conn).

Cell Culture
Mouse peritoneal macrophages (MPMs) were prepared as previously described.¹⁶ J774 macrophages (J774A.1 from American Type Culture Collection, Manassas, Va) were routinely grown in RPMI 1640 medium containing 50 mmol/L HEPES buffer and 50 µg/mL gentamicin (RPMI) and supplemented with 10% FBS. Cells were plated in 12-well tissue-culture plates at a density of 500 000 cells per well. To cholesterol load the macrophages, RPMI containing 1% FBS, acetylated LDL (acLDL, 100 µg of protein per mL), and FC/PC dispersions (250 µg of FC per mL) were added to the incubation medium for 48 hours. Monolayers were then washed 3 times with minimum essential medium containing 2 g/L NaHCO₃ and 50 µg/mL gentamicin. Monolayers were equilibrated in RPMI containing 0.2% BSA for 18 hours. After this period, cells were incubated with the ACAT inhibitor (2 µg/mL CP-113,818), c₂-ceramide (10 µg/mL), or progesterone (20 µg/mL). Control incubations contained RPMI with 0.2% BSA. Some incubations contained CPT-cAMP (0.3 mmol/L) and/or apoA-I or apoE4/4 (50 µg/mL). Cells were incubated in a humidified atmosphere containing 95% air and 5% CO₂ at 37°C.

Microscopy
After cholesterol loading and equilibration, MPMs were washed and then placed on media containing either the ACAT inhibitor in dimethyl sulfoxide (DMSO) or DMSO alone. At the times indicated in the text, the cells were washed and fixed for microscopy. Fluorescence microscopy was used to visualize cellular filipin-stained FC, transmission electron microscopy (TEM) was utilized to detect the presence of intracellular cholesterol crystals, and scanning electron microscopy (SEM) was employed to analyze extracellular cholesterol crystals. To visualize cellular FC, macrophages were grown on sterile glass coverslips placed in the bottom of the wells of 6-well culture plates. Before being stained, the coverslips were washed in PBS and fixed for 1 hour in 10% neutral buffered formalin. After fixation, the cells were washed in PBS and stained for 3 hours at 37°C in filipin stain solution. The stain solution consisted of 1.25 mg of filipin dissolved in 0.5 mL of DMSO diluted with 25 mL of PBS.¹⁷ After being stained, the coverslips were washed in distilled water and mounted on glass slides with a phenylenediamine-glycerol solution to inhibit photobleaching. Slides were kept in the dark until viewing. The presence of filipin-positive unesterified cholesterol was detected by epi-illumination with UV (UG-1 filter) and viewed through a 510-nm barrier filter.

For TEM, cells were grown on sterile coverslips coated with Formvar plastic. At the appropriate times, the cells were washed in 0.1 mol/L cacodylate buffer and then fixed overnight at 4°C in 2.5% glutaraldehyde in cacodylate buffer. The cells were then washed again, postfixed in 1% cacodylate-buffered OsO₄, and dehydrated in a graded series of ethanol to 70% ethanol in water solution. At this point, the Formvar with attached cells was carefully removed from the coverslips and gently rolled up, and dehydration was completed by 2 changes of 100% ethanol. After dehydration, the cells on Formvar were embedded in epoxy resin. In our experience, this embedding procedure is gentler than scraping the cells from the culture dishes and maintains the integrity of fragile, lipid-engorged cells. Thin (60-nm) and thick (1-µm) sections of embedded cells were collected on copper mesh grids and viewed at 80 and 300 keV, respectively.

For SEM, cells were grown on sterile coverslips placed in the wells of 6-well culture plates. To prepare cells for SEM, the coverslips were washed in 0.1 mol/L cacodylate, and the cells were fixed in 4% cacodylate-buffered glutaraldehyde. After fixation, half of the samples were dehydrated in a graded series of ethanol/water solutions, infiltrated with liquid CO₂, and dried using the critical-point method. An identical set of cells was air dried after fixation and washing. Quantitatively there were no differences in size or number of crystals between the air-dried and critical point–dried samples. However, critical-point drying better maintained the cellular ultrastructure.

Quantitation of Extracellular Crystal Number and Size
Crystals were classified as plates or needles depending on their morphology. Needles were long and narrow, having a length-to-width ratio >10. In contrast, plates were wider, with a smaller length-to-width ratio (<10). For each sample, the total number of plates and needles in 1645 fields was calculated. Each field represented 6556 µm² of area, which is the same as the viewing area of the SEM at x1250 magnification. In addition, the lengths and widths of crystals were determined for randomly selected crystals from each sample. The microscope was routinely calibrated to ensure the accuracy and reproducibility of the measurements.

Preparation of Oriented Cellular Membranes for X-Ray Diffraction Analysis
J774 macrophage foam cells were scraped into a buffer containing 0.5 mmol/L HEPES and 150 mmol/L NaCl, after which the cells were disrupted by N₂ cavitation (250 psi for 30 minutes). This step was followed by a low-speed spin (1000 rpm) to pellet and remove the nuclei. Oriented cellular membrane samples from J774 macrophages were prepared for x-ray diffraction analysis by centrifugation, as previously described.^{18 19} In brief, pooled, cellular membrane samples, each consisting of 200 µg of phospholipid, were transferred to Lucite sedimentation cells containing an aluminum foil substrate. The sedimentation chambers were placed in a Sorvall AH-629 swinging-bucket ultracentrifuge (Dupont Corp) and centrifuged at 30 000g for 50 minutes at 5°C. After completion of the spin, the supernatants were immediately removed and the membrane pellets equilibrated overnight in glass vials containing a saturated salt solution (NH₄H₂PO₄), which defines a relative humidity of 93% at 20°C. The samples were placed on a curved glass substrate and placed into sealed brass canisters with thin aluminum foil in which the relative humidity was controlled. The temperature (20°C) was maintained during the x-ray diffraction experiments by placing the brass chambers into a brass water jacket regulated with a Neslab RTE-1 II water bath. The samples were exposed to the x-ray beam until 2x10⁶ photon diffraction counts had been collected by the electronic detector; the average time for each experiment was 30 minutes. The samples produced highly reproducible diffraction patterns in which each peak had a signal-to-noise ratio of >10³.

X-Ray Diffraction Data Collection and Analysis
Small-angle x-ray diffraction analyses were carried out by aligning the oriented membrane samples at grazing incidence with respect to the high-brilliance x-ray beam. The radiation source was a collimated, monochromatic x-ray beam (CuK =1.54 Å) from a Rigaku RU200 rotating-anode microfocus generator. The fixed-geometry beam line utilized a single Franks mirror, providing nickel-filtered radiation (K₁ and K₂ unresolved) at the detection plane. Coherent scattering from the curved samples was recorded on a 1-dimensional, position-sensitive electronic detector (Innovative Technologies) that had been calibrated with cholesterol monohydrate crystals. The sample-to-detector distance used in these experiments was 150 mm.

The unit cell periodicity, or d space, of the membrane is the measured distance from the center of 1 membrane bilayer to the next, including surface hydration. The d space for the membrane multilayer samples was calculated from Bragg's Law, n=2dsin, in which n is the diffraction order number (assumed to be 1), is the wavelength of radiation (1.54 Å), and is the Bragg angle equal to 1/2 of the angle between the incident beam and the scattered beam.

Cellular Cholesterol Quantification
J774 macrophages were treated as described under Cell Culture. To terminate the incubation, the treatment medium was removed and the monolayer washed 3 times with cold PBS. The lipids were extracted from the monolayers with isopropanol with the addition of cholesteryl methyl ether as an internal standard. Unesterified (FC) and total cholesterol was quantitated by gas-liquid chromatography as previously described.¹⁶ After lipid extraction, the cell monolayers were solubilized in SDS, and protein was determined by Lowry assay, as modified by Markwell et al.²⁰

Cellular Toxicity
Cellular toxicity was assessed through the release of adenine as described by Shirhatti and Krishna.²¹ After the 48-hour cholesterol-enrichment period, cultured macrophages were incubated with 1.0 µCi of [³H]adenine in RPMI containing 0.2% BSA for 2 hours. This medium was removed, and the monolayers were then equilibrated for 10 minutes in RPMI containing 0.2% BSA. After equilibration, a treatment medium was added for 24 hours unless otherwise indicated. After this time, 150 µL of medium was removed and filtered (Multiscreen filtration system, Millipore Corp). Aliquots were analyzed for the release of cellular tritium.

	Results

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Microscopy of FC Crystallization in MPMs
CE-loaded MPMs were incubated with or without CP-113,818 for up to 5 days. Crystals were visible by light microscopy in the ACAT-inhibited cells only at times >30 hours (Figure 1). The crystals appeared as plates, needles, and helixes. Quantitation of crystal number in each sample (Figure 2) showed that the number of plates increased over time after ACAT inhibition. After 72 hours, there was a >3.5-fold increase in the number of plates seen compared with those present at 6 hours. Occasionally, plates were also seen in the non–ACAT-inhibited samples, but these numbers remained small and constant throughout the duration of the experiment. Cholesterol needles were less numerous than plates, with only 8 needles seen in all examined fields after 12 hours of ACAT inhibition. At 72 hours, this number had increased to 25. As with plates, however, in non–ACAT-inhibited samples the number of needles was low and remained constant (only 2 or 3 per prepared slide).

View larger version (0K): [in this window] [in a new window]   Figure 1. Light photomicrograph showing the various shapes of FC crystals appearing in model MPM foam cell incubations: plate (A), rod (B), and helix (C). MPMs were loaded with cholesterol by using acLDL and FC/phospholipid dispersions as described in Methods and then treated for 30 hours with the ACAT inhibitor CP-113,818 (2 µg/mL).

View larger version (0K): [in this window] [in a new window]   Figure 2. Effect of the ACAT inhibitor CP-113,818 on the number of cholesterol plates seen in each treatment group. MPMs were treated as described in the legend to Figure 1. In the presence of the ACAT inhibitor (), the number of plates increased, reaching a peak at 48 hours. In contrast, without the ACAT inhibitor (), the number of plates remained low and relatively constant.

The size of the crystals, particularly the plates, also increased over time. Figure 3 compares the distribution of plate lengths for samples at 30 hours and 184 hours after ACAT inhibition was initiated. Whereas the majority (68%) of plates at 30 hours were <20 µm long, almost 50% of plates at 184 hours had a length >60 µm. Similar increases in width were also detected.

View larger version (0K): [in this window] [in a new window]   Figure 3. Effect of the ACAT inhibitor CP-113,818 on the distribution of plate lengths seen in MPM foam cell incubations at 30 and 184 hours of treatment with CP-113,818 (2 µg/mL).

Examination of monolayers treated for 30 hours or more by light microscopy revealed many extracellular crystals, some partially contained within cells. The abundance of extracellular crystals was confirmed by quantitative SEM. In general, the cholesterol crystals seen by SEM could be classified as either long, thin needlelike crystals (Figure 4) or as plates of cholesterol having at least 1 smooth, wide surface (Figure 5). Often, the crystals, particularly the plates, were partially covered by adherent macrophages that had extended pseudopods over the crystal surface (Figures 6A and 6B).

View larger version (0K): [in this window] [in a new window]   Figure 4. SEM photomicrograph of cultured MPM foam cells. After cholesterol loading and equilibration, the cells were incubated in the presence of the ACAT inhibitor CP-113,818 for 72 hours. In the center of the field a large, extracellular cholesterol needle is present. Magnification=x500; bar=10 µm.

View larger version (0K): [in this window] [in a new window]   Figure 5. SEM photomicrograph of cultured MPM foam cells. After cholesterol loading and equilibration, the cells were incubated in the presence of the ACAT inhibitor CP-113,818 for 24 hours. A plate of crystallized cholesterol is prominent in the center of the field. Magnification=x1600; bar=5 µm.

View larger version (0K): [in this window] [in a new window]   Figure 6. SEM photomicrograph of macrophages after 72 hours in the presence of the ACAT inhibitor. Several cells have attached to and spread over the cholesterol crystal. In addition, 2 cells near the crystal in A show a loss of membrane integrity, indicating that the cell is dead (arrowheads). Magnification=x1170; bar=5 µm. B, A cholesterol needle that appears in the center of the field of view has been colonized by multiple macrophages, which have become attached to and spread over the surface of the crystal. Magnification=x1100; bar=5 µm.

Filipin fluorescence revealed that many cells incubated with the ACAT inhibitor contained large, filipin-positive intracellular stores of unesterified cholesterol (ie, FC), as indicated by the punctate staining pattern (Figure 7A). In contrast, in the absence of the ACAT inhibitor, only cellular membranes showed positive fluorescence (Figure 7B). Despite the presence of unesterified cholesterol in the cells, TEM showed no evidence of intracellular crystals. To further confirm the lack of intracellular crystals, thick (0.5-µm) sections were observed by intermediate-voltage EM. This procedure allowed viewing of a greater cell volume and ensured that the crystals had simply not been missed owing to plane-of-section artifacts inherent in thin sections.

View larger version (0K): [in this window] [in a new window]   Figure 7. Fluorescence photomicrograph of filipin-stained MPMs after 48 hours in the presence (A) or absence (B) of an ACAT inhibitor. MPMs were treated as described in the legend to Figure 1, fixed, and stained with filipin. In addition to membrane staining, very bright fluorescence is shown associated with large, unesterified cholesterol accumulations in ACAT-inhibited cells. Only membrane staining is evident in cells with no ACAT inhibitor. Magnification=x1250; bar=4 µm.

FC Crystallization in J774 Macrophages
CE-loaded J774 macrophages were incubated under the same conditions as the MPMs to induce FC crystallization. This protocol failed to produce FC crystals in the J774 cells.¹¹ However, cAMP analogues such as CPT-cAMP have been shown to increase the rate of CE hydrolysis in these cells.²² Therefore, treatment with both CPT-cAMP and CP-113,818 should maximize FC accumulation in J774 cells. Table 1 demonstrates that the FC level in cells treated with CP-113,818 plus CPT-cAMP was 40% greater than the FC level in cells treated with the ACAT inhibitor alone. Interestingly, after 3 days of combined treatment with CPT-cAMP and CP-113,818, FC crystals were visible by light microscopy. Similar to previous studies, however, treatment with CP-113,818 alone did not stimulate FC crystallization (data not shown). In addition, treatment with CP-113,818 and/or CPT-cAMP inhibited cell growth, as evidenced by cellular protein values (Table 1).

View this table: [in this window] [in a new window]   Table 1. Cholesterol Content in J774 Macrophage Foam Cells

Small-Angle X-Ray Diffraction Analysis of J774 Macrophage Cellular Membranes
Small-angle x-ray diffraction analysis was used to examine the physical state of cholesterol in J774 macrophage cellular membranes as a function of cholesterol enrichment at 20°C and 93% relative humidity. After a 31-hour exposure to CP-113,818 plus CPT-cAMP, the meridional diffraction patterns from the membrane samples were consistent with the presence of 2 separate lipid phases in the plane of a membrane bilayer (Table 2): a heterogeneous liquid-crystalline phase (66 to 67 Å) and an immiscible cholesterol monohydrate phase (34 Å). The liquid-crystalline phase was not detected at times earlier than 31 hours (Table 2). The periodicity of 34 Å corresponds to a tail-to-tail cholesterol bilayer, as the long axis of an individual cholesterol molecule is 17 Å in the crystalline state.²³ These results demonstrate a crystalline cholesterol phase at a time when crystals were visible by light microscopy.

View this table: [in this window] [in a new window]   Table 2. Effect of Cholesterol Enrichment on the Structure of J774 Macrophage Cell Membranes

Apolipoprotein Effects on FC Crystallization
During the course of these experiments, we observed that in MPM cultures, low cell density and frequent changes of the treatment medium were conducive to FC crystallization. We speculated that apoE secreted by the MPMs may have acted as an extracellular FC acceptor and prevented crystallization; both low cell density and changes of medium could dilute the concentration of apoE secreted by the cells. To test this hypothesis, CE-loaded J774 macrophages were incubated in the presence of CPT-cAMP plus CP-113,818. Some incubations also contained apoA-I or apoE. The presence of either apoA-I or apoE reduced the number of FC crystals in the incubations (Figure 8).

View larger version (0K): [in this window] [in a new window]   Figure 8. Inhibition of FC crystallization by addition of apolipoproteins to the extracellular medium. J774 macrophages were cholesterol enriched as described in Methods. After an equilibration period, the monolayers were treated for 96 hours with CPT-cAMP (0.3 mmol/L) plus CP-113,818 (2 µg/mL, medium A), medium A plus apoA-I (50 µg/mL), or medium A plus apoE (50 µg/mL). The monolayers were examined by light microscopy by 2 independent researchers. Crystals were counted in 10 random fields at x100 magnification and are reported as means. The average error in reading the slides between investigators was 10%.

Effect of ACAT Inhibition on Cellular Toxicity
Previously, we reported that inhibition of ACAT with pharmacological agents resulted in cellular toxicity in addition to FC crystal formation.¹¹ This cellular toxicity appeared directly related to the accumulation of excess FC.²⁴ Short-chain ceramides are reported inhibitors of ACAT. Progesterone also inhibits cholesterol reesterification,²⁵ although it is not clear whether the progesterone effect is due to direct enzyme inhibition or the restriction of FC substrate. In our studies, MPMs accumulated FC when treated with either c₂-ceramide or progesterone. Figure 9 demonstrates that significant toxicity accompanied this accumulation. Progesterone or c₂-ceramide plus CPT-cAMP also caused cholesterol crystal formation in J774 macrophage foam cells (data not shown).

View larger version (0K): [in this window] [in a new window]   Figure 9. Effect of c2-ceramide and progesterone on cellular toxicity as measured by [3H]adenine release. J774 macrophages were loaded with cholesterol by using acLDL and FC/phospholipid dispersions as described in Methods and then treated for 24 hours with c2-ceramide (10 µg/mL), progesterone (20 µg/mL), or 0.2% BSA as a control. *Significantly different from negative control at P<0.001.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Crystalline cholesterol is a major component of advanced lesions in humans and animal models.^{7 8 9 10} In fact, lesions with a large amount of extracellular lipid are among the most vulnerable to plaque rupture, with the associated cardiovascular event.⁴ Despite the prevalence of crystalline cholesterol in atheroma, relatively little is known about its origins, although it is thought that crystalline cholesterol arises from either extracellular lipoproteins or intracellularly derived cholesterol.^{8 11} Recently, we demonstrated that FC crystallizes in model MPM foam cells in which ACAT has been inhibited.¹¹ In this system, the only FC available is from intracellular sources; therefore, crystallization occurs at some intracellular site. We have extended these studies to characterize and examine the formation of intracellular FC crystals.

Characterization of Cholesterol Crystals
The FC crystals observed in these studies had various shapes and sizes. Crystalline cholesterol in atheroma as well as in our studies predominantly appears as plates, an indication that they are cholesterol monohydrate.^{26 27} The crystals also formed needles and helixes. A needlelike structure is indicative of anhydrous crystalline cholesterol, which forms in a nonaqueous environment.²⁷ The mechanism of needle and plate cholesterol crystal formation has been well documented.^{1 26} However, the formation of cholesterol into helixes is less understood.²⁷ Konikoff et al²⁷ reported helical and tubular cholesterol crystals in human bile. These authors suggested that the crystals take on this form either because of the chirality of added cholesterol (anhydrous or not) or that the crystals may contort into helixes because of phospholipid interactions with the crystal's surface. The presence of helical crystals in our incubations may indicate that they form in a phospholipid environment, and as they grow, phospholipid may coat a face of the crystal and prevent uniform crystal growth in all directions.

The number of crystalline plates in the ACAT-inhibited cells increased in time from 0 to 48 hours of treatment, after which the number of crystals remained constant; however, the mean crystal size continued to increase over the 184-hour treatment period. This demonstrates that extracellular crystals continue to add molecular cholesterol to their surfaces. This additional cholesterol must come from either (1) lipid pools released by dead or dying macrophages or (2) cholesterol donated to the crystal by viable macrophages whose membranes are saturated with FC. In support of the latter possibility, we observed many macrophages attached to the crystals in culture, indicating that the crystal face and cellular membrane are juxtaposed. This interaction may allow for transfer of cholesterol from cell to crystal. It is probable that FC crystals within an atheroma originate inside cells, are then expelled, and continue to grow by adding cholesterol from either cellular debris and/or FC derived from lipoproteins modified by extracellular phospholipases or CE hydrolases.

Location of Crystals
Because light-microscopic examination revealed crystals partially contained within individual cells in this system and there is no extracellular source of FC, the nucleation event likely occurs within the cell. Consistent with this concept, filipin staining indicated that there was massive intracellular FC accumulation in the CP-113,818–treated cells. Based on our EM data, once nucleation occurs, the crystal must rapidly be expelled from the cell or quickly grow too large to be contained within the cell. Moreover, quantitative examination of the crystals revealed that the extracellular crystals increase in size. This finding suggests that the cells continued to remodel the crystals even after they had been expelled.

Cholesterol domains have been previously described in model membrane systems at elevated ratios of cholesterol to phospholipid.^{28 29 30} The present studies with the use of small-angle x-ray diffraction identified crystalline domains in the plane of the membrane, suggesting that the site of nucleation may be within some cellular membrane. However, these results do not preclude the possibility that the crystalline domains detected by x-ray diffraction may be extracellular crystals trapped within layers of membranes during sedimentation. Further investigation is needed to determine the exact intracellular site of nucleation.

Factors Affecting FC Crystallization
Previously, we reported that crystals do not form in J774 macrophages under the conditions that cause crystal formation in MPMs.¹¹ J774 macrophages are a rapidly dividing cell line, and the rate of CE hydrolysis is slower in J774 macrophages than in the nondividing MPMs.^{23 31} Thus, J774 macrophages would be expected to have lower FC levels. However, when CE-enriched J774 macrophage cells were incubated with a cAMP analogue, CPT-cAMP, which slows cell division and stimulates CE hydrolysis,²³ intracellular FC levels were higher than in non–cAMP-treated cells.

The cycle of cholesterol esterification, hydrolysis, and reesterification is necessary for cholesterol homeostasis and ultimately for proper cell function.²⁴ Perturbing the balance between EC and FC in macrophage foam cells by inhibiting ACAT with a pharmacological agent and forcing the accumulation of intracellular excess FC result in cell toxicity and eventually, FC crystallization. We have investigated additional factors that modulate intracellular FC concentrations in model foam cells and measured their impact on FC crystal formation.³² During the course of the experiments with MPM foam cells, which secrete apoE, we observed that low cell density and frequent medium changes promoted crystal formation. Both of these parameters serve to dilute any secreted apoE, thus reducing its ability to act as an acceptor and preventing the removal of cellular FC.³³ Furthermore, our studies indicate that exogenously added apolipoproteins (human apoA-I and E) inhibit crystal formation, presumably by removing excess intracellular FC and preventing a nucleation event from occurring.

Two physiologically relevant compounds that directly inhibit the ACAT enzyme,^{34 35} c₂-ceramide and progesterone,²⁵ were used in our model macrophage foam cell system to determine their effect on cellular toxicity and FC crystal formation. We found that incubation of J774 macrophage foam cells with c₂-ceramide or progesterone caused cellular toxicity, and, when coincubated with CPT-cAMP, resulted in FC crystal formation. The effect of progesterone on cellular CE metabolism is complex. It appears that progesterone can both inhibit ACAT directly^{35 36} and inhibit transport of cholesterol to the enzyme.^{37 38 39} In the present study, progesterone at a concentration of 20 µg/mL in J774 macrophage foam cells caused toxicity. In contrast, we previously found that cotreatment of MPM foam cells with progesterone at a concentration of 5 µg/mL, together with a pharmacological ACAT inhibitor, actually prevented the toxicity seen when cells were treated with an ACAT inhibitor only.¹⁶ Furthermore, in the present studies, progesterone alone at 5 µg/mL did not cause any cellular toxicity when incubated with J774 macrophage foam cells (data not shown). A possible explanation for this apparent dichotomy is that progesterone may affect different metabolic processes when present in different concentrations. For example, at lower concentrations (5 µg/mL), progesterone may inhibit transport of FC generated from the hydrolysis of CEs to the pool of cholesterol that is causing toxicity, and at higher concentrations (20 µg/mL), progesterone may inhibit ACAT directly and cause massive accumulation of FC, resulting in toxicity.

In summary, we can reproducibly induce formation of FC crystals in 2 macrophage foam cell models by increasing intracellular FC levels. Perturbation of cholesterol homeostasis by adding an exogenous ACAT inhibitor promotes cholesterol crystallization, and cholesterol crystal formation can be produced by exposure of foam cells to physiologically relevant inhibitors of ACAT. Our studies do not preclude the possibility that extracellular, lipoprotein-derived cholesterol may contribute to cholesterol crystallization within the atheroma. However, even if this were to occur, lesion macrophages may act to expand these crystals, just as they appear to do for the macrophage-produced crystals in our studies. We hypothesize that within the lesion, where concentrations of extracellular cholesterol acceptors may be low, initial nucleation of cholesterol occurs within foam cells, followed by expulsion of the crystal, cell death, and subsequent crystal growth by the addition of molecular cholesterol from cellular and extracellular sources.

	Acknowledgments

 
This study was supported by National Institutes of Health (Bethesda, Md) grants HL22633 (to M.C.P. and G.H.R.), HL07443 (to G.K.-W.), and RO1-HL49148 (to W.G.J.); and American Heart Association, Southeastern Pennsylvania Affiliate Grant 198305E (to G.K.-W.) and North Carolina Affiliate Grant 97-FW05 (to P.G.Y.). Additional funding from Pfizer Central Research (to G.H.R.) is also gratefully acknowledged. The assistance of Dr M. Page Haynes of MCP Hahnemann University and of Ken Grant and Paula Moore of the Wake Forest University School of Medicine EM laboratory is gratefully acknowledged. Ultraviolet fluorescence microscopy was carried out in Dr Mark Willingham's laboratory at Wake Forest University School of Medicine.

Received October 28, 1998; accepted January 22, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 
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