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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:809-814.)
© 1996 American Heart Association, Inc.

Articles

Gene Expression of Acyl Coenzyme A

Cholesterol Acyltransferase Is Upregulated in Human Monocytes During Differentiation and Foam Cell Formation

Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 15, 1994.

Hongxing Wang; Sandra J. Germain; Pamela P. Benfield; Peter J. Gillies

From the DuPont Merck Research Laboratories, Cardiovascular Diseases Research, Wilmington, Del.

Correspondence to Dr Peter J. Gillies, DuPont Merck Research Laboratories, Experimental Station, PO Box 80400, Wilmington, DE 19880-0400.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract The gene expression and enzyme kinetics of acyl coenzyme A:cholesterol acyltransferase (ACAT) were investigated in human monocytes, macrophages, and foam cells. Northern blot analysis using a 1.65-kb coding region of human ACAT cDNA as the probe showed that each of the cell types exhibited four mRNA transcripts. The levels of the 4.2- and 3.7-kb ACAT transcripts were three- and sixfold higher, respectively, in macrophages than monocytes. These transcripts were expressed at the same high levels after conversion of macrophages to foam cells. In contrast, the 6.3- and 4.4-kb transcripts for ACAT were expressed at a relatively constant level in all three cell types. The expression of mRNA for glyceraldehyde phosphate dehydrogenase, the control gene in this study, was also expressed at a constant level in each of the cell types. The increase in ACAT mRNA was accompanied by changes in the kinetic properties of the enzyme. Specifically, there was a 14-fold increase in V_max and a 71% decrease in K_m with respect to oleoyl coenzyme A. Although not definitive, the concomitant changes in mRNA and V_max strongly suggest that the amount of ACAT protein increases upon conversion of monocytes to macrophages. The data show that ACAT in monocytes can be regulated by both substrate and gene expression.

Key Words: human acyl coenzyme A:cholesterol acyltransferase • monocyte • macrophage • foam cell • mRNA

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The presence of lipid-laden FCs is one of the pathological hallmarks of atherosclerosis.¹ It is generally believed that circulating MCs are a significant source of the MP-derived FCs in atherosclerotic lesions.² In addition, it is believed that the foamy phenotype of these cells is due to the excessive accumulation of cholesteryl ester in the form of large cytoplasmic lipid droplets.³ Much of the cholesteryl ester represents reesterified cholesterol formed by the action of the enzyme ACAT (EC 2.3.1.26). A dramatic increase in ACAT activity is one of the biochemical hallmarks of atherogenesis. Although well documented, the mechanisms responsible for the increase in ACAT activity in atherogenesis are poorly understood.^{4 5}

ACAT activity is dependent upon the availability of oleoyl CoA and cholesterol. Several investigators have proposed the existence of a small pool of cholesterol that directly supplies cholesterol to the catalytic site of the enzyme.^{6 7} Notably, the amount of cholesterol in this pool often limits ACAT activity. As a result of an influx of cholesterol into the cell, the amount of cholesterol in the cholesterol substrate pool increases, and ACAT activity increases accordingly.⁸ Whether changes in substrate availability can fully account for changes in ACAT activity is uncertain, and resolution of this issue has been hampered by the lack of molecular probes to study the enzyme at the level of the gene. The cDNA for human ACAT has recently been reported.⁹ We have used this new information to explore the molecular regulation of human ACAT in freshly isolated MCs, MPs (ie, MCs maintained in culture for several days), and FCs (ie, MPs incubated in the presence of Ac-LDL). The data reported here show that ACAT activity in human MCs is regulated not only by substrate availability but also by changes in gene expression.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
[³H]oleoyl CoA (444 GBq/mmol), [¹⁴C]cholesteryl linoleate (2960 MBq/µmol), and [-³²P]dCTP (111 TBq/mmol) were purchased from DuPont New England Nuclear. RPMI 1640 medium, human serum, FBS, antibiotic/antimycotic mixture, and L-glutamine were obtained from GIBCO BRL. Human serum and FBS were heat treated at 56°C for 30 minutes before use. Ficoll-Hypaque was from Pharmacia. Solvents were high-performance liquid chromatography grade, and all chemicals were of the highest grade commercially available. Unless indicated otherwise, all reagents were purchased from Sigma Chemical Co. Human leukocytes were purchased from Biological Specialty Corp. Human Ac-LDL was purchased from Biotechnology Research Institute. Silica gel–impregnated glass fiber chromatography plates were obtained from Gelman Sciences Inc. The insulin, transferrin, and selenium mixture was obtained from Collaborative Research Inc.

cDNA Probe
The human ACAT cDNA probe used for Northern blot analysis was a 1.65-kb fragment corresponding to the coding region of nucleotides 1393 through 3049 of the published cDNA sequence.⁹ The probe was synthesized by PCR amplification by using human leukocyte cDNA as the template. The amplification was performed by using a DNA thermal cycler (Perkin-Elmer Cetus Instruments). The PCR reaction mixture contained 250 µmol/L dNTPs, 25 µmol/L oligonucleotide primers, 1 µL template cDNA, 10 mmol/L Tris, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.01% gelatin, and 0.5 U AmpliTaq DNA polymerase (Perkin-Elmer Cetus). Not I and Apa I restriction sites as underlined below were included in each of the PCR primers, respectively (1138: 5'-TACGG CGT ACA ATG GTG GGT GAA GAG AAG ATG TCT C-3'; 1139: 5'-CTA CCC CTA AAA CAC GTA ACG ACA AGT CCA GGA ACG TGG-3'). PCR reactions were performed with 40 cycles of 95°C, 55°C, and 72°C for 1 minute each. The PCR product was ligated into a plasmid vector, the transformation was performed (TA Cloning Kit, Invitrogen), and the cDNA fragment inserted into the vector (clone 795) was sequenced (Sequenase 2.0, US Biochemical Corp). Sequence analysis indicated that the insert DNA was identical to the published ACAT cDNA sequence. For Northern blot hybridization, the insert was digested with appropriate restriction enzymes and then isolated by agarose gel electrophoresis from the plasmid DNA; this ensured the elimination of any nonspecific binding during the hybridization procedure.

Isolation of Total RNA and Northern Blot Analysis
Total RNA from monocytic cells was extracted by using a guanidinium thiocyanate–based procedure.¹⁰ RNA (10 µg) was denatured by heating at 65°C for 15 minutes in 0.63 mol/L formaldehyde and 38.4% formamide. The RNA was then electrophoresed on a 1% agarose gel containing 0.63 mol/L formaldehyde. The gel was soaked for 10 to 20 minutes in 0.05 mol/L NaOH and 1x SSC (0.15 mol/L NaCl and 0.015 mol/L sodium citrate) and then washed twice in 10x SSC for 20 minutes. RNA was transferred to nylon membranes (Zetaprobe, Bio-Rad Laboratories) in 10x SSC by capillary blotting and then UV–cross-linked by using Stratalinker (Stratagene). The membranes were prehybridized at 42°C for 2 hours in 50% deionized formamide, 10x SSC, 50 mmol/L sodium phosphate (pH 6.5), 10x Denhardt's reagent, 0.5% SDS, and 500 µg/mL denatured salmon sperm DNA. Hybridization was performed at 42°C overnight in 50% deionized formamide, 5x SSC, 20 mmol/L sodium phosphate (pH 6.5), 2x Denhardt's reagent, 5% dextran sulfate, and 100 µg/mL denatured salmon sperm DNA with an appropriate [³²P]-labeled cDNA probe. The cDNA probe radiolabeled with [³²P]dCTP was synthesized by random priming (Boehringer Biochemica GmbH). Blots were washed twice in 2x SSC and 0.1% SDS at room temperature and twice in 0.1x SSC and 0.1% SDS at 55°C. The blots were briefly dried and then exposed to x-ray film with intensifying screens at -70°C. The intensity of the radioactive signal was determined by phosphoimage scanning (PhosphoImager, Molecular Dynamics). After each run of the hybridization procedure for ACAT, radioactive labels were removed from the blot, which was then rehybridized with a GAPDH cDNA probe labeled with [³²P]dCTP. The removal of radioactive labels was performed by strip-washing the blot with boiling buffer (0.1x SSC and 0.1% SDS) for 10 minutes. The sizes of the transcripts were calculated by using RNA markers (0.24 to 9.49 kb) purchased from Life Technology Inc.

Cell Isolation and Culture Conditions
Human lymphocytes were obtained by hemapheresis/plasmapheresis of 3 L of blood from healthy, medication-free donors. Mononuclear cells were isolated from the sample on the same day.¹¹ After isolation on a Ficoll-Hypaque gradient for 30 minutes at 1500g, mononuclear cells were collected from the interface. MCs were obtained by elutriation by using a Beckman J-6M elutriator according to the manufacturer's instructions. Purity was approximately 93%. For MC-derived MP and FC preparations, mononuclear cells obtained from the Ficoll-Hypaque gradient were plated at a density of 1.5 to 2.0x10⁶ cells/cm² in growth media. The growth media consisted of RPMI 1640 containing 2 g/L sodium bicarbonate, 5 mg/mL insulin, 5 mg/mL transferrin, 5 ng/mL selenious acid, and a 1% antibiotic/antimycotic mixture (penicillin and streptomycin sulfate [10 000 U/mL each], amphotericin B [25 µg/mL]), 10% FBS, and 10% pooled human serum. After a 3-hour incubation at 37°C in a humidified cell-culture incubator maintained at 5% CO₂, the medium was removed, and the monolayer of adherent cells was washed three times with phosphate-buffered saline containing 10% FBS. The nonmonocytic cells were washed away during this procedure. Fresh medium was added to MCs, which were then maintained in culture for 7 days; the medium was replaced every other day. On day 6, the differentiated MCs were changed to growth medium devoid of human serum and used as MPs on day 7. FCs were formed from day-6 MPs incubated 18 hours with 100 µg/mL Ac-LDL. The uptake of Ac-LDL by MPs and subsequent formation of lipid droplets in the cells were confirmed by oil red O staining.

Preparation of Subcellular Fractions
To prepare MPs and FCs, cells were scraped from plates and pelleted by centrifugation for 10 minutes at 200g. Cells were resuspended in a buffer of 0.1 mol/L Tris-HCl, pH 7.4, 2.0 mmol/L EDTA, and 2.0 mmol/L glutathione. Cells were disrupted by sonication (three 10-second bursts with a Virsonic ultrasound probe set at 15%) and then homogenized with five strokes of a ground-glass pestle. Sucrose was added to the homogenate to give a final concentration of 0.25 mol/L. The homogenate was then centrifuged at 1000g for 5 minutes to remove unbroken cells and nuclei. Upon assay, this fraction contained <5% of the total ACAT activity observed in the different monocytic preparations. The pellet obtained by centrifugation of the postnuclear supernate at 105 000g for 60 minutes was resuspended in assay buffer (0.1 mol/L potassium phosphate buffer, pH 7.4, containing 2 mmol/L glutathione) and used as the protein source for the ACAT assay.

ACAT Assays
At the outset of these studies, it was first verified that the rate of reaction as measured in each of the different monocytic preparations was linear with respect to protein and assay time. Given the low level of ACAT activity found in freshly isolated MCs, it was preferable to use more protein and to run the assay longer than was necessary for MP and FC preparations. This ensured sufficient incorporation of radiolabeled oleoyl CoA into cholesteryl ester to allow for a statistically significant determination of radioactivity. For the determination of ACAT activity in MCs, 100 µg protein was used; for MPs and FCs, 20 µg sufficed. Protein was determined by using a protein dye-binding assay.¹² Prior to the addition of oleoyl CoA, exogenous cholesterol suspended in Triton WR-1339 was incubated with the protein for 30 minutes at 37°C to saturate ACAT. The addition of 40 µg cholesterol was more than adequate to ensure saturation of ACAT activity. Under these conditions, ACAT activity increased approximately fivefold in MCs, threefold in MPs, but little at all in FC preparations compared with the activity observed in the absence of exogenous cholesterol. A substrate curve for oleoyl CoA was generated for each of the different monocytic preparations under saturating concentrations of cholesterol. In all the assays, oleoyl CoA was complexed 1:1 with bovine serum albumin to circumvent inhibition of enzyme activity due to the detergent effect of the acyl CoA. In the standard assay, the reaction was initiated by the addition of 150 µmol/L [³H]oleoyl CoA (11470 MBq/mmol) and run 30 minutes for MC preparations or 5 minutes for MP and FC preparations (final reaction volume, 200 µL). The reaction was stopped by adding 4 mL chloroform/methanol (2:1). An internal standard of [¹⁴C]cholesteryl linoleate (0.17 kBq) plus 1 mg/mL each of cholesterol, cholesteryl oleate, oleic acid, and triolein were added as carrier. Lipids were extracted by using standard procedures.¹³ Lipid classes were fractionated on thin-layer chromatography sheets by using a mobile phase of hexane/diethyl ether/glacial acetic acid (170:30:1). Lipid bands were visualized by staining with iodine vapor. The amount of cholesteryl [³H]oleate formed was quantified by using standard liquid scintillation counting techniques. The experimental rationale for the various assay conditions is available.⁵

Calculation of Enzyme Kinetic Parameters
The calculation of Michaelis Menten parameters was done by using a nonlinear least-squares regression analysis based on the sequential application of Nelder-Mead simplex and Marquardt algorithms. The computer program used for these calculations (EZ-FIT) was specifically designed for studies of enzyme kinetics.¹⁴ V_max and K_m were calculated from fitted curves that passed a Runs test and the Akaike information criterion for goodness of fit and a Student's t test to show the parameters were significantly different from zero. SEs were calculated as linear approximations of parameter dispersion rather than actual variation in the fitted parameter.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Comparison of ACAT mRNA Levels and ACAT Activity in MCs, MPs, and FCs
Fig 1 shows a Northern blot analysis with the ACAT probe that compares freshly isolated MCs with MPs maintained in culture for 7 days and MPs maintained in culture for 6 days that were then converted to FCs on the seventh day of the experiment. The ACAT probe revealed four bands representing transcripts of 6.3, 4.4, 4.2, and 3.7 kb; these values were calculated by comparison with RNA standards run in the same experiment. The expression of mRNA for GAPDH was similar in the three monocytic preparations; in contrast, the expression of the ACAT mRNAs was much higher in MPs and FCs than in MCs. Fig 2 provides a quantitative comparison of the mRNA levels based on phosphoimage scanning of the Northern blots from four separate experiments. The intensities of the 4.2- and 3.7-kb transcripts were approximately three- and sixfold higher, respectively, in MPs than MCs; MPs and FCs exhibited similar intensities. In contrast, the intensities of the 6.3- and 4.4-kb transcripts and the mRNA level for GAPDH were similar in all the cells.

View larger version (28K): [in this window] [in a new window]   Figure 1. Freshly isolated human mononuclear cells were plated in RPMI 1640 medium containing 10% pooled human serum and 10% FBS. After a 3-hour incubation, the cells were washed three times with phosphate-buffered saline containing 1% bovine serum albumin to remove nonmonocytic cells. A portion of the adherent MCs was subjected to immediate RNA isolation procedures; the remaining MCs were maintained in culture for 7 days in the same medium as above. On day 7, the cells were washed twice with serum-free RPMI 1640 and overlaid with RPMI 1640 in the presence or absence of Ac-LDL (100 µg/mL). After incubation for another 18 hours at 37°C, the cells were washed and subjected to RNA isolation procedures. Top, Northern blot probed with [32P]-labeled human ACAT cDNA; markers on the right are the calculated sizes of the ACAT transcripts. Bottom, Northern blot from top panel was strip-washed to remove radioactive labels and then reprobed with [32P]-labeled GAPDH cDNA. Lanes 1, 2, and 3 indicate MCs, MPs, and FCs, respectively.

View larger version (29K): [in this window] [in a new window]   Figure 2. After hybridization with the [32P]dCTP-labeled ACAT cDNA probe, the ACAT transcripts were quantified by phosphoimage scanning. Bar graph shows data from four separate experiments, each done in duplicate and expressed as mean±SEM. Experiments and Northern blot analysis are described in Fig 1 legend.

In parallel studies ACAT activity was measured in MCs, MPs, and FCs (Fig 3). Cells were prepared under identical conditions to those described in Figs 1 and 2, and the assay was run under conditions of substrate saturation for both oleoyl CoA and cholesterol. The level of ACAT activity in these cells mirrored the levels of the 4.2- and 3.7-kb transcripts shown in Fig 2. ACAT activity was approximately 16-fold higher in MPs than MCs, with no further increase in activity observed after conversion of MPs to FCs.

View larger version (57K): [in this window] [in a new window]   Figure 3. Cells were prepared and ACAT activity was determined at the times described in the Fig 1 legend. The ACAT assay was run under saturating conditions for both oleoyl CoA and cholesterol. Data are expressed as mean±SEM for n=5 donors. CE indicates cholesteryl ester.

Time Course of ACAT mRNA Expression in Culture
Northern blot analyses were performed on cells at selected times for up to 160 hours in culture to explore the increase in ACAT mRNA as MCs differentiate into MPs. The results of these studies are presented qualitatively in Fig 4 and quantitatively in Fig 5. After a lag period of approximately 40 hours, the expression levels of the 4.2- and 3.7-kb ACAT transcripts steadily increased over time; there was no attenuation in the rate of rise for up to 160 hours. In contrast, the expression levels of the 6.3- and 4.4-kb ACAT transcripts and the message level for GAPDH were relatively constant over the course of the experiment.

View larger version (46K): [in this window] [in a new window]   Figure 4. Mononuclear cells were plated in RPMI 1640 medium containing 10% human serum and 10% FBS. After a 3-hour incubation, the cells were washed three times with phosphate-buffered saline containing 1% bovine serum albumin to remove nonadherent cells. A portion of the adherent cells was subjected to immediate RNA isolation procedures; this portion was used in the lane labeled hour 0. The remaining MCs were maintained in culture for the times indicated and then subjected to RNA isolation procedures. Top, Northern blot probed with [32P]-labeled human ACAT cDNA; markers on the right are the expected sizes of the ACAT transcripts. Bottom, Northern blot from top panel was strip-washed to remove radioactive labels and then reprobed with [32P]-labeled GAPDH cDNA.

View larger version (17K): [in this window] [in a new window]   Figure 5. Line graph. Experiments and Northern blot analysis are described in Fig 4 legend. Data are from three separate experiments, each done in duplicate and expressed as mean±SEM.

ACAT Kinetics
Given the differences observed in the expression of the ACAT transcripts in MCs compared with MPs and FCs, studies were undertaken to look for possible differences in the kinetic properties of ACAT as well. Thus, ACAT activity was measured under saturating conditions of cholesterol but with variable concentrations of oleoyl CoA to enable the calculation of the Michaelis Menten parameters V_max and K_m. Although the kinetic parameters were calculated by using a nonlinear least-squares regression analysis of the raw data to circumvent the biases associated with graphical techniques, the results of the study are presented in a standard Lineweaver-Burk format in Fig 6. For oleoyl CoA, there was a 14-fold increase in V_max and a 71% decrease in K_m in MPs and FCs compared with MCs. Similar studies using cholesterol as the variable substrate were not conducted because the true concentration of cholesterol in the ACAT substrate pool is unknown.

View larger version (18K): [in this window] [in a new window]   Figure 6. Lineweaver-Burk plot of ACAT activity in MCs, MPs, and FCs. Vmax (in picomoles cholesteryl ester per minute per milligram) and Km (in micromoles per liter) were calculated by using the kinetic computational program EZ-FIT. Note that the best-fit lines for the MP and FC data overlap. For MC data, n=8 donors; for MP and FC data, n=5 donors.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The purpose of this study was to elucidate the mechanisms responsible for the high level of ACAT activity commonly observed in FCs compared with MCs. To this end, we measured the mRNA and kinetic properties of ACAT in freshly isolated human MCs, in MCs differentiated into MPs, and in MPs converted to FCs. The results of these experiments show that the gene expression of ACAT is upregulated during the differentiation of MCs into MPs and that the upregulation of the ACAT gene is associated with changes in the kinetic properties of the enzyme that favor cholesteryl ester formation.

Each of the three monocytic lineages exhibited four transcripts encoded by human ACAT cDNA, which is consistent with observations in other human tissues using the same cDNA probe.⁹ The sizes of the transcripts observed in this study are similar but not identical to those previously reported. The differences are small, however, and may simply reflect minor variations in electrophoresis conditions between laboratories or in the calculation of the kilobase values. Two of the transcripts (3.7 and 4.2 kb) increased dramatically during the differentiation of MCs into MPs. Since only the coding region of the ACAT cDNA was used as the probe for the Northern blot analysis, it is likely that these transcripts are responsible for the dramatic increase in ACAT activity observed in MPs and FCs compared with MCs. Although speculation about the significance of the various ACAT transcripts is premature, it may be germane to note that the gene for the scavenger receptor also exhibits differential expression of transcripts as MCs differentiate into MPs and form FCs.¹⁵ The scavenger receptor may serve as a paradigm for future studies on the nature and origin of the ACAT transcripts.

The data presented here provide strong evidence that ACAT in MCs is regulated at the gene level during the process of differentiation. A similar claim has been made for ACAT in epithelial cells on the basis of studies in HT29 cells, a human colon carcinoma cell line.¹⁶ Whether gene regulation is important in cells not involved in the differentiation process is less clear. For instance, gene regulation may not be a significant control mechanism in the liver and adrenal glands of normal or hypercholesterolemic rabbits.¹⁷ Further studies of specific cell types within the various organs in which ACAT is physiologically important will help to clarify this issue. A cell type of particular interest in this regard is the arterial smooth muscle cell, as this cell also plays a role in FC formation and can modulate its own phenotypic expression.¹⁸

Changes in the expression of the ACAT gene were associated with changes in the kinetic properties of the enzyme. Specifically, an increase in the mRNA for ACAT was associated with an increase in V_max and a decrease in K_m with respect to oleoyl CoA. An important caveat to the enzyme data is that the calculated values of the kinetic parameters represent averages of all the ACAT activity in the cell, and they cannot yet be ascribed to a particular transcript. Nonetheless, the kinetic analysis still affords some new insights into the regulation of ACAT. The increase in V_max is consistent with the hypothesis that increased expression of the ACAT gene leads to an increase in the amount of ACAT protein; studies using antibodies to ACAT are needed to confirm this hypothesis. The biochemical basis for the decrease in K_m is less clear; it could result from a change in the membrane environment in which the enzyme resides or a change in enzyme subtype. It is notable that the increase in message level and the change in enzymatic properties of ACAT precede rather than result from the cholesterol loading caused by Ac-LDL. In this regard, the MP may either be viewed as being primed to handle an influx of cholesterol via the scavenger receptor or as being predisposed to FC formation.

ACAT may now be added to the list of genes involved in cholesterol metabolism that are upregulated as MCs differentiate into MPs. Other genes in this category include the scavenger receptor,¹⁵ the LDL receptor–related protein,¹⁹ lipoprotein lipase,²⁰ and apoE.²¹ With the development of molecular probes for each of these genes, it now becomes possible to explore their interplay and relative roles in the regulation of FC formation.

	Selected Abbreviations and Acronyms

 

ACAT = acyl coenzyme A:cholesterol acyltransferase Ac-LDL = acetylated LDL acyl CoA = acyl coenzyme A FBS = fetal bovine serum FC = foam cell MC = monocyte MP = macrophage oleoyl CoA = oleoyl coenzyme A PCR = polymerase chain reaction SDS = sodium dodecyl sulfate SSC = saline–sodium citrate

	Acknowledgments

 
The authors wish to acknowledge the expert technical assistance of Linda Santomenna and Maureen Kearney during the course of this research.

Received August 22, 1995; accepted March 5, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW. A definition of intimal, fatty streak, and intermediate lesions of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb. 1994;14:840-856. [Abstract/Free Full Text]

2. Gown AM, Tsukada T, Ross R. Human atherosclerosis, II: immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am J Pathol. 1986;125:191-207. [Abstract]

3. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem. 1983;52:223-261. [Medline] [Order article via Infotrieve]

4. Bell FP. Arterial cholesterol esterification by acylCoA-cholesterol acyltransferase: its possible significance in atherogenesis and its inhibition by drugs. In: Fears R, ed. Pharmacological Control of Hyperlipidaemia. Barcelona, Spain: JR Prous Science Publishers SA; 1986:409-422.

5. Billheimer JT, Gillies PJ. Intracellular cholesterol esterification. In: Esfahani M, Swaney J, eds. Advances in Cholesterol Research. Caldwell, NJ: Telford Press; 1990:7-45.

6. Synouri-Yrettakou S, Mitropoulos KA. Acyl-CoA: cholesterol acyltransferase: transfer of cholesterol to its substrate pool and modulation of activity. Eur J Biochem. 1983;133:299-307. [Medline] [Order article via Infotrieve]

7. Gillies PJ, Rathgeb KA, Perri MA, Robinson CS. Regulation of acyl-CoA: cholesterol acyltransferase activity in normal and atherosclerotic rabbit aortas: role of a cholesterol substrate pool. Exp Mol Pathol. 1986;44:329-339. [Medline] [Order article via Infotrieve]

8. Gillies PJ, Robinson CS, Rathgeb KA. Regulation of ACAT activity by a cholesterol substrate pool during the progression and regression phases of atherosclerosis: implications for drug discovery. Atherosclerosis. 1990;83:177-185. [Medline] [Order article via Infotrieve]

9. Chang CY, Huh HY, Cadigan KM, Chang TY. Molecular cloning and functional expression of human acyl-coenzyme A: cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells. J Biol Chem. 1993;268:20747-20755. [Abstract/Free Full Text]

10. Wang H, Chen X, Fisher EA. N-3 fatty acids stimulate intracellular degradation of apolipoprotein B in rat hepatocytes. J Clin Invest. 1993;91:1380-1389.

11. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest. 1968;suppl 21:77-89.

12. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem.. 1976;72:248-254. [Medline] [Order article via Infotrieve]

13. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497-509. [Free Full Text]

14. Perrella F. A practical curve-fitting microcomputer program for the analysis of enzyme kinetic data on IBM-PC compatible computers. Anal Biochem. 1988;174:437-447. [Medline] [Order article via Infotrieve]

15. Geng Y, Kodama T, Hansson GK. Differential expression of scavenger receptor isoforms during monocyte-macrophage differentiation and foam cell formation. Arterioscler Thromb. 1994;14:798-806. [Abstract/Free Full Text]

16. Viallard V, Lacombe C, Nebot C, Caston I, Trocheris V. Acyl-CoA: cholesterol acyltransferase in HT29 cell subpopulations: defect of activity in the undifferentiated cells. Biochim Biophys Acta. 1991;1085:265-271. [Medline] [Order article via Infotrieve]

17. Pape ME, Schultz PA, Rea TJ, DeMattos RB, Kieft K, Bisgaier CL, Newton RS, Krause BR. Tissue specific changes in acyl-CoA: cholesterol acyltransferase (ACAT) mRNA levels in rabbits. J Lipid Res. 1995;36:823-838. [Abstract]

18. Mosse PRL, Campbell GR, Wang Z-L, Campbell JH. Smooth muscle phenotypic expression in human carotid arteries, I: comparison of cells from diffuse intimal thickening adjacent to atheromatous plaques with those of the media. Lab Invest. 1985;53:556-562. [Medline] [Order article via Infotrieve]

19. Watanabe Y, Inaba T, Shimano H, Gotoda T, Yamamoto K, Mokuno H, Sato H, Yazaki Y, Yamada N. Induction of LDL receptor–related protein during the differentiation of monocyte-macrophages: possible involvement in the atherosclerotic process. Arterioscler Thromb. 1994;14:1000-1006. [Abstract/Free Full Text]

20. Inaba T, Kawamura M, Gotoda T, Harada K, Shimada M, Ohsuga J, Shimano H, Akanuma Y, Yazaki Y, Yamada N. Effects of platelet-derived growth factor on the synthesis of lipoprotein lipase in human monocyte–derived macrophages. Arterioscler Thromb.. 1995;15:522-528. [Abstract/Free Full Text]

21. Werb Z, Chin JR. Endotoxin suppresses expression of apoprotein E by mouse macrophages in vivo and in culture: a biochemical and genetic study. J Biol Chem. 1983;258:10640-10648.

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				L. O'Rourke, L. M. Gronning, S. J. Yeaman, and P. R. Shepherd Glucose-dependent Regulation of Cholesterol Ester Metabolism in Macrophages by Insulin and Leptin J. Biol. Chem., November 1, 2002; 277(45): 42557 - 42562. [Abstract] [Full Text] [PDF]

				T. Y. Chang, C. C. Y. Chang, X. Lu, and S. Lin Catalysis of ACAT may be completed within the plane of the membrane: a working hypothesis J. Lipid Res., December 1, 2001; 42(12): 1933 - 1938. [Abstract] [Full Text] [PDF]

				K. K. Maung, A. Miyazaki, H. Nomiyama, C. C. Y. Chang, T.-Y. Chang, and S. Horiuchi Induction of acyl-coenzyme A:cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D3 or 9-cis-retinoic acid in undifferentiated THP-1 cells J. Lipid Res., February 1, 2001; 42(2): 181 - 187. [Abstract] [Full Text]

				L. Li and H. J. Pownall Regulation of Acyl-Coenzyme A:Cholesterol Acyltransferase (ACAT) Synthesis, Degradation, and Translocation by High-Density Lipoprotein2 at a Low Concentration Arterioscler Thromb Vasc Biol, December 1, 2000; 20(12): 2636 - 2642. [Abstract] [Full Text] [PDF]

				C. G. Panousis and S. H. Zuckerman Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-{gamma} J. Lipid Res., January 1, 2000; 41(1): 75 - 83. [Abstract] [Full Text]

				B.-L. Li, X.-L. Li, Z.-J. Duan, O. Lee, S. Lin, Z.-M. Ma, C. C. Y. Chang, X.-Y. Yang, J. P. Park, T. K. Mohandas, et al. Human Acyl-CoA:Cholesterol Acyltransferase-1 (ACAT-1) Gene Organization and Evidence That the 4.3-Kilobase ACAT-1 mRNA Is Produced from Two Different Chromosomes J. Biol. Chem., April 16, 1999; 274(16): 11060 - 11071. [Abstract] [Full Text] [PDF]

				J.-L. Escary, H. A. Choy, K. Reue, X.-P. Wang, L. W. Castellani, C. K. Glass, A. J. Lusis, and M. C. Schotz Paradoxical effect on atherosclerosis of hormone-sensitive lipase overexpression in macrophages J. Lipid Res., March 1, 1999; 40(3): 397 - 404. [Abstract] [Full Text]

				W. Cheng, O. D. Lau, and N. A. Abumrad Two Antiatherogenic Effects of Progesterone on Human Macrophages; Inhibition of Cholesteryl Ester Synthesis and Block of Its Enhancement by Glucocorticoids J. Clin. Endocrinol. Metab., January 1, 1999; 84(1): 265 - 271. [Abstract] [Full Text]

				A. Miyazaki, N. Sakashita, O. Lee, K. Takahashi, S. Horiuchi, H. Hakamata, P. M. Morganelli, C. C. Y. Chang, and T.-Y. Chang Expression of ACAT-1 Protein in Human Atherosclerotic Lesions and Cultured Human Monocytes-Macrophages Arterioscler Thromb Vasc Biol, October 1, 1998; 18(10): 1568 - 1574. [Abstract] [Full Text] [PDF]

				O. Lee, C. C. Y. Chang, W. Lee, and T.-Y. Chang Immunodepletion experiments suggest that acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) protein plays a major catalytic role in adult human liver, adrenal gland, macrophages, and kidney, but not in intestines J. Lipid Res., August 1, 1998; 39(8): 1722 - 1727. [Abstract] [Full Text]

				N. Khelef, X. Buton, N. Beatini, H. Wang, V. Meiner, T.-Y. Chang, R. V. Farese Jr., F. R. Maxfield, and I. Tabas Immunolocalization of Acyl-Coenzyme A:Cholesterol O-Acyltransferase in Macrophages J. Biol. Chem., May 1, 1998; 273(18): 11218 - 11224. [Abstract] [Full Text] [PDF]

				J. R. Burnett, L. J. Wilcox, D. E. Telford, S. J. Kleinstiver, P. H. R. Barrett, R. S. Newton, and M. W. Huff Inhibition of HMG-CoA Reductase by Atorvastatin Decreases Both VLDL and LDL Apolipoprotein B Production in Miniature Pigs Arterioscler Thromb Vasc Biol, November 1, 1997; 17(11): 2589 - 2600. [Abstract] [Full Text]

				H. Yang, D. Cromley, H. Wang, J. T. Billheimer, and S. L. Sturley Functional Expression of a cDNA to Human Acyl-coenzyme A:Cholesterol Acyltransferase in Yeast. SPECIES-DEPENDENT SUBSTRATE SPECIFICITY AND INHIBITOR SENSITIVITY J. Biol. Chem., February 14, 1997; 272(7): 3980 - 3985. [Abstract] [Full Text] [PDF]

				J.-B. Yang, Z.-J. Duan, W. Yao, O. Lee, L. Yang, X.-Y. Yang, X. Sun, C. C. Y. Chang, T.-Y. Chang, and B.-L. Li Synergistic Transcriptional Activation of Human Acyl-coenzyme A: Cholesterol Acyltransterase-1 Gene by Interferon-gamma and All-trans-Retinoic Acid THP-1 Cells J. Biol. Chem., June 8, 2001; 276(24): 20989 - 20998. [Abstract] [Full Text] [PDF]

				K. K. Buhman, H. C. Chen, and R. V. Farese Jr. The Enzymes of Neutral Lipid Synthesis J. Biol. Chem., October 26, 2001; 276(44): 40369 - 40372. [Full Text] [PDF]

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