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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1968-1975

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1968.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Expression of Class A Scavenger Receptor Inhibits Apoptosis of Macrophages Triggered by Oxidized Low Density Lipoprotein and Oxysterol

Hai-Sun Liao; Tatsuhiko Kodama; Yong-Jian Geng

From the Cardiovascular and Pulmonary Research Institute (H.-S.L., Y.-J.G.), Allegheny General Hospital, Pittsburgh, Pa; Molecular Biology and Medicine (T.K.), University of Tokyo, Tokyo, Japan; and the Cardiology Division (Y.-J.G.), Department of Internal Medicine, University of Texas School of Medicine at Houston.

Correspondence to Yong-Jian Geng, MD, PhD, Cardiology Division, Department of Internal Medicine, University of Texas School of Medicine at Houston, 6431 Fannin St, MSB 1.240, Houston, TX 77030. E-mail ygeng{at}heart.med.uth.tmc.edu


*    Abstract
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Abstract—The class A macrophage scavenger receptor (MSR-A) is a multifunctional trimeric glycoprotein involved in innate immune response as well as the development of lipid-laden foam cells during atherosclerosis. The MSR ligand, oxidized low density lipoprotein (oxLDL), is known to be cytotoxic to macrophages and other cell types. This study examined whether MSR mediates or modulates oxLDL-induced apoptosis. Treatment with oxLDL and its cytotoxic oxysterol, 7-ketocholesterol (7-KC), reduced viability and increased DNA fragmentation in human THP-1 cells, Chinese hamster ovary cells, and mouse peritoneal macrophages. However, cell death and DNA fragmentation were markedly diminished in the phorbol ester–differentiated MSR-expressing THP-1 cells and Chinese hamster ovary cells, with stable expression of MSR-AI after cDNA transfection when exposed to the same concentrations of oxLDL and 7-KC. Moreover, treatment with oxLDL and 7-KC induced much greater death and DNA fragmentation in MSR-A–deficient peritoneal macrophages compared with wild-type macrophages. Thus, MSR-A does not act as a receptor responsible for the apoptotic effect of oxLDL, and instead, expression of this receptor confers resistance of macrophages to the apoptotic stimulation by oxLDL and its cytotoxic lipid component. These results suggest that by preventing apoptosis, MSR-A may contribute to the long-term survival of macrophages and macrophage-derived lipid-laden foam cells in atherosclerotic lesions.


Key Words: macrophages • scavenger receptors • atherosclerosis • lipoproteins • cell death


*    Introduction
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Atherosclerosis is a chronic arterial disease with 2 life-threatening complications, myocardial and cerebral infarcts. The long-lasting process of atherogenesis involves dramatic alterations in cellularity, extracellular matrix, and lipid components of the arterial wall, resulting in intimal thickening, vessel lumen narrowing, and increased susceptibility to thrombosis. Cell proliferation has been traditionally regarded as a key factor that increases the number of cells in the arterial intima. However, analysis of proliferative markers has revealed a low degree of cell replication.1 On the other hand, recent studies have documented that deregulation of apoptosis, a form of genetically programmed cell death, occurs in atherosclerotic lesions.2 3 Imbalance between cell survival and death may contribute to dramatic alterations in cellularity of the arterial wall with atherosclerosis.

Formation of lipid-laden foam cells from macrophages and, to a less extent, from smooth muscle cells represents a landmark for atherosclerosis. The development of foam cells is mainly due to overloading of lipids, particularly cholesterol and cholesterol ester, into the cells through a scavenger receptor–mediated process. It is believed that the lipids within the foam cells largely come from the lipid-rich proteins in blood, particularly LDLs, which enter the cells after chemical modification, such as oxidation and converting to the ligand for macrophage scavenger receptors (MSRs). Three different classes of MSR have been identified.4 Among them, the class A MSR (MSR-A) is the first one to be cloned and characterized for its ligand binding specificity and capacity.5 Its expression is limited to differentiated or mature monomyeloid cells and is thus characterized for the monocyte-to-macrophage differentiation. Our previous studies have shown that circulating monocytes elaborate MSR-A at undetectable levels but that when the cells differentiate into tissue macrophages, they express high levels of MSR-A.6 7

The MSR ligand, oxidized LDL (oxLDL), has been identified as an apoptosis-promoting agent to a variety of cell types, including vascular smooth muscle cells,8 9 endothelial cells,10 11 12 and macrophages.13 14 Other modified LDLs, such as enzymatically degraded LDLs (E-LDL), can selectively induce the expression of monocyte chemotactic protein-1 and exert cytotoxic effects on human macrophages.15 16 In vitro treatment with oxLDL may activate the cysteinyl protease, caspase-3,9 which acts as an effector in the downstream caspase death cascade. However, despite the well-defined ligand-receptor relationship between oxLDL and MSR, it remains largely unclear as to whether MSR-A mediates or modulates apoptotic effects of oxLDL and associated lipids. Our recent work17 has shown that differentiated MSR-A–expressing human THP-1 macrophages become resistant to apoptosis triggered by fluoride anions, a global activator of G protein. Chinese hamster ovary (CHO) cells with stable expression of MSR-A also exhibit increased resistance to the fluoride apoptotic stimulation. Moreover, recent studies have shown that expression of a scavenger receptor–like gene, the cellular stress response gene, prevents cellular damage by UV irradiation and oxidative stress18 and that thymocytes lacking the apoptosis inhibitor of macrophages, a member of the scavenger receptor cysteine-rich domain family, are more susceptible to apoptosis.19 Thus, these data seem argue against the notion that oxLDL triggers apoptosis in an MSR-A–dependent manner.

The present study was aimed to determine (1) whether the expression of MSR-A prevents oxLDL-induced apoptosis in differentiated human THP-1 monocytic macrophages, (2) whether overexpression of MSR-A in CHO cells reduces apoptosis triggered by oxLDL and oxysterol, and (3) whether resident peritoneal macrophages from the MSR-A–null (MSR-/-) mice are more sensitive to oxLDL and oxysterol.


*    Methods
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Reagents
Free cholesterol and 7-ketocholesterol (7-KC) were purchased from Sigma Chemical Co and dissolved in dimethyl sulfoxide at a stock solution of 10 mg/mL. Native LDL and oxLDL were obtained from Perlmmune Inc. Acridine orange and ethidium bromide (nucleic acid–binding dyes) were obtained from Sigma. RPMI-1640 and F-12 medium were purchased from GIBCO. Phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma and dissolved in dimethyl sulfoxide at a stock concentration of 10 mmol/L.

Cell Culture and Treatment
THP-1 and CHO cells were obtained from American Type Culture Collection. THP-1 cells were maintained in RPMI-1640 supplemented with 10% heat-inactivated FBS, 100-U/mL penicillin, and 100 µg/mL streptomycin. CHO cells were maintained in F-12 medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. THP-1 cells were pretreated with or without 100 nmol/L of PMA, washed with PBS, and then treated with various concentrations of oxLDL and 7-KC, respectively. Control experiments were performed by using the same concentration of the vehicle, dimethyl sulfoxide.

Resident Peritoneal Macrophage Isolation and Treatment
Resident peritoneal macrophages were isolated from wild-type (MSR+/+) and homozygous MSR-deficient (MSR-/-) mice by peritoneal lavage as described previously.20 Briefly, mice injected with 0.2 mL of ketamine (100 µg/mL) underwent lavage with 8 mL of serum-free RPMI-1640 media into the peritoneal cavity. Peritoneal macrophages were obtained by centrifugation of the lavage fluid collected from the peritoneal cavity. The collected macrophages were resuspended in RPMI-1640 with 10% heat-inactivated FBS and incubated at 1x105 cells per milliliter in 8-well chamber slides for 48 hours. The cells were then stimulated with oxLDL or 7-KC. After stimulation, the cells were fixed for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining as shown below.

Cell Viability Assay
Cell viability was determined by staining with a combination of the nucleic acid–binding dyes acridine orange (10 µg/mL) and ethidium bromide (10 µg/mL) for 5 minutes on ice. After staining, viable and nonviable cells were counted on the basis of color and appearance under a fluorescence microscope. Acridine orange and ethidium bromide dyes can intercalate into DNA. Viable cells are stained with green nuclei by acridine orange, whereas nonviable cells are stained with ethidium bromide, producing a red or orange color in nuclei. At least 200 cells were counted. The percentage of dead cells was calculated by dividing the number of dead cells by total cell number.

Analysis of DNA Fragmentation
DNA was extracted from the cells stimulated by oxLDL and 7-KC in various concentrations for 24 hours in 6-well plates. After treatment, 5x106 cells were lysed in 1 mL of DNA extract solution containing 100 mmol/L NaCl, 20 mmol/L Tris-HCl (pH. 8.0), and 10 mmol/L EDTA at room temperature for 15 minutes. The cell lysates were incubated overnight with proteinase K (200 µg/mL) at 37°C, extracted for DNA with phenol/chloroform isoamyl alcohol, and precipitated with the same volume of isopropanol. Remaining RNA was removed by digestion with RNase A (25 µg/mL) at 37°C for 30 minutes. After reextraction and precipitation, DNA concentration was determined by spectrophotometer at a wavelength of 260 nmol/L. DNA at 20 µg per lane was loaded into 2% agarose gels with 0.5 µg/mL ethidium bromide and separated by electrophoresis at a constant voltage of 100 V for 1 to 2 hours at room temperature. The DNA ladder was visualized under UV light.

Total RNA Isolation and RT-PCR
Total RNA was extracted from THP-1 cells treated with or without PMA (100 nmol/L) for 48 hours by using the phenol/guanidinium thiocyanate method.21 cDNA was generated by reverse transcription (RT) from 1 µg of total RNA and a 30-cycle polymerase chain reaction (PCR) with specific primers for MSR-A types I and II (MSR-AI and MSR-AII, respectively), as described previously.6 RT-PCR for ß-actin mRNA was used as an internal control. The PCR products were analyzed by electrophoresis on 2% agarose gels containing 0.5 µg/mL ethidium bromide.

In Situ 3' End Labeling of DNA Fragments (TUNEL)
Mouse peritoneal macrophages treated with and without oxLDL or 7-KC in 8-well chamber slides for 24 hours were fixed with 3% paraformaldehyde in PBS. After they were washed in PBS, the cells were incubated with 2% H2O2 in methanol to inactivate endogenous peroxidase. Digoxigenin-conjugated dUTP was incorporated into 3' ends of DNA with TdT by using an ApoTag in situ apoptosis detection kit (Oncor, Inc). The presence of digoxigenin-labeled DNA fragments was determined by using a peroxidase-conjugated antibody against digoxigenin. The chromogenic substance diaminobenzidine was used as a substrate for visualization of the immunostaining. The percentage of TUNEL-positive cells was calculated by dividing the number of positive cells by the total cell number.

CHO Cell Line With Stable Overexpression of MSR-A
CHO cells were transfected with MSR-A type I cDNA by a standard calcium phosphate precipitate method.17 After transfection, CHO cells were selected with G-418 medium for 4 weeks. The establishment of the CHO cell line with stable expression of MSR-A was confirmed by Northern and Western blotting.

Flow Cytometry
THP-1 cells were analyzed by flow cytometry for the expression of different isoforms of MSR, such as MSR-A, CD36, and CD68. The cells were pretreated with PMA and then exposed to 25 µg/ml oxLDL at 37°C for 24 hours. After they were washed in PBS, the cells were stained with monoclonal antibodies against MSR-A, CD36, and CD68, followed by incubating with FITC-conjugated anti-mouse IgG. The immunostains were detected by using a FACScalibur flow cytometer (Becton Dickinson) with Cytoquest software.

Statistic Analysis
The experimental data were analyzed by using Excell software (Microsoft), and ANOVA was used for statistical analysis of multiple groups. Significant difference was established at a value of P<0.05.


*    Results
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 *Results
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MSR+ THP-1 Cells Resist Apoptosis Triggered by OxLDL
Human THP-1 monocytic cells expressed high levels of mRNA for MSR-AI and MSR-AII after stimulation with PMA (Figure 1Down). To determine the extent of apoptosis in the MSR+ THP-1 cells, the cells were pretreated with PMA and then treated with various concentrations of oxLDL for 24 hours. We observed that treatment with oxLDL significantly reduced the viability of MSR- and MSR+ THP-1 cells in a concentration-dependent manner, as determined by staining with acridine orange and ethidium bromide (TableDown). In contrast, under the same concentration, native LDL did not alter cell viability. Interestingly, the number of nonviable MSR+ THP-1 cells appeared significantly lower than the number of nonviable MSR- THP-1 cells ( TableDown). Because internucleosomal DNA fragmentation biochemically characterizes apoptosis, we analyzed the sizes of DNA isolated from oxLDL-treated and untreated THP-1 cells by agarose gel electrophoresis. As shown in Figure 1bDown, PMA-treated THP-1 cells showed decreased fragmentation of DNA at the internucleosomal sizes compared with the untreated MSR- THP-1 cells. Morphological changes, including cell shrinkage, blebbing, chromatin condensation, and nucleus fragmentation, also occurred, suggesting that the cells underwent apoptosis.



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  		Figure 1. RT-PCR for MSR mRNA and agarose gel electrophoresis of DNA in human THP-1 cells. Panel A, RT-PCR analysis of MSR-AI and MSR-AII mRNA in PMA-treated and untreated THP-1 cells. Total RNA was isolated from THP-1 cells treated with or without 100 nmol/L PMA. cDNA reverse-transcribed from RNA was amplified by 30-cycle PCR with a set of primers that detect MSR-AI and MSR-AII. ß-Actin primers were used as an internal control (panel a, bottom). M indicates DNA size markers; B, no template (blank). Panel B, Agarose gel electrophoresis of DNA isolated from PMA-pretreated THP-1 cells after exposure to oxLDL (200 µg/mL) for 24 hours. Lanes are as follows: 1, DNA from control cells treated with oxLDL; 2, DNA from PMA-differentiated cells treated with oxLDL; and 3, DNA from cells treated without oxLDL.


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  		Table 1. Difference in Viability of MSR+ and MSR- THP-1 Cells Treated With and Without OxLDL and 7-KC

MSR+ THP-1 Cells Resist Apoptosis Triggered by 7-KC
OxLDL contains many different lipid components, eg, lipid peroxides, hydroxides, and oxidized cholesterol derivatives (eg, oxysterol), many of which are potentially cytotoxic. We examined whether 7-KC, a major oxysterol component found in oxLDL,22 23 promotes apoptosis of THP-1 cells. We observed that similar to oxLDL, 7-KC also reduced the viability of THP-1 cells in a concentration-dependent fashion ( TableUp). Increasing concentrations of 7-KC from 5 µg/mL to 20 µg/mL led to 50% cell death ( TableUp). Thus, 7-KC appeared to exert a cytotoxic effect on THP-1 cells. The 7-KC–induced cell death occurred via an apoptotic mechanism that was evidenced by internucleosomal DNA fragmentation, as shown by the appearance of DNA ladder in agarose gel electrophoresis in the 7-KC–treated cells (Figure 2Down). In control experiments, we treated the cells with the same concentrations of free cholesterol. We observed no effect of free cholesterol on the cell viability, because it neither increased the percent cell death (4.3±0.4% in the free cholesterol–treated cells versus 3.9±0.5% in the control cells, n=3) nor caused DNA fragmentation (data not shown). To determine whether MSR-expressing cells resist apoptosis induced by the oxysterol, we analyzed the differences in viability and DNA fragmentation between MSR- and MSR+ THP-1 cells. We found that the percentage of cell death and the internucleosomal DNA fragmentation were diminished in the MSR+ cells compared with the MSR- THP-1 cells ( TableUp and FigureUp 2). These results indicate that PMA-pretreated MSR-A–expressing THP-1 macrophages were resistant to apoptosis induced by 7-KC.



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  		Figure 2. Agarose gel electrophoresis of DNA from PMA-differentiated THP-1 cells after exposure to 7-KC. DNA was extracted from THP-1 cells pretreated with (lanes 4 to 6) and without (lanes 1 to 3) 100 nmol/L PMA for 48 hours and then exposed to 7-KC for 24 hours. DNA (20 µg per lane) was loaded and fractionated on 2% agarose gels containing 0.5 µg/mL of ethidium bromide. Lanes are as follows: 1 and 4, control; 2 and 5, 10 µg/mL 7-KC; and 3 and 6, 20 µg/mL 7-KC.

To determine whether other isoforms of MSR participate in the regulation of apoptosis induced by oxLDL and oxysterols, we analyzed the expression of CD36 and CD68, 2 isoforms of MSR, in the PMA-differentiated THP-1 cells by flow cytometry after stimulation with oxLDL. We observed that compared with untreated control cells, there were no significant increases in the numbers of CD36-positive cells (11% versus 12% in control cells) and CD68-positive cells (20% versus 19% in control cells) in the PMA-pretreated cultures 24 hours after stimulation with oxLDL (50 µg/mL). Similarly, we found that the 7-KC–treated cells exhibited no changes in the expression of the 2 proteins. This suggests that MSR-A other than CD36 and CD68 contributed to the resistance of the cells to apoptosis induced by oxLDL and 7-KC.

Overexpression of MSR-A by cDNA Transfection Inhibits Apoptosis Induced by OxLDL in CHO Cells
To further confirm that expression of MSR itself prevents apoptosis induced by oxLDL and 7-KC, we recently established a CHO cell line with stable overexpressed MSR-AI by transfection with the MSR-AI cDNA. Under baseline conditions, CHO cells expressed little MSR activities, but transfection with a cDNA coding for this receptor dramatically increased MSR expression in the cells, as shown previously.5 24 Figure 3aDown demonstrates that the CHO cells with stable expression of MSR showed significantly higher viability than untransfected wild-type CHO cells when treated with the same concentrations of oxLDL. Agarose gel electrophoresis of DNA revealed a decrease in internucleosomal fragmentation of DNA in the MSR-A–expressing CHO cells compared with wild-type control cells (Figure 3bDown). These results suggest that overexpression of MSR-AI might increase the resistance of CHO cells to apoptosis induced by oxLDL.



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  		Figure 3. CHO cell death and internucleosomal DNA fragmentation after treatment with oxLDL. Wild-type and MSR-A cDNA-transfected CHO cells were stimulated with oxLDL in various concentrations for 24 hours. a, Cell viability. After they were stained with nucleic acid–binding dyes acridine orange (10 µg/mL) and ethidium bromide (10 µg/mL) for 5 minutes on ice, cells were observed under a fluorescence microscope. Cell viability was determined by dividing the number of dead cells by the total number of cells. Data indicate mean±SD of at least 3 independent experiments. *P<0.05 (ANOVA). b, Internucleosomal DNA fragmentation. DNA extracted from CHO cell transfected with or without MSR-A cDNA after exposure to oxLDL was loaded on 2% agarose gel containing ethidium bromide for electrophoresis. Lanes are as follows: 1 and 4, control; 2 and 5, 100 µg/mL oxLDL; and 3 and 6, 200 µg/mL oxLDL.

Expression of MSR by cDNA Transfection Inhibits Apoptosis Induced by 7-KC in CHO Cells
To determine whether MSR expression confers resistance of CHO cells to apoptosis induced by the oxysterol, 7-KC, we examined the viability of wild-type and MSR-AI–transfected CHO cells after treatment with 7-KC. We observed a concentration-dependent increase in the percentage of cell death in untransfected wild-type cells treated with 7-KC for 24 hours (Figure 4aDown). However, the percentage of cell death was much lower in the transfected cells than in the wild-type control cells. Agarose gel electrophoresis also showed a reduction in DNA cleavage in the CHO cells with overexpressed MSR (FigureUp 4b). Thus, the MSR-overexpressing CHO cells were also resistant to apoptotic stimulation by 7-KC.



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  		Figure 4. CHO cell death and internucleosomal DNA fragmentation after treatment with 7-KC. a, Cell viability. CHO cells transfected with or without MSR-A cDNA were treated with 7-KC for 24 hours and stained with acridine orange and ethidium bromide. After the staining procedure, the cells were observed under a fluorescence microscope. The percentage of cell death was determined by dividing the number of dead cells by the total number of cells. Data represent mean±SD of at least 3 independent experiments. *P<0.05 (ANOVA). b, Internucleosomal DNA fragmentation. DNA extracted from 7-KC–treated and untreated CHO cells with (lanes 4 to 6) or without (lanes 1 to 3) overexpressed MSR-AI was loaded onto 2% agarose gel containing 0.5 µg/mL ethidium bromide. After electrophoresis, lanes were as follows: 1 and 4, untreated control; 2 and 5, 10 µg/mL 7-KC; and 3 and 6, 20 µg/mL 7-KC.

MSR-A–Null Macrophages Exhibit Increased Sensitivity to Apoptosis Induced by OxLDL and 7-KC
To further verify the antiapoptotic effect of MSR-A on macrophages, we treated resident peritoneal macrophages isolated from wild-type (MSR+/+) and MSR-knockout (MSR-/-) mice with oxLDL and 7-KC. We observed that treatment with oxLDL led to increased numbers of cell death in MSR-/- macrophages compared with wild-type cells in a concentration-dependent manner (Figure 5Down), as determined by staining with acridine orange and ethidium bromide.25 26 In situ 3' end labeling of DNA fragments by use of the TUNEL technique demonstrated an increase in nuclear DNA fragmentation in the treated cells (Figure 6Down). There were significantly higher numbers of TUNEL-positive nuclei present in the MSR-/- cells compared with wild-type macrophages treated with the same concentration of oxLDL (Figure 7aDown). The MSR-/- macrophages also exhibited higher TUNEL positivity than did the wild-type cells when exposed to 7-KC for 24 hours (Figures 6Down and Figure 7bDown). Thus, the MSR-A deficiency appeared to increase the susceptibility of the peritoneal macrophages to apoptotic stimulation by oxLDL and the oxysterol 7-KC.



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  		Figure 5. Death of oxLDL-treated peritoneal macrophages from MSR-/- and MSR+/+ mice. Peritoneal macrophages from MSR-/- and MSR+/+ mice were treated with and without oxLDL. The numbers of nonviable cells were determined by staining with acridine orange and ethidium bromide. The percentage of dead cells was calculated by dividing the number of dead cells by the total cell number. Data represent mean±SD (n=3). *P<0.05 (ANOVA).



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  		Figure 6. In situ detection of DNA fragments by TUNEL in MSR+/+ and MSR-/- peritoneal macrophages treated with 7-KC. Wild-type (WT, a and b) and MSR-/- (c and d) peritoneal macrophages treated with 7-KC in an 8-well chamber slide were labeled with digoxigenin-conjugated dUTP and TdT by use of an ApoTag in situ apoptosis detection kit (Oncor, Inc). The presence of digoxigenin-labeled DNA fragments was determined with a peroxidase-conjugated antibody against digoxigenin, with diaminobenzidine as a substrate. a and c, Phase-contrast microscopy. b and d, Light microscopy. Note an increased number of MSR-/- macrophages with shrunken and TUNEL-positive nuclei stained brown.



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  		Figure 7. Quantification of the TUNEL positivity in MSR+/+ and MSR-/- peritoneal macrophages treated with oxLDL and 7-KC. Peritoneal macrophages treated with or without oxLDL and 7-KC, respectively, were fixed with 3% formaldehyde in PBS. TUNEL staining was performed with an ApoTag in situ apoptosis detection kit (Oncor, Inc). The percentage of TUNEL-positive cells was calculated by dividing the number of positive cells by the total cell number. a, Macrophages treated with oxLDL. b, Macrophages treated with 7-KC. Data represent mean±SD of at least 3 independent experiments. *P<0.05 (ANOVA).


*    Discussion
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 *Discussion
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As part of the innate immune system, macrophages recognize and remove denatured macromolecules, pathogenic microorganisms, and damaged tissue or apoptotic cells through their scavenger system. Despite the high frequency of exposure to those environmental cytostatic or cytotoxic factors, the lifespan of tissue macrophages remains considerably longer. This is particularly the case in the atherosclerotic lesions, in which numerous macrophages survive an extremely harsh microenvironment that contains numerous toxic substances, such as oxLDL and E-LDL.15 16 27 This mystery has led us to explore the possibility that tissue macrophages may express a protein(s) with the potential of preventing apoptosis, a form of genetically programmed cell death.

MSR, an abundant glycoprotein expressed on the surface of macrophages, mediates the uptake of chemically modified LDL by macrophages. This raises an interesting question as to whether oxLDL exerts its cytotoxic effect through the scavenger receptor pathway. In the present study, we used 3 different in vitro models to define the role for MSR-A in the modulation of macrophage apoptosis triggered by oxLDL and its cytotoxic lipid component, 7-KC. The first model uses PMA-differentiated human THP-1 cells, which express MSR-A at high levels. The second model uses a CHO cell line with stably overexpressed MSR-A. The third or last model focuses on peritoneal macrophages from the MSR-A knockout mice. Our observations clearly indicate that MSR-A does not act as a signaling receptor for oxLDL and 7-KC to achieve their cytotoxic effects. Instead, the expression of MSR-A reduces the extent of apoptosis in macrophages exposed to oxLDL and 7-KC, whereas MSR-A deficiency increases the sensitivity to apoptosis. Thus, MSR-A may function as an inhibitor of apoptosis triggered by the oxidized lipoprotein and its toxic lipid component rather than as a signal transducer responsible for the oxLDL-induced cell death. The cytotoxicity of oxLDL appears to be nonselective or independent of MSR-A, inasmuch as it causes apoptosis in a broad range of cell types, including MSR-A–negative and –positive cells.

However, we cannot exclude other scavenger receptor families, which may serve as death-signaling receptors for oxLDL. In fact, MSR-A has a relatively lower affinity to oxLDL compared with other forms of MSR, such as CD3628 and CD 68.29 30 The major ligand used for evaluating MSR activity is acetylated LDL, an artificially modified lipoprotein that may not exist in vivo. Hence, the physiological ligand for MSR-A has not been characterized at this time point. Nevertheless, the observation that oxLDL and 7-KC induce apoptosis of THP-1 and CHO cells confirms and extends previous findings8 9 by other groups that oxLDL treatment can trigger vascular cell apoptosis. Because we observed no major change in the expression of CD36 and CD68 in the PMA-differentiated THP-1 cells after stimulation with oxLDL and because oxLDL can trigger apoptosis in MSR-negative cells, it is unlikely that CD36 and CD68 are the principal contributors to the resistance of macrophages to apoptosis induced by oxLDL and oxysterols.

The mechanism underlying the inhibitory effect of MSR on oxLDL-mediated apoptosis remains to be determined. OxLDL is composed of various cytotoxic components, including oxysterols. 7-KC is the second most abundant oxysterol found in human atherosclerotic lesions after 27-hydroxycholesterol. Whether 7-KC is a major cytotoxic component in oxLDL remains to be investigated. The intracellular domains of MSR do not contain the sequences of any known signaling factor–binding sites. Although ligand transporting to lysosomes via the MSR pathway can help to remove and degrade oxLDL, the MSR-mediated endocytosis may not be the sole or major mechanism by which MSR prevents apoptosis. This notion is supported by the fact that there is a low affinity between oxLDL and MSR-A and no 7-KC binding to MSR-A. Our recent studies17 have provided an additional interpretation for the protective effect of MSR, showing that expression of MSR-A blocks apoptosis induced by the activation of membrane GTP-binding proteins. In this regard, MSR-A may participate in G-protein–mediated cross-membrane signal transduction in macrophages.

THP-1 cells, a human monocytic leukemia cell line, can differentiate into macrophage-like cells, after exposure to the transforming agent, phorbol esters. The differentiated THP-1 macrophages express high levels of MSR and behave in a manner highly similar to that of native monocyte-derived macrophages.31 PMA is a potent activator of protein kinase C. Regulation of apoptotic death by the protein kinase C pathway has been recently reported in the cells.32 33 34 35 However, in the present study, we observed no difference in cell death between PMA-treated and untreated THP-1 cells. Very recently, we have shown increased expression of apoptosis-promoting genes, such as FasL, and CPP-32 (caspase-3) in PMA-differentiated THP-1 cells, but there is no increase in the number of cells bearing markers of apoptosis.17 It is reasonable to expect that the expression of MSR-A plays a role in the regulation of apoptosis of macrophages. Nishio and Watanabe9 reported that oxysterols induced apoptosis in cultured smooth muscle cells through CPP-32 activation and bcl-2 protein downregulation. However, it is unclear whether 7-KC exerts a similar effect on macrophages and whether MSR can block the regulatory effects of 7-KC on the cells. Munn et al36 reported that macrophage colony-stimulating factor (CSF)–differentiated macrophages are resistant to apoptosis, whereas granulocyte-macrophage CSF–differentiated and interferon-{gamma}–differentiated macrophages are sensitive to apoptosis. Interestingly, macrophage CSF increases but interferon-{gamma} and granulocyte-macrophage CSF decrease the expression of MSR.37 38 39

Recently, several proteins with the MSR cysteine-rich domain (SRCR), such as cellular stress response elements18 and apoptosis inhibitors of macrophages,19 have been reported to exert protective effects on apoptosis of fibroblasts and macrophages, respectively. The mechanism underlying the protective effects of the SRCR-containing proteins is unknown. The mRNA and protein levels of MSR-AI with the SRCR domain are not appreciable in most peripheral blood monocytes, but MSR-AI expression elevates dramatically when monocytes differentiate into macrophages. The increased expression of MSR-AI concurs with the development of resistance to apoptosis in the monocyte-derived macrophages. Further investigation is needed to delineate the relationship of MSR-AI expression with the macrophage resistance to apoptosis.

It has been shown that macrophages of atherosclerotic lesions express high levels of MSR-A,6 suggesting that the expression of MSR is associated with maturation of monocytes and resistance of certain macrophages to apoptosis in atherosclerosis. Most of the plaque macrophages contain substantial amounts of lipids, mainly cholesterol, that are derived from modified lipoproteins internalized via the scavenger pathway. The increased MSR-A expression may not only promote the uptake of those lipid components but also contribute to the longevity of the lipid-rich foam cells by increasing the resistance of the cells to cytotoxic effects of oxLDL and oxysterols. Recently, Kockx and colleagues40 41 reported lower levels of apoptosis in certain atherosclerotic lesions than previously thought, suggesting that nonspecific staining for the cells undergoing apoptosis or attenuation of apoptosis might occur in the lesions. Our present data appear to support the notion that in atherosclerotic plaque, there is a mechanism that is responsible for the inhibition of apoptosis and that leads to long-term accumulation of lipid-laden macrophage-derived foam cells during atherogenesis.


*    Acknowledgments
 
This study was supported by National Institutes of the Health grant HL-59249–01 (Y.-J. Geng) and an American Heart Association, Pennsylvania affiliate, Grant-in-Aid award (Y.-J. Geng). We thank Dr Hiroshi Suzuki, Chugaii Pharmaceutical Co, Japan, for the generation of MSR-A knockout mice. We also thank Tamara Pittman and Qi Wu for their skilled technical assistance.

Received December 31, 1999; accepted January 17, 2000.


*    References
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 *References
 

  1. Gordon D, Reidy MA, Benditt EP, Schwartz SM. Cell proliferation in human coronary arteries. Proc Natl Acad Sci U S A. 1990;87:4600–4604.[Abstract/Free Full Text]
  2. Geng Y-J, Libby P. Evidence for apoptosis in advanced human atheroma: colocalization with interleukin-1ß converting enzyme. Am J Pathol. 1995;147:251–266.[Abstract]
  3. Kockx MM, De MG, Muhring J, Bult H, Bultinck J, Herman AG. Distribution of cell replication and apoptosis in atherosclerotic plaques of cholesterol-fed rabbits. Atherosclerosis. 1996;120:115–124.[Medline] [Order article via Infotrieve]
  4. Freeman MW. Scavenger receptors in atherosclerosis. Curr Opin Hematol. 1997;4:41–47.[Medline] [Order article via Infotrieve]
  5. Kodama T, Freeman M, Rohrer L, Zabrecky J, Matsudaira P, Krieger M. Type I macrophage scavenger receptor contains alpha-helical and collagen-like coiled coils. Nature. 1990;343:531–535.[Medline] [Order article via Infotrieve]
  6. Geng Y-J, 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]
  7. Kodama T, Doi T, Suzuki H, Takahashi K, Wada Y, Gordon S. Collagenous macrophage scavenger receptors. Curr Opin Lipidol. 1996;7:287–291.[Medline] [Order article via Infotrieve]
  8. Nishio E, Arimura S, Watanabe Y. Oxidized LDL induces apoptosis in cultured smooth muscle cells: a possible role for 7-ketocholesterol. Biochem Biophys Res Commun. 1996;223:413–418.[Medline] [Order article via Infotrieve]
  9. Nishio E, Watanabe Y. Oxysterols induced apoptosis in cultured smooth muscle cells through CPP32 protease activation and bcl-2 protein downregulation. Biochem Biophys Res Commun. 1996;226:928–934.[Medline] [Order article via Infotrieve]
  10. Li D, Yang B, Mehta JL. Ox-LDL induces apoptosis in human coronary artery endothelial cells: role of PKC, PTK, bcl-2, and Fas. Am J Physiol. 1998;275:H568–H576.
  11. Seelos C, Gmeiner B, Kapiotis S, Cerni C. Oxidized low-density-lipoprotein triggers programmed death of endothelial cells. Biochem Mol Biol Int. 1997;42:1015–1022.[Medline] [Order article via Infotrieve]
  12. Harada Shiba M, Kinoshita M, Kamido H, Shimokado K. Oxidized low density lipoprotein induces apoptosis in cultured human umbilical vein endothelial cells by common and unique mechanisms. J Biol Chem. 1998;273:9681–9687.[Abstract/Free Full Text]
  13. Kinscherf R, Claus R, Wagner M, Gehrke C, Kamencic H, Hou D, Nauen O, Schmiedt W, Kovacs G, Pill J, Metz J, Deigner HP. Apoptosis caused by oxidized LDL is manganese superoxide dismutase and p53 dependent. FASEB J. 1998;12:461–467.[Abstract/Free Full Text]
  14. Hardwick SJ, Hegyi L, Clare K, Law NS, Carpenter KL, Mitchinson MJ, Skepper JN. Apoptosis in human monocyte-macrophages exposed to oxidized low density lipoprotein. J Pathol. 1996;179:294–302. (Correction. 1996;180:114.)[Medline] [Order article via Infotrieve]
  15. Klouche M, Gottschling S, Gerl V, Hell W, Husmann M, Dorweiler B, Messner M, Bhakdi S. Atherogenic properties of enzymatically degraded LDL: selective induction of MCP-1 and cytotoxic effects on human macrophages. Arterioscler Thromb Vasc Biol. 1998;18:1376–1385.[Abstract/Free Full Text]
  16. Klouche M, May AE, Hemmes M, Messner M, Kanse SM, Preissner KT, Bhakdi S. Enzymatically modified, nonoxidized LDL induces selective adhesion and transmigration of monocytes and T-lymphocytes through human endothelial cell monolayers. Arterioscler Thromb Vasc Biol. 1999;19:784–793.[Abstract/Free Full Text]
  17. Liao HS, Matsumoto A, Itakura H, Pittman T, Kodama T, Geng Y-J. De novo expression of the class-A macrophage scavenger receptor conferring resistance to apoptosis in differentiated human THP-1 monocytic cells. Cell Death Differ. 1999;6:245–255.[Medline] [Order article via Infotrieve]
  18. Han HJ, Tokino T, Nakamura Y. CSR, a scavenger receptor-like protein with a protective role against cellular damage caused by UV irradiation and oxidative stress. Hum Mol Genet. 1998;7:1039–1046.[Abstract/Free Full Text]
  19. Miyazaki T, Hirokami Y, Matsuhashi N, Takatsuka H, Naito M. Increased susceptibility of thymocytes to apoptosis in mice lacking AIM, a novel murine macrophage-derived soluble factor belonging to the scavenger receptor cysteine-rich domain superfamily. J Exp Med. 1999;189:413–422.[Abstract/Free Full Text]
  20. Suzuki H, Kurihara Y, Takeya M, Kamada N, Kataoka M, Jishage K, Ueda O, Sakaguchi H, Higashi T, Suzuki T, et al. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature. 1997;386:292–296.[Medline] [Order article via Infotrieve]
  21. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987;162:156–160.[Medline] [Order article via Infotrieve]
  22. Lyons MA, Brown AJ. 7-Ketocholesterol. Int J Biochem Cell Biol. 1999;31:369–375.[Medline] [Order article via Infotrieve]
  23. Lyons TJ. Oxidized low density lipoproteins: a role in the pathogenesis of atherosclerosis in diabetes? Diabet Med. 1991;8:411–419.[Medline] [Order article via Infotrieve]
  24. Freeman MW. Macrophage scavenger receptors. Curr Opin Lipidol. 1994;5:143–148.[Medline] [Order article via Infotrieve]
  25. Geng Y-J, Hellstrand K, Wennmalm A, Hansson GK. Apoptotic death of human leukemic cells induced by vascular cells expressing nitric oxide synthase in response to gamma-interferon and tumor necrosis factor-alpha. Cancer Res. 1996;56:866–874.[Abstract/Free Full Text]
  26. Geng Y-J, Wu Q, Muszynski M, Hansson GK, Libby P. Apoptosis of vascular smooth muscle cells induced by in vitro stimulation with interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta. Arterioscler Thromb Vasc Biol. 1996;16:19–27.[Abstract/Free Full Text]
  27. Bhakdi S, Dorweiler B, Kirchmann R, Torzewski J, Weise E, Tranum-Jensen J, Walev I, Wieland E. On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety. J Exp Med. 1995;182:1959–1971.[Abstract/Free Full Text]
  28. Endemann G, Stanton LW, Madden KS, Bryant CM, White RT, Protter AA. CD36 is a receptor for oxidized low density lipoprotein. J Biol Chem. 1993;268:11811–11816.[Abstract/Free Full Text]
  29. Yamada Y, Doi T, Hamakubo T, Kodama T. Scavenger receptor family proteins: roles for atherosclerosis, host defence and disorders of the central nervous system. Cell Mol Life Sci. 1998;54:628–640.[Medline] [Order article via Infotrieve]
  30. Martinez-Pomares L, Platt N, McKnight AJ, da Silva RP, Gordon S. Macrophage membrane molecules: markers of tissue differentiation and heterogeneity. Immunobiology. 1996;195:407–416.[Medline] [Order article via Infotrieve]
  31. Auwerx J. The human leukemia cell line, THP-1: a multifaceted model for the study of monocyte-macrophage differentiation. Experientia. 1991;47:22–31.[Medline] [Order article via Infotrieve]
  32. Kizaki H, Tadakuma T, Odaka C, Muramatsu J, Ishimura Y. Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters. J Immunol. 1989;143:1790–1794.[Abstract]
  33. Day ML, Zhao X, Wu S, Swanson PE, Humphrey PA. Phorbol ester-induced apoptosis is accompanied by NGFI-A and c-fos activation in androgen-sensitive prostate cancer cells. Cell Growth Differ. 1994;5:735–741.[Abstract]
  34. Uckun FM, Schieven GL, Tuel-Ahlgren LM, Dibirdik I, Myers DE, Ledbetter JA, Song CW. Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors. Proc Natl Acad Sci U S A. 1993;90:252–256. (Correction. 1993;90:3775.)[Abstract/Free Full Text]
  35. McConkey DJ, Orrenius S, Jondal M. Cellular signalling in programmed cell death (apoptosis). Immunol Today. 1990;11:120–121.[Medline] [Order article via Infotrieve]
  36. Munn DH, Beall AC, Song D, Wrenn RW, Throckmorton DC. Activation-induced apoptosis in human macrophages: developmental regulation of a novel cell death pathway by macrophage colony-stimulating factor and interferon gamma. J Exp Med. 1995;181:127–136.[Abstract/Free Full Text]
  37. Ishibashi S, Inaba T, Shimano H, Harada K, Inoue I, Mokuno H, Mori N, Gotoda T, Takaku F, Yamada N. Monocyte colony-stimulating factor enhances uptake and degradation of acetylated low density lipoproteins and cholesterol esterification in human monocyte-derived macrophages. J Biol Chem. 1990;265:14109–14117.[Abstract/Free Full Text]
  38. Geng Y-J, Hansson GK. Interferon-gamma inhibits scavenger receptor expression and foam cell formation in human monocyte-derived macrophages. J Clin Invest. 1992;89:1322–1330.
  39. van der Kooij MA, Morand OH, Kempen HJ, van Berkel TJ. Decrease in scavenger receptor expression in human monocyte-derived macrophages treated with granulocyte macrophage colony-stimulating factor. Arterioscler Thromb Vasc Biol. 1996;16:106–114.[Abstract/Free Full Text]
  40. Kockx MM. Apoptosis in the atherosclerotic plaque: quantitative and qualitative aspects. Arterioscler Thromb Vasc Biol. 1998;18:1519–1522.[Abstract/Free Full Text]
  41. Kockx MM, Muhring J, Knaapen MW, de Meyer GR. RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis. Am J Pathol. 1998;152:885–888.[Abstract]



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