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(Circulation. 2001;103:1142.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Evidence of Macrophage Foam Cell Formation by Very Low-Density Lipoprotein Receptor

Interferon- Inhibition of Very Low-Density Lipoprotein Receptor Expression and Foam Cell Formation in Macrophages

Shotaro Kosaka, MD; Sadao Takahashi, MD, PhD; Katsuhiko Masamura, MD; Hideo Kanehara, MD; Juro Sakai, MD, PhD; Gen Tohda, MD, PhD; Eiko Okada, MD, PhD; Koji Oida, MD, PhD; Tadao Iwasaki, MS; Hiroaki Hattori, PhD; Tatsuhiko Kodama, MD, PhD; Tokuo Yamamoto, PhD; Isamu Miyamori, MD, PhD

From the Third Department of Internal Medicine (S.K., S.T., K.M., H.K., G.T., E.O., K.O., I.M.), Fukui Medical University, Fukui, Japan; Second Department of Internal Medicine (J.S.), Tohoku University School of Medicine, Sendai, Japan; Research Department (T.I., H.H.), R & D Center, BML, Inc, Kawagoe, Japan; Department of Molecular Biology and Medicine (T.K.), Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan; and Tohoku University Gene Research Center (T.Y.), Sendai, Japan.

Correspondence to Sadao Takahashi, MD, PhD, the Third Department of Internal Medicine, Faculty of Medicine, Fukui Medical University, Matsuoka-cho, Fukui, Japan. E-mail sadaost{at}fmsrsa.fukui-med.ac.jp

	Abstract

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Background—Expression of the VLDL receptor, primarily in macrophages, has been confirmed in human and rabbit atherosclerotic lesions. The high binding affinity of the VLDL receptor for remnant particles implicates the VLDL receptor pathway in the foam cell formation mechanism in macrophages. This study investigates the effect of interferon (IFN)- on VLDL receptor expression in phorbol-12-myristate-13-acetate (PMA)-treated THP-1, HL-60 macrophages, and human monocyte-derived macrophages.

Methods and Results—THP-1 cells were induced to differentiate into macrophages by PMA treatment. IFN- was added to the medium, and expression of the VLDL receptor was determined. ¹²⁵I-ß-VLDL degradation study and oil red O staining were examined. In THP-1 macrophages, VLDL receptor protein expression decreased at 2 days after PMA treatment but increased at 3 days and increased up to 5 days. Scavenger receptor proteins, which were not originally present, appeared at 3 days after PMA treatment. IFN- inhibited VLDL receptor expression in a dose-and time-dependent manner in macrophages. However, no inhibitory effect was observed in monocytes. Moreover, IFN- receptor mRNA increased during differentiation to macrophages. ¹²⁵I-ß-VLDL degradation study and oil red O staining showed that IFN- significantly inhibited foam cell formation after the uptake of ß-VLDL. LDL receptor–related protein (LRP) and LDL receptor mRNAs were not expressed in macrophages. In PMA-treated HL-60 macrophages and human monocyte-derived macrophages, IFN- also inhibited VLDL receptor expression and foam cell formation by ß-VLDL.

Conclusions—VLDL receptor expression is upregulated during monocyte-macrophage differentiation. IFN- inhibits VLDL receptor expression and foam cell formation only in macrophages. Remnant particles induce macrophage foam cell formation through the VLDL receptor pathway.

Key Words: lipoproteins • interferon- • cells • receptors • proteins

	Introduction

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VLDL receptor cDNA was cloned and characterized from rabbit heart and human THP-1 monocytic leukemia cells.^{1 2} The amino acid sequence of the VLDL receptor, which is abundant in extrahepatic tissues, is strikingly homologous with the LDL receptor.^{1 2 3} However, the physiological significance of the VLDL receptor remains unclear. The VLDL receptor binds only apoE-containing particles such as VLDL and IDL obtained from Watanabe heritable hyperlipidemic (WHHL) rabbits, as well as ß-VLDL obtained from cholesterol-fed rabbits.¹ Furthermore, VLDL from patients homozygous for apo E2/2 and apo E3/3 binds the human VLDL receptor identically.⁴ Both apoE and lipoprotein lipase (LPL), which are secreted by macrophages, accelerate the binding of triglyceride-rich lipoprotein to the VLDL receptor.⁵ It was recently reported that the atherogenic lipoprotein Lp(a) was also recognized by the human VLDL receptor.⁶ Transformation of the macrophage into a lipid-laden foam cell likely occurs due to receptor-mediated uptake of cholesterol-rich particles.⁷ VLDL receptor expression, primarily in macrophages, has been confirmed in human and rabbit atherosclerotic lesions.^{6 8 9 10} The VLDL receptor on THP-1 cells and rabbit alveolar macrophages reportedly was not downregulated by incubation with ß-VLDL.^{2 11} Moreover, incubation of ß-VLDL with LDL-deficient Chinese hamster ovary (CHO) cells (ldlA-7) transfected with the rabbit VLDL receptor enabled these cells to accumulate cholesteryl ester, resulting in foam cell formation.¹¹ 1,25-Dihydroxyvitamin D₃ induces VLDL receptor mRNA expression in HL-60 cells in association with monocytic differentiation.¹² We proposed the VLDL receptor pathway as a candidate mechanism of foam cell formation in macrophages.⁵ Activated T-lymphocytes produce interferon (IFN)- appear in early atherosclerotic lesions.¹³ In the present study, we examine the effect of IFN- on VLDL receptor expression in macrophages.

	Methods

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Materials
Human THP-1 monocytic leukemia cells, HL-60 cells, and HepG2 cells were obtained from American Type Culture Collection. A mutant CHO cells that lack LDL receptors (ldlA-7) was kindly provided by Dr Monty Krieger (Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass). Phorbol-12-myristate-13-acetate (PMA) was purchased from Wako Pure Chemical Industries. RPMI 1640, DMEM, and FBS were obtained from GIBCO BRL. Serum-free medium (SFM-101) was obtained from Nissui. IFN- was a gift of Shionogi Co Ltd. [-³²P]dCTP was purchased from Amersham Pharmacia Biotech.

Cell Culture
Cells were maintained in RPMI 1640 medium for THP-1 and HL-60 cells or DMEM for HepG2 cells supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, 100 µg/mL streptomycin sulfate, and 2 mmol/L L-glutamine in a humidified atmosphere of 5% CO₂ at 37°C.

Isolation and Culture of Human Monocyte-Derived Macrophages
Buffy coat cells were separated from the fresh anticoagulated blood of healthy normolipidemic volunteers. Mononuclear cells were then isolated through centrifugation of the diluted buffy coats in Lymphocyte separation solution (Muto Pure Chemicals Co) at 1500 rpm for 30 minutes at room temperature as described by Böyum et al.¹⁴ The cell pellets were pooled and washed twice with RPMI 1640 serum-free medium. The cells were suspended at a concentration of 1xx10⁷ cells/mL and seeded onto 100x20-mm dishes (Falcon; Becton Dickinson). The seeded cells were allowed to adhere for 60 minutes in a humidified atmosphere of 5% CO₂ at 37°C. Nonadherent cells were removed through 3 vigorous washes with RPMI 1640 serum-free medium, and then adhered cells were incubated in RPMI 1640 10% type AB serum medium. After incubation for 4 days, human monocyte-derived macrophages were used for the experiments.

Antibodies Against Human VLDL Receptor, LDL Receptor, LRP, and Class A Scavenger Receptor
A synthetic peptide, GRKAKCEPAQFQCTNGRC, which represents 1 to 18 amino acid residues in the NH₂ terminus of the human VLDL receptor, and a synthetic peptide, WPQRCRGLYVFQGDSSPC, which represents 158 to 175 amino acid residues of the human LDL receptor, were conjugated to keyhole limpet hemocyanin (Calbiochem). BALB/c mice were immunized with the conjugate in Freund’s complete adjuvant and subsequently received 6 booster injections at 2-week intervals. After the sixth booster, spleen cells of the mice were fused with Sp2/0.¹⁵ The supernatants of hybridoma cells were screened through the use of ELISA with plates coated with the synthetic peptides mentioned (50 ng/well) and through immunoblotting against membrane fraction from ldlA-7 cells expressing human VLDL or LDL receptor. Positive hybridoma cells were cloned through limiting dilution and injected into BALB/c mice. We also generated rabbit polyclonal antibody against a synthetic peptide CASVGHTYPAISVVST DDDL, which encodes carboxyl terminus of the human VLDL receptor. The specificity of monoclonal antibody VP1-5C3, polyclonal antibody VR (human VLDL receptor), and monoclonal antibody LL-8H6 (human LDL receptor) was confirmed through immunoblotting against membrane fraction from ldlA-7 cells expressing human type I VLDL receptor,² human LDL receptor,³ and human apoE receptor 2¹⁶ (data not shown). Hybridoma cells producing monoclonal anti-human LRP were purchased from American Type Culture Collection (CRL-1936). Polyclonal antibodies against human class A scavenger receptor were prepared as previously described.¹⁷

Protein Isolation and Western Blot Analysis
Membrane fractions were prepared according to a standard method.¹⁸ Cellular protein was measured according to the method of Lowry et al.¹⁹ SDS-PAGE was performed on the fractions with 7.0% slab gels that contained 0.1% SDS. Total cell proteins for THP-1, HL-60 cell (200 µg/lane), and human monocyte-derived macrophage (230 µg/lane) were applied. Proteins were transferred from the gels to Immobilon-P membranes (Millipore Corp) with Trans-Blot Cell (Bio-Rad Laboratories). The detection of antibodies was performed with a second antibody and visualized with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). We determined VLDL receptor protein with both monoclonal antibody against the NH₂ terminus and polyclonal antibody against the carboxyl terminus of VLDL receptor.

RNA Isolation and Northern Blot Analysis
Total cellular RNA was isolated according to the guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski et al.²⁰ Total RNA (15 µg) was electrophoresed in denaturing formaldehyde-agarose gel and transferred to a Zeta-probe (Bio-Rad Laboratories) filter via capillary transfer. The filter was hybridized with cDNA fragments that were labeled with [-³²P]dCTP according to the random primer method with the Bca BESTTM Labeling Kit (Takara Shuzo Co, Ltd). The VLDL receptor probe was prepared from digested human VLDL receptor cDNA.² The primers used for RT-PCR were designed on the basis of the human VLDL receptor sequence: forward primer, 5'-CTAGTCAACAACCTGAATGATG-3', nucleotides 2041 to 2062; and reverse primer, 5'-AAGAATGGCCCATGCGGCAGAA-3', nucleotides 2388 to 2409. To obtain a fragment of human IFN- receptor²¹ and LRP²² cDNA, RT-PCR was performed on total RNA prepared from THP-1 and HepG2 cells, respectively. The cDNA fragment obtained through RT-PCR was subcloned with use of the pGEM-T Vector System (Promega Corporation). The sequences of the cDNA fragments were confirmed with the dideoxy sequence.

Lipoprotein Preparation
Rabbit ß-VLDL (d<1.006 g/mL) was isolated from the plasma of Japanese white rabbits that fed a diet that contained 0.5% cholesterol for 4 weeks. Lipoproteins were subjected to second ultracentrifugation, isolated at the same density, and exhaustively dialyzed against 150 mmol/L NaCl and 0.24 mmol/L EDTA (pH 7.4).²³ Proteins were measured according to the method of Lowry.¹⁹

Oil Red O Staining and ¹²⁵I-ß-VLDL Degradation Study
THP-1 cells, HL-60 cells, and human monocyte-derived macrophages were seeded onto multiwell slides at 5x10⁴ cells (Nunc). The cells were washed 3 times with PBS, fixed with formaldehyde, and stained with oil red O and hematoxylin.^{24 125}I-ß-VLDL degradation was performed for 24 hours as previously reported.⁵ Nonspecific degradation was determined in parallel incubations in the presence of excess unlabeled ß-VLDL (x25), and specific degradation was calculated through subtracting from total degradation.

	Results

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VLDL Receptor Expression During THP-1 Cell Differentiation
To evaluate changes in VLDL receptor expression, THP-1 cells (1x10⁷ cells/dish) were incubated with PMA at a concentration of 200 nmol/L for 0 to 5 days. VLDL receptor protein levels were estimated with Western blot analysis. VLDL receptor protein expression (130 kDa for type I VLDL receptor, 105 kDa for type II VLDL receptor) decreased at 2 days after PMA treatment but increased at 3 days and continued to increase up to 5 days. Class A scavenger receptor proteins, which were not originally present, appeared at 3 days after PMA treatment (Figure 1A). THP-1 cells were incubated with various concentrations of PMA at 0 day. The VLDL receptor protein levels were determined at 2 and 5 days. The VLDL receptor protein levels were not changed in a dose-dependent manner through treatment with PMA (Figure 1B). These results showed that the transient decrease of VLDL receptor expression was due to the differentiation into macrophages.

View larger version (55K): [in this window] [in a new window]   Figure 1. A, Western blot analysis of VLDL receptor and class A scavenger receptor expression during differentiation of THP-1 cells to macrophages. B, THP-1 cells were incubated with varying concentrations of PMA (10, 50, 100, and 200 nmol/L) at 0 day. VLDL receptor protein levels were determined after 2 and 5 days.

Effect of IFN- on VLDL Receptor Expression in THP-1 Macrophages
We also investigated the effect of IFN- on VLDL receptor expression. THP-1 cells (1x10⁷ cells per dish) were incubated with PMA at concentration of 200 nmol/L for 2 days and then removed from medium and washed with PBS. PMA-treated THP-1 macrophages were then cultured with IFN- at varying times and concentrations. IFN- inhibited VLDL receptor protein expression from 6 to 24 hours, with the greatest effect noted at 12 hours (Figure 2A), and inhibited VLDL receptor mRNA (3.8 kb) expression at 6 hours (Figure 3A). THP-1 cells were subsequently incubated with varying concentrations of IFN- for 12 hours. The VLDL receptor protein levels in THP-1 macrophages were markedly inhibited in a dose-dependent manner through treatment with IFN- (Figure 2C). Under the same conditions, IFN- had no effect on THP-1 monocytes with respect to VLDL receptor protein levels (Figure 2B).

View larger version (56K): [in this window] [in a new window]   Figure 2. A, Western blot analysis of effect of IFN- on VLDL receptor expression in THP-1 macrophages. PMA-treated THP-1 macrophages (2days) were cultured with IFN- (1000 U/mL) for varying periods. B, Untreated THP-1 monocytes were cultured with IFN- for 24 hours. C, Western blot analysis of dose-dependent effect of IFN- on VLDL receptor expression in PMA-treated THP-1 macrophages for 12 hours. INF- indicates IFN-.

View larger version (44K): [in this window] [in a new window]   Figure 3. Northern blot analysis of effect of IFN- on VLDL receptor expression in THP-1 macrophages. A, PMA-treated THP-1 macrophages (2days) were cultured with IFN- at varying periods. B, Northern blot analysis of IFN- receptor expression during differentiation of THP-1 cells to macrophages. INF- indicates IFN-.

IFN- Receptor Expression During THP-1 Cell Differentiation
Next, we examined why IFN- effectively inhibited VLDL receptor expression in PMA-treated THP-1 macrophages but not untreated THP-1 cells (monocytes). We investigated changes in IFN- receptor expression during the differentiation of THP-1 cells to macrophages. THP-1 cells were incubated with PMA at a concentration of 200 nmol/L for 2 to 5 days. After PMA treatment, IFN- receptor expression increased during the differentiation to macrophages (Figure 3B).

Effect of IFN- on Foam Cell Formation in THP-1 Macrophages
VLDL receptor–mediated uptake of ß-VLDL (remnant particles) represents 1 route for lipid accumulation and foam cell transformation of macrophages. Thus, if IFN- inhibits VLDL receptor expression, this might in turn inhibit the transformation of macrophages into foam cells by ß-VLDL. We examined the role of the VLDL receptor in foam cell formation through ¹²⁵I-ß-VLDL degradation study and oil red O staining. First, THP-1 cells (5x10⁴ or 1x10⁶ cells per dish) were incubated with RPMI 1640 medium supplemented with PMA at a concentration of 200 nmol/L for 2 or 4 days. The cells were then cultured with SFM with or without IFN- for 1 day, and the medium was replaced with fresh SFM and rabbit ß-VLDL with or without IFN-. After 24 hours at 37°C, ¹²⁵I-ß-VLDL degradation study and oil red O staining showed that IFN- significantly inhibited ¹²⁵I-ß-VLDL degradation and foam cell formation by ß-VLDL in PMA-treated THP-1 macrophages. In fully differentiated THP-1 macrophages, ¹²⁵I-ß-VLDL degradation was 8 to 9 times higher than that in early differentiated THP-1 macrophages. The value of ¹²⁵I-ß-VLDL degradation in fully differentiated cells with IFN- (1000 U/mL) was 3 times higher than that in early differentiated cells without IFN-. The result was compatible in the oil red O stain study (Figures 4 and 5).

View larger version (14K): [in this window] [in a new window]   Figure 4. Effect of IFN- on 125I-ß-VLDL degradation in early differentiated PMA-treated THP-1 cells (2day) or fully differentiated PMA-treated THP-1 cells (4day). INF- indicates IFN-.

View larger version (121K): [in this window] [in a new window]   Figure 5. Oil red O staining for effect of IFN- on macrophage foam cell formation by rabbit ß-VLDL in early differentiated PMA-treated THP-1 cells (2day) or fully differentiated PMA-treated THP-1 cells (4day). Magnification x100. INF- indicates IFN-.

LRP and LDL Receptor Expression During THP-1 Cell Differentiation
LRP, a candidate for ß-VLDL receptors, was examined during the differentiation of THP-1 cells to macrophages. LRP mRNA was not detected with Northern blot analysis. HepG2 cells, which show LRP expression, were used as controls. The expression of LDL receptor, which also binds ß-VLDL, almost exclusively disappeared during differentiation to macrophages (Figure 6).

View larger version (56K): [in this window] [in a new window]   Figure 6. Northern blot analysis of LRP and LDL receptor expression during differentiation of THP-1 cells to macrophages. HepG2 cells, which express LDL receptor and LRP, were used as control.

Effect of IFN- on VLDL Receptor Expression and Foam Cell Formation in HL-60 Macrophages
We further determined the effect of IFN- in PMA-treated HL-60 macrophages. The VLDL receptor protein increased after PMA (10 nmol/L for 2 days) treatment. IFN- inhibited the VLDL receptor protein and mRNA levels (Figures 7A and 7B). Oil red O staining showed that the inhibitory effect of IFN- for foam cell formation was a dose-dependent manner (Figure 7C).

View larger version (44K): [in this window] [in a new window]   Figure 7. A, Western blot analysis of effect of IFN- (1000 U/mL) on VLDL receptor expression in PMA-treated HL-60 macrophages. B, Northern blot analysis of effect of IFN- (1000 U/mL) on VLDL receptor expression in PMA-treated HL-60 macrophages. C, Oil red O staining for dose-dependent effect of IFN- on foam cell formation by rabbit ß-VLDL in PMA-treated HL-60 macrophages. Magnification x100. INF- indicates IFN-.

Effect of IFN- on VLDL Receptor Expression and Foam Cell Formation in Human Monocyte-Derived Macrophages
We finally determined the effect of IFN- in human monocyte-derived macrophages. The VLDL receptor protein was not present in monocytes and appeared in macrophages. Otherwise, LDL receptor and LRP protein were present in monocytes and decreased in macrophages (Figure 8A). Western blot analysis and RT-PCR showed that IFN- (1000 U/mL) inhibited VLDL receptor protein and mRNA levels and slightly inhibited LRP protein, and there was little LDL receptor expression in macrophages (Figure 8B). Oil red O staining showed that monocytes did not accumulate ß-VLDL and that IFN- significantly inhibited foam cell formation by ß-VLDL in a dose-dependent manner (Figure 8C).

View larger version (49K): [in this window] [in a new window]   Figure 8. A, Western blot analysis of VLDL receptor, LDL receptor, and LRP protein and RT-PCR of VLDL receptor mRNA during differentiation (1, human monocytes; 2, human monocyte-derived macrophages after incubation for 5 days). B, Western blot analysis of VLDL receptor, LDL receptor, and LRP and RT-PCR of VLDL receptor of effect of IFN- (1000 U/mL for 24 hours) in human monocyte-derived macrophages. C, Oil red O staining for monocytes and dose-dependent effect of IFN- on foam cell formation by rabbit ß-VLDL in human monocyte-derived macrophages. Magnification x100. INF- indicates IFN-.

	Discussion

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The appearance of lipid-laden foam cells is 1 of the features of atherosclerotic plaques.^{13 25} The transformation of macrophages into lipid-laden foam cells is most likely the result of receptor-mediated uptake of cholesterol-rich particles.⁷ Familial hypercholesterolemia is a hereditary disease that caused by LDL receptor deficiency and characterized by severe atherosclerotic lesions.²⁶ This suggests that the development of atherosclerotic lesions is not related to the LDL receptor pathway. Therefore, in macrophage foam cell formation, there may be other lipoprotein receptors that mediate the uptake of cholesterol-rich particles. One of the pathways is the scavenger receptors, which mediate the uptake of acetyl LDL or oxidized LDL.^{7 27} Another possible pathway is the ß-VLDL receptor, which mediates the uptake of ß-VLDL and IDL, and there is a route for lipid accumulation and foam cell transformation of macrophages.²⁸ Clinically, several studies have reported a relationship between coronary heart disease and remnant lipoprotein disorder such as diabetes mellitus or type III hyperlipidemia.^{29 30 31}

In the present study, we investigated the VLDL receptor as a characteristic ß-VLDL receptor. First, we investigated VLDL receptor expression during the differentiation of THP-1 cells to macrophages. VLDL receptor protein expression decreased at 2 days after PMA treatment but increased at 3 days and continued to increase up to 5 days. This effect was dependent on differentiation to macrophage, not independent of the concentration of PMA. These results suggest the importance of the VLDL receptor in lipid accumulation and foam cell transformation in macrophages, as well as its possible role in the development of atherosclerotic lesions. Fujioka et al³² reported that the metabolism of chylomicron remnants by PMA-treated THP-1 macrophages did not involve the VLDL receptor. However, they reported values for only 2 days after PMA treatment. Thus, the relationship between the metabolism of chylomicron remnants and the VLDL receptor should be carried out for 3 days after PMA treatment in THP-1 cells. In addition, the role of T-lymphocytes in the development of atherosclerotic lesions remains unclear. Several studies have investigated the role of early colocalization of T-lymphocytes and macrophages in atherosclerotic lesion formation.¹³ In the present study, we found that IFN- inhibited not only VLDL receptor mRNA expression but also protein expression in macrophages. Furthermore, IFN- simultaneously inhibited the degradation and foam cell formation by ß-VLDL in PMA-treated THP-1 macrophages. However, the inhibitory effect of IFN- on foam cell formation was adequate in early differentiated macrophages. We believe that once the VLDL receptor increased in fully differentiated macrophages, the inhibitory effect of IFN- on the VLDL receptor expression never exceeded foam formation through the VLDL receptor pathway by ß-VLDL. We subsequently examined LRP and LDL receptor expressions. Northern blot analysis did not reveal LRP mRNA during this period, and LDL receptor mRNA decreased after differentiation into macrophages. IFN- inhibited VLDL receptor expression only in macrophages, not in untreated THP-1 cells (monocytes). Thus, we examined changes in IFN- receptor expression during differentiation to macrophages. IFN- receptor expression increased during the differentiation to macrophages. In PMA-treated HL-60 macrophages and human monocyte-derived macrophages, the same inhibitory effect of IFN- on the VLDL receptor expression and foam cell formation by ß-VLDL was observed. Human monocytes expressed LDL receptor and LRP, but not VLDL receptor, even though ß-VLDL could not induce foam cell formation. In contrast, human monocyte-derived macrophages started to possess the VLDL receptor and LDL receptor and LRP protein decreased. Human monocyte-derived macrophages induced foam cell formation by ß-VLDL. Furthermore, the foam cell formation was inhibited by IFN-, which also inhibited VLDL receptor expression. These results indicate that the VLDL receptor pathway is a major route for foam cell formation by ß-VLDL in macrophages. In vivo treatment with rabbit interferon purified from RK13 rabbit kidney cultures stimulated with parainfluenza-1 virus resulted in the suppression of aortic atherosclerosis in cholesterol-fed rabbits,³³ and the absence of autoantibodies and T-lymphocytes did not influence the extent of aortic atherosclerotic lesions in apoE^-/- mice.³⁴ IFN- also reportedly inhibits arterial stenosis after injury,³⁵ inhibits scavenger receptor in human monocyte-derived macrophages,³⁶ and blocks smooth muscle cell proliferation,^{37 38} indicating that IFN- has antiatherogenic properties. On the other hand, another studies have shown that IFN- possesses atherogenic effects, such as the stimulation of vascular cell adhesion molecule-1 expression in rabbit aortic endothelium,³⁹ MHC II on macrophages and vascular smooth muscle cells,^{39 40} and scavenger receptor in smooth muscle cells.⁴¹ IFN- also reportedly potentiates atherosclerosis in apoE knockout mice,⁴² and IFN- elicits arteriosclerosis in the absence of leukocytes.⁴³ We believe that the effect of IFN- on atherosclerotic lesions might depend on the stage of lesion or the cell type under investigation. The true effect of IFN- will be elucidated in the future.

In summary, we confirmed that remnant particles, with high binding affinity for the VLDL receptor, induce macrophage foam cell formation through the VLDL receptor pathway.

	Acknowledgments

 
This work was supported by the Japan Diabetes Foundation and Takeda Science Foundation. We thank Naoyo Yamaguchi for her expert technical assistance.

Received August 9, 2000; revision received September 7, 2000; accepted September 12, 2000.

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