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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1961.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Low Density Lipoprotein Receptor of Macrophages Facilitates Atherosclerotic Lesion Formation in C57Bl/6 Mice

Nicole Herijgers; Miranda Van Eck; Pieter H. E. Groot; Peter M. Hoogerbrugge; Theo J. C. Van Berkel

From the Division of Biopharmaceutics (N.H., M.V.E., T.J.C.V.B.), Leiden/Amsterdam Center for Drug Research, Sylvius Laboratories, Leiden University, Leiden, the Netherlands; the Department of Vascular Biology (P.H.E.G.), SmithKline Beecham Research and Development, Harlow, Essex, UK; and the Department of Pediatric Oncology (P.M.H.), St. Radboud University Hospital, Nijmegen, the Netherlands.

Correspondence to M. Van Eck, Division of Biopharmaceutics, Sylvius Laboratories, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, Netherlands. E-mail M.Eck{at}LACDR.LeidenUniv.nl

	Abstract

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Abstract—Macrophage-derived foam cells play an important role in the initiation and progression of atherosclerosis. To examine the role of the macrophage low density lipoprotein receptor (LDLr) in atherosclerotic lesion formation, bone marrow from LDLr knockout [LDLr(-/-)] mice was transplanted into irradiated wild-type C57Bl/6 [LDLr(+/+)] mice. After 3 months on an atherogenic diet, C57Bl/6 mice, reconstituted with LDLr(-/-) bone marrow, showed a mean lesion area of 34.7x10³±22.4x10³ µm² compared with 100.8x10³±33.0x10³ µm² (P<0.001) in control C57Bl/6 mice that were transplanted with LDLr(+/+) bone marrow. There were no significant differences in total serum cholesterol, triglyceride levels, and lipoprotein profiles between the 2 groups. Histochemical analysis of macrophage LDLr expression in the atherosclerotic lesions indicated that C57Bl/6 mice, reconstituted with LDLr(+/+) bone marrow, showed extensive staining of the foam cells in the atherosclerotic lesions, whereas mice reconstituted with LDLr(-/-) bone marrow showed only a few LDLr-positive foam cells. In vitro, peritoneal macrophages isolated from wild-type C57Bl/6 mice were, respectively, 4.7- and 10.7-fold more effective in cell association and degradation of atherogenic ¹²⁵I-ß-very low density lipoprotein than were LDLr(-/-) peritoneal macrophages, establishing that the LDLr on macrophages is important for the interaction of macrophages with ß-very low density lipoprotein. It is concluded that the LDLr on macrophages can facilitate the development of atherosclerosis, possibly by mediating the uptake of atherogenic lipoproteins.

Key Words: LDL receptor • atherosclerosis • macrophages • gene transfer

	Introduction

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The LDL receptor (LDLr) plays a crucial role in the clearance of atherogenic LDL and its precursors, IDL and VLDL.¹ The LDLr is expressed in a wide variety of tissues, with the liver responsible for 70% of the total body LDLr activity.² The importance of the LDLr in lipoprotein metabolism is demonstrated in patients with the genetic disorder familial hypercholesterolemia. Familial hypercholesterolemia is characterized by the lack of functional LDLr, causing an elevation of the plasma levels of LDL and, as a result, hypercholesterolemia and premature atherosclerosis.^{3 4} Ishibashi et al⁵ developed a mouse model for homozygous familial hypercholesterolemia through targeted disruption of the LDLr gene: the LDLr(-/-) mouse. The total cholesterol level in these mice appeared to be 2-fold higher than that in their wild-type littermates [LDLr(+/+) mice], mainly because of a 7- to 9-fold increase in IDL and LDL. On a cholesterol-rich diet, these mice exhibit massive xanthomatosis and atherosclerosis.⁶ Therefore, LDLr(-/-) mice form an excellent model to investigate the role of the LDLr in lipoprotein metabolism and atherosclerosis.

Macrophage-derived foam cells play an important role in the initiation and progression of atherosclerosis.⁷ One of the best characterized mechanisms by which macrophages in the artery wall become foam cells is the uptake of modified LDL via the scavenger receptor–mediated pathway.⁸ The direct role of the LDLr on macrophages in the formation of atherosclerosis is not yet established, because it is assumed that downregulation of the LDLr by cellular cholesterol will limit its role in foam cell formation.⁹ In vitro, however, the expression of the LDLr was demonstrated on foam cells extracted from the human artery wall.¹⁰ Furthermore, it has been shown that the LDLr on cultured macrophages is involved in the internalization of atherogenic ß-VLDL, which can induce cholesteryl ester (CE) accumulation.^{11 12} We and others have recently transferred wild-type LDLr(+/+) cells into LDLr(-/-) mice via bone marrow transplantation (BMT) in vivo and investigated the effects on hypercholesterolemia and atherosclerosis.^{13 14 15} Transplantation of wild-type bone marrow into LDLr(-/-) mice resulted in a temporary decrease of total serum cholesterol. The mean atherosclerotic lesion area of these transplanted mice fed a cholesterol-rich diet was not different from that of control LDLr(-/-) mice that were transplanted with LDLr(-/-) bone marrow. We speculated that in LDLr(-/-) mice transplanted with wild-type bone marrow, the LDLr is downregulated by the relatively high concentrations of circulating cholesterol.

In the present study, we investigated the role of the macrophage LDLr under less extreme cholesterol conditions to circumvent the possible downregulation of the LDLr. Therefore, we performed the complementary experiment of transplanting LDLr(-/-) bone marrow into C57Bl/6 mice recipients. The absence of the LDLr on macrophages caused a 2.9-fold decrease of mean atherosclerotic lesion area, whereas the total serum cholesterol and triglyceride levels were not significantly different from those of the control transplanted mice.

Thus, our results indicate that the LDLr on macrophages can facilitate foam cell formation and, therefore, can contribute to the atherosclerotic process under conditions of modestly high cholesterol levels.

	Methods

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Mice
Homozygous LDLr knockout [LDLr(-/-)] mice were obtained from Jackson Laboratory, Bar Harbor, Me, as mating pairs and were bred in the Gaubius Laboratory, Leiden, the Netherlands. These mice were hybrids between the C57Bl/6 and 129 Sv strains (F4 generation of backcrosses to C57Bl/6). C57Bl/6 mice were obtained from Broekman, Someren, the Netherlands. The animals were housed in sterile filter-top cages. They were fed standard rat/mouse chow (SMR-A, Hope Farms). For the analysis of atherosclerosis, animals were fed a diet containing 1% (wt/wt) cholesterol and 0.5% (wt/wt) cholic acid as described by Nishina et al¹⁶ (diet N, Hope Farms) for up to 3 months, starting at 2 months after transplantation. The sterile drinking water contained 67 mg/L polymyxin B, 84 mg/L ciprofloxacin, 5x10⁴ mol/L HCl, and 6 g/L sugar. The experiments were approved by the ethical committee on animal experiments of the University of Leiden.

Irradiation and BMT
Female recipient mice, aged 6 to 8 weeks, were subjected to 13 Gy of total body irradiation (roentgen source). Bone marrow was harvested by flushing femurs and tibias of 8- to 10-week-old male donor mice with cold PBS. The cells were washed twice with PBS. Recipients received 1x10⁷ bone marrow cells by tail vein injection 24 hours after irradiation.

Serum Cholesterol and Triglyceride Analysis
After an overnight fasting period, small blood samples (50 µL) were obtained by tail bleeding. The concentrations of total cholesterol and triglycerides in the sera were determined by using enzymatic procedures (Boehringer-Mannheim). Precipath (standardized serum, Boehringer-Mannheim) was used as an internal standard.

The distribution of cholesterol over the different lipoproteins was determined by loading 30 µL serum of each individual mouse onto a Sepharose 6 column (3.2x30 mm, Smart-system, Pharmacia). The serum was fractionated at a constant flow rate of 50 µL/min by use of PBS containing 1 mmol/L EDTA. Twenty-eight fractions of 50 µL were collected. The fractions were assayed for their total cholesterol content.

Analysis of Atherosclerosis
The mice were killed, and the hearts and vascular trees were perfused in situ with oxygenated Krebs-Ringer bicarbonate buffer at 37°C under a pressure of 100 mm Hg for 30 minutes via a cannula in the left ventricle. The buffer was then replaced by 3.7% neutral-buffered formalin (3.7% formaldehyde, Formal-fix, Shandon Scientific Ltd), and the tissue was fixated during a perfusion of 30 minutes. Hearts and aortas were excised and stored in formalin. The hearts were bisected just below the atria, and the base of the heart and aortic root were taken for analysis. Cryostat 10-µm cross sections of the aortic root were taken and stained with oil red O as described before.¹⁷ Atherosclerotic lesions in the sections were quantified by using a light microscope connected with a video camera and by running Optimas software, version 6.1 (Bioscan Inc). Mean lesion area was calculated from the first 10 sections in the direction of the aortic arch from the point at which all 3 aortic valve leaflets first appeared as previously described.¹⁷

Immunohistochemistry
Macrophages were detected in atherosclerotic lesions by immunolocalization of the MOMA-2 macrophage marker. Formaldehyde-fixated cryostat sections were permeabilized, washed with PBS, and incubated for 1 hour in blocking buffer (PBS, 1% blocking reagent for ELISA, and 5% normal goat serum, pH 7.4). Subsequently, the sections were incubated with rat anti-mouse MOMA-2 antiserum (dilution 1:10 in blocking buffer) for 1 hour at room temperature and overnight at 4°C. After they were washed, the sections were exposed for 1 hour to goat anti-rabbit IgG conjugated to alkaline phosphatase (dilution 1:200 in blocking buffer, Sigma Chemical Co). Thereafter, sections were extensively washed with washing buffer, and MOMA-2–positive macrophages were visualized by incubation with BCIP/NBT alkaline phosphatase substrate (Sigma) in 0.2 mol/L Tris-HCl and 10 mmol/L MgCl₂, pH 9.6.

The presence of the LDLr in atherosclerotic lesions was assessed immunohistochemically with a rabbit anti-rat LDLr antiserum, which cross-reacts with the murine LDLr (kindly provided by Dr A. Cooper, Palo Alto Medical Foundation, Palo Alto, Calif). Formaldehyde-fixated cryostat sections were permeabilized, washed with PBS, and subsequently incubated for 0.5 hours with 0.3% H₂O₂ and 1 hour with blocking buffer. Subsequently, the sections were incubated with rabbit anti-rat LDLr antiserum (1:50 in blocking buffer) for 1 hour at room temperature and overnight at 4°C. After they were washed, the sections were exposed to biotinylated goat anti-rabbit IgG (dilution 1:200 in blocking buffer, Dako), washed, and incubated with biotinylated horseradish peroxidase–conjugated streptavidin (Amersham International Plc). Thereafter, sections were extensively washed with PBS, and LDLr-positive areas were visualized by incubation with 3,3'-diaminobenzidine (Sigma) as a horseradish peroxidase substrate in 0.05 mol/L Tris-HCl (pH 7.4), 7% sucrose, and 0.03% H₂O₂.

Isolation and Labeling of Lipoproteins
ß-VLDL was obtained from rats that were fed a diet containing 2% cholesterol, 5% olive oil, and 0.5% bile acid for 2 weeks. The rats were fasted overnight, after which blood was collected by puncture of the abdominal aorta. The sera were pooled and centrifuged at 250 000g in a discontinuous KBr gradient for 18 hours as reported by Redgrave et al.¹⁸ The top fraction (density <1.006 g/mL) was dialyzed against PBS containing 1 mmol/L EDTA. The composition of ß-VLDL was 14.6±2.1% triacylglycerols, 15.8±1.1% phospholipids, 49.4±3.1% CEs, 9.9±1.0% free cholesterol, and 10.3±0.7% protein. ß-VLDL was labeled with ¹²⁵I at pH 10.0 according to McFarlane,¹⁹ as described earlier.²⁰ Free ¹²⁵I was removed by Sephadex G50 gel filtration, followed by dialysis against PBS containing 1 mmol/L EDTA. Human LDL was isolated from healthy volunteers as described by Redgrave et al¹⁸ and subsequently acetylated according to Basu et al.²¹

Peritoneal Macrophage Harvesting
Five days after peritoneal injection of 1 mL of 3% Brewer thioglycollate medium (Difco), peritoneal macrophages were harvested by lavage of the peritoneal cavity with 10 mL PBS/1 mmol/L EDTA. After 3 washing steps, the cells (0.5x10⁶) were plated out in 25-mm multiwell culture dishes with DMEM (Bio-Whittaker) containing 10% FCS, 2 mmol/L glutamine, 100 µg/mL streptomycin, and 100 IU/mL penicillin. After 4 hours, the nonadherent cells were removed by washing, and the culture medium was replaced by DMEM containing 10% human lipoprotein-deficient serum, 2 mmol/L glutamine, 100 µg/mL streptomycin, and 100 IU/mL penicillin. The cells were cultured for 2 days.

Peritoneal Macrophage Metabolism Studies
ß-VLDL association and degradation studies were carried out with the indicated amounts of ¹²⁵I-ß-VLDL for 3 hours at 37°C. Incubations of the cells were performed in DMEM (Bio-Whittaker) containing 2% (wt/vol) BSA (Sigma) in a total volume of 0.5 mL. After incubation, the cells were washed 3 times with washing buffer (50 mmol/L Tris-HCl, pH 7.4, containing 0.9% NaCl, 1 mmol/L EDTA, 5 mmol/L CaCl₂, and 0.2% [wt/vol] BSA), followed by 2 washing steps with washing buffer without BSA. The cells were lysed in 0.1 mol/L NaOH, and the radioactivity was determined. Cell protein was measured by the method of Lowry et al²² with BSA as a standard. Degradation of the radiolabeled ß-VLDL was determined by precipitation with trichloroacetic acid as described by Henriksen et al.²³ Intracellular lipid accumulation in macrophages after a 24-hour incubation with 50 µg/mL ß-VLDL or 100 µg/mL acetylated LDL was determined as described by Havekes et al.²⁴

Statistical Analysis
Statistical analysis of the data was performed by unpaired Student t test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
To examine the role of the LDLr on macrophages in atherosclerotic lesion formation, we transplanted irradiated C57Bl/6 mice with the bone marrow of LDLr(-/-) mice. In this way, we created mice that are only LDLr(-/-) in their macrophages and other cells of hemopoietic origin. Previously, we showed that the method of BMT in mice results in an almost complete replacement of recipient hemopoietic cells by those of donor origin.¹³

During the weeks after BMT, the total serum cholesterol level was repeatedly determined. No significant differences between LDLr(-/-)C57Bl/6 and C57Bl/6C57Bl/6 could be observed at 4 and 8 weeks after BMT. The cholesterol level at these time points did not differ significantly from the level measured before transplantation (data not shown). To induce atherosclerotic lesion formation, the transplanted mice were fed an atherogenic diet containing 1% (wt/wt) cholesterol and 0.5% (wt/wt) cholic acid, starting at 8 weeks after transplantation. To induce atherosclerosis in this animal model, the mice were fed an atherogenic diet containing 1% (wt/wt) cholesterol and 0.5% (wt/wt) cholic acid. Although the use of such diets has been criticized for their possible inflammatory effects,²⁵ their use is commonly accepted as an experimental tool to evaluate atherosclerotic lesion development in resistant mouse strains.^{26 27 28} As a result, the total serum cholesterol levels in the control and experimental groups increased 3-fold (Figure 1). The triglyceride levels decreased 4-fold in both groups (data not shown). The distribution of cholesterol among serum lipoproteins was analyzed by liquid chromatography. The lipoprotein profiles of the 2 groups were essentially identical on a standard chow diet (Figure 2A). After 12 weeks on the atherogenic diet, in both groups, cholesterol in the VLDL and IDL/LDL fractions increased 10-fold, and there was a 2-fold reduction of cholesterol in the HDL fraction (Figure 2B). No significant differences between the lipoprotein profiles of the 2 groups were observed. To determine the effects of LDLr deficiency in macrophages on the formation of atherosclerotic lesions, the hearts and aortas of the mice were perfused and fixed after 3 months of the atherogenic diet. Cryostat cross sections of the aortic root were stained with oil red O. Representative photomicrographs of the aortic valves of control transplanted mice and mice transplanted with LDLr-deficient bone marrow are shown in Figure 3. The mean atherosclerotic lesion area in the aortic root was calculated and is presented in Figure 4. The mean±SD lesion area was 2.9-fold smaller in LDLr(-/-)C57Bl/6 mice (n=14) than in C57Bl/6C57Bl/6 mice (n=16); the respective areas were 34 680±22 401 and 100 846±33 043 µm² (P<0.001). No correlation between the individual serum cholesterol levels and the mean lesion area could be observed. Staining of the atherosclerotic lesion areas for MOMA-2, a specific marker for macrophages, revealed that in both transplantation groups, the atherosclerotic lesion area consisted primarily of lipid-filled macrophages (Figure 5A and 5B). No qualitative difference in lesion composition was observed between the 2 groups. Furthermore, serial sections of the aortic root were analyzed for the presence of the macrophage LDLr (with the use of rabbit anti-rat LDLr antiserum) that cross-reacts with the murine LDLr. Foam cells in lesions of C57Bl/6C57Bl/6 mice stained extensively for the presence of the LDLr, whereas only some foam cells stained for the presence of the LDLr in LDLr(-/-)C57Bl/6 mice (Figure 5C and 5D). Analysis of more advanced foam cell–rich lesions of C57Bl/6C57Bl/6 mice revealed that the LDLr is downregulated during the progression of lesion development (data not shown). Thus, it appears that the introduction of LDLr(-/-) bone marrow into C57Bl/6 mice results in a reduction of atherosclerotic lesion development that is due to decreased numbers of LDLr-expressing macrophages without affecting cholesterol and triglyceride levels significantly.

View larger version (24K): [in this window] [in a new window]   Figure 1. Effect of a 3-month atherogenic diet containing 1% cholesterol and 0.5% cholic acid on total serum cholesterol levels. The diet was started 2 months after BMT. Before the atherogenic diet was given, the mice were fed a standard chow diet (SMR-A). Open bars indicate C57Bl/6 mice transplanted with C57Bl/6 bone marrow (n=19); solid bars, C57Bl/6 mice transplanted with LDLr(-/-) bone marrow (n=15). Values are mean±SD. No significant differences in cholesterol values between the experimental and control mice were observed at the different time points.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of BMT on the distribution of serum lipoprotein cholesterol over various lipoprotein fractions in female C57Bl/6 mice. Mice were transplanted with C57Bl/6 (, n=19) or LDLr(-/-) (•, n=15) bone marrow. Sera of individual fasted mice were analyzed on a Pharmacia Smart column 2 months after BMT (standard chow diet, A) and 5 months after BMT (3 months on an atherogenic diet containing 1% cholesterol and 0.5% cholic acid, B). Fractions 3 through 7 represent VLDL and chylomicrons; fractions 8 through 14, IDL and LDL; and fractions 15 through 21, HDL. Values are mean±SEM.

View larger version (58K): [in this window] [in a new window]   Figure 3. Photomicrographs of cross sections of the aortic root of transplanted C57Bl/6 mice. These mice were fed an atherogenic diet containing 1% cholesterol and 0.5% cholic acid for 3 months. The sections were stained with oil red O and hematoxylin. Representative sections of C57Bl/6 mice transplanted with C57Bl/6 (A) or LDLr(-/-) (B) bone marrow are shown. Magnification x40.

View larger version (14K): [in this window] [in a new window]   Figure 4. Effect of BMT on mean atherosclerotic lesion area. C57Bl/6 mice transplanted with C57Bl/6 (n=16) or LDLr(-/-) (n=14) bone marrow were fed an atherogenic diet containing 1% cholesterol and 0.5% cholic acid for 3 months. Mean lesion area was calculated from cross sections of aortic root that were stained with oil red O. Values are mean±SD. *Significant difference vs C57Bl/6 transplanted with C57Bl/6 bone marrow (P<0.001).

View larger version (114K): [in this window] [in a new window]   Figure 5. Photomicrographs of cross sections of the aortic root of C57Bl/6 mice transplanted with either control C57Bl/6 (A and C) or LDLr(-/-) (B and D) bone marrow. After 3 months on an atherogenic diet containing 1% cholesterol and 0.5% cholic acid, the mice were euthanized, and their hearts were perfused and fixed with neutral-buffered formalin. Cryostat sections of the aortic root were stained with an antibody against MOMA-2 to visualize macrophages (A and B) and with antiserum against the rat LDLr to visualize the presence of the LDLr in the atherosclerotic lesions (C and D). Magnification x100.

To elucidate the potential mechanism by which the LDLr on macrophages can contribute to foam cell formation, we studied the in vitro uptake of atherogenic ß-VLDL by thioglycollate-elicited peritoneal macrophages of C57Bl/6 and LDLr(-/-) mice. Previous studies showed that the uptake of ß-VLDL by murine peritoneal macrophages is mediated predominantly by a so-called unusual LDLr. This unusual LDLr differs from the classic LDLr, as described in human fibroblasts, in its low affinity for LDL and its relative resistance to downregulation by extracellular cholesterol.^{12 29} C57Bl/6 macrophages appeared to be much more efficient in ß-VLDL association and degradation than were LDLr(-/-) macrophages (B_max 3730 versus 797 ng/mg for association [Figure 6A] and B_max 2163 versus 202 ng/mg for degradation [Figure 6B]). CE accumulation after 24 hours of incubation with 50 µg/mL ß-VLDL was decreased from 69.1±4.17 µg CE per milligram cell protein in wild-type macrophages to 40.6±2.97 µg CE per milligram cell protein in LDLr(-/-) macrophages. No significant difference in cellular CEs could be observed without the addition of lipoproteins [2.2±1.9 µg CE per milligram cell protein for wild-type macrophages compared with 1.6±0 µg CE per milligram cell protein for LDLr(-/-) macrophages] or after 24 hours of incubation with 100 µg/mL acetylated LDL [22.2±3.7 µg CE per milligram protein for wild-type macrophages compared with 23.2±6.1 µg CE per milligram protein for LDLr(-/-) macrophages]. The observed large difference in ß-VLDL metabolism and ß-VLDL–induced CE accumulation between C57Bl/6 and LDLr(-/-) macrophages thus suggests that the so-called unusual LDLr originates from the same gene as the classic LDLr. Furthermore, the significant role of the LDLr in the interaction of the atherogenic ß-VLDL with macrophages may explain its facilitating role in atherogenic lesion formation.

View larger version (16K): [in this window] [in a new window]   Figure 6. Metabolism of 125I-ß-VLDL by C57Bl/6 and LDLr(-/-) peritoneal macrophages. Thioglycollate-elicited peritoneal macrophages of C57Bl/6 () and LDLr-/- (•) mice were isolated and cultured in lipoprotein-deficient medium for 2 days in a concentration of 0.5x106 cells per 25-mm well. Association (A) and degradation (B) of 125I-ß-VLDL by the macrophages (3 hours at 37°C) were determined and expressed as nanograms 125I-ß-VLDL per milligram cell protein. The data are results of 3 independent experiments. Values are mean±SD.

	Discussion

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The LDLr is expressed in a wide variety of cell types. Hepatic cells contain the majority of the LDLr activity and play a prominent role in the clearance of LDL from the circulation.² However, the function of the LDLr expressed on bone marrow–derived cells in lipoprotein metabolism and atherosclerosis is not well known. Bone marrow–derived cells give rise to lymphoid and erythroid tissues and to monocytes, which become resident macrophages in various tissues, including the liver, lungs, peritoneal cavity, and vessel wall. Expression of the LDLr on macrophages may influence atherosclerotic lesion development when these cells migrate into the intima of the vessel wall. Recently, we¹³ and others^{14 15} transplanted C57Bl/6 bone marrow into LDLr(-/-) mice to study specifically the role of macrophage-expressed LDLr. The recipient hemopoietic cells were almost completely replaced by donor cells. The transplantation resulted in a temporary decrease of total serum cholesterol. The mean atherosclerotic lesion area of these transplanted mice fed a cholesterol-rich diet did not reveal differences compared with the lesion area of control LDLr(-/-) mice that were transplanted with LDLr(-/-) bone marrow. Because of the relatively high concentrations of circulating cholesterol, we speculated that the newly acquired LDLr is downregulated in the LDLr(-/-) mice transplanted with wild-type bone marrow. In the present study, we investigated the role of the LDLr on macrophages under less extreme cholesterol conditions to circumvent the possible downregulation of the LDLr. Therefore, we performed the complementary experiment of transplanting LDLr(-/-) bone marrow into C57Bl/6 mice. The absence of the LDLr on macrophages resulted in a significant reduction in susceptibility to diet-induced atherosclerosis, whereas total cholesterol and triglyceride levels were not altered significantly. Furthermore, no correlation was observed between individual serum cholesterol levels and lesion area in the transplanted mice.

Using antiserum directed against the LDLr, we showed that most of the foam cells in the atherosclerotic lesions of LDLr(-/-)C57Bl/6 mice were unable to express the LDLr, which is in contrast to the situation in control transplanted mice. Therefore, it is concluded that the decreased susceptibility of C57Bl/6 mice reconstituted with LDLr(-/-) bone marrow to diet-induced atherosclerosis is due to the inability of arterial macrophages to express the LDLr. Thus, although the role of the macrophage LDLr in atherosclerotic lesion development has been thought to be limited because it is subject to downregulation by intracellular cholesterol,^{9 30 31 32} we now clearly demonstrate that in vivo the macrophage LDLr does play a role in diet-induced atherosclerotic lesion development in C57Bl/6 mice. The effect of the macrophage LDLr on diet-induced atherosclerosis can be considered a direct effect: the LDLr contributes to the progression of lesion development in the arterial wall without affecting serum cholesterol levels. We previously demonstrated that transplantation of C57Bl/6 bone marrow into LDLr(-/-) mice, which had serum cholesterol levels 9-fold higher than those of the mice in the present study even without cholate added to the diet, did not result in an increase of the atherosclerotic lesion area.¹³ Although different animal models were used to analyze diet-induced (with and without the addition of cholate) atherosclerosis in these studies, which could make direct comparisons difficult, the present data strongly suggest that the LDLr can facilitate foam cell formation only under conditions of moderate cholesterol levels. Analysis of LDLr expression in more advanced foam cell–rich lesions of control transplanted C57Bl/6C57Bl/6 mice revealed that in these mice, the macrophage LDLr is downregulated during the progression of lesion development. Therefore, we would like to suggest that the macrophage LDLr is involved in the initial development of foam cell–rich atherosclerotic lesions.

To induce atherosclerosis, the transplanted mice were fed an atherogenic diet containing 1% cholesterol and 0.5% cholic acid. As a result, cholesterol in VLDL and IDL/LDL increased 10-fold. It has previously been shown that although murine macrophages take up relatively little unmodified LDL, they take up ß-VLDL and chylomicron remnants via the LDLr.^{12 29} The uptake of ß-VLDL results in CE accumulation in macrophages, which is the first step in atherosclerosis.³¹ The LDLr on mouse peritoneal macrophages was identified as being different from the classic LDLr, described in human fibroblasts, because of its low affinity for LDL and its relative resistance to downregulation by extracellular cholesterol.^{12 29} In human monocyte–derived macrophages, however, Koo et al¹¹ demonstrated that ß-VLDL uptake is mediated by the classic LDLr, as described for human fibroblasts. The availability of wild-type and LDLr(-/-) mice makes it possible to further identify the unusual LDLr on mouse peritoneal macrophages. In the present study, the association and degradation of ¹²⁵I-ß-VLDL appeared to be much higher in C57Bl/6 than in LDLr(-/-) peritoneal macrophages, indicating that the unusual LDLr originates from the same LDLr gene as the classic LDLr. The different properties of the unusual LDLr compared with the classic LDLr may be related to the fact that macrophages are metabolically different from fibroblasts. Alternatively, the murine LDLr may differ from the human LDLr. At present, we can conclude that LDLr-containing macrophages show a 2.2-fold increase in cell association and a 4.7-fold increase in degradation of ß-VLDL, suggesting a significant role for the LDLr in ß-VLDL catabolism by the macrophages. The decreased ß-VLDL catabolism by LDLr(-/-) macrophages also resulted in a decreased CE accumulation by these macrophages. No difference in accumulation of CEs from acetylated LDL was observed between wild-type and LDLr(-/-) macrophages, indicating that the reduced susceptibility to lesion formation on reconstitution of C57Bl/6 mice with LDLr(-/-) bone marrow cannot be attributed to differences in scavenger receptor expression by LDLr(-/-) macrophages. Thus, only the decreased ß-VLDL catabolism and CE accumulation in the absence of the macrophage LDLr may explain the facilitating role of the LDLr in atherogenic lesion formation.

During the revision period of the present article, a study by Linton et al³³ was published that also evaluated the effect of BMT of LDLr-negative bone marrow into C57Bl/6 mice, confirming the role of the LDLr in facilitating atherosclerotic lesion formation. The reported effect of the LDLr on atherosclerosis between both studies is highly comparable: 65% reduction in lesion formation in the absence of the macrophage LDLr in the present study compared with 63% smaller lesions in the study of Linton et al. However, in contrast to our present findings, Linton et al observed a significant (40%) increase in serum cholesterol levels in C57Bl/6 mice transplanted with LDLr-/- bone marrow after consuming a high cholesterol diet for 13 weeks, whereas at 6 weeks, no difference was noticed. The reason for the increase in serum cholesterol levels in mice reconstituted with LDLr(-/-) bone marrow, as observed by Linton et al, between 6 weeks and 13 weeks on a high cholesterol diet is unknown. Previously, we reported that the reconstitution of LDLr(-/-) mice with wild-type bone marrow resulted in only a small and transient reduction in serum cholesterol levels. In comparable studies performed by Boisvert et al,¹⁵ a minimal decrease in serum cholesterol levels was also observed, whereas Fazio et al¹⁴ did not observe any differences at all. Thus, our present findings confirm the initial conclusions that the leukocyte LDLr does not play an important role in the clearance of lipoproteins from the circulation in mice with intrinsic high cholesterol levels or in mice on a high cholesterol diet.

In conclusion, by transplanting LDLr(-/-) bone marrow into C57Bl/6 mice, we showed that the macrophage LDLr plays a significant role in diet-induced atherosclerosis under conditions of modestly high serum cholesterol. The observation that C57Bl/6 peritoneal macrophages have a much higher association and degradation of ¹²⁵I-ß-VLDL than do LDLr(-/-) peritoneal macrophages provides a possible mechanism for the contribution of the LDLr to lesion development in the arterial wall.

	Acknowledgments

 
This research was supported by the Netherlands Heart Foundation (project 93188). We would like to thank Dr G.M. Benson for helpful assistance with the image analysis of atherosclerotic lesions. The rabbit anti-rat LDLr antiserum was kindly provided by Dr A. Cooper (Stanford University).

Received September 21, 1998; accepted March 21, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science. 1986;232:34–47.[Free Full Text]

2. Brown MS, Goldstein JL. Lipoprotein receptors in the liver: control signals for plasma cholesterol traffic. J Clin Invest. 1983;72:743–747.

3. Hobbs HH, Brown MS, Goldstein JL. Molecular genetics of the LDL receptor gene in familial hypercholesterolemia. Hum Mutat. 1992;1:445–466.[Medline] [Order article via Infotrieve]

4. Goldstein JL, Brown MS. Familial hypercholesterolemia. In: Stanbury JB, Wyngaarden JB, Frederickson DS, Goldstein JL, Brown MS. The Metabolic Basis of Inherited Diseases. New York, NY: McGraw-Hill; 1983:622–730.

5. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest. 1993;92:883–893.

6. Ishibashi S, Goldstein JL, Brown MS, Herz J, Burns DK. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice. J Clin Invest. 1994;93:1885–1893.

7. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]

8. Steinberg D. Modified forms of low-density lipoprotein and atherosclerosis. J Intern Med. 1993;233:227–232.[Medline] [Order article via Infotrieve]

9. Brown MS, Goldstein JL. Regulation of the activity of low density lipoprotein receptor in human fibroblasts. Cell. 1975;6:307–316.[Medline] [Order article via Infotrieve]

10. Jaakkola O, Ylä-Herttuala S, Sarkioja T, Nikkari T. Macrophage foam cells from human aortic fatty streaks take up beta-VLDL and acetylated LDL in primary culture. Atherosclerosis. 1989;79:173–182.[Medline] [Order article via Infotrieve]

11. Koo C, Wernette-Hammond ME, Garcia Z, Malloy MJ, Uauy R, East C, Bilheimer DW, Mahley RW. The uptake of cholesterol-rich remnant lipoproteins by human monocyte-derived macrophages is mediated by low density lipoprotein receptors. J Clin Invest. 1988;81:1332–1340.

12. Ellsworth JL, Kraemer FB, Cooper AD. Transport of ß-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway. J Biol Chem. 1987;262:2316–2325.[Abstract/Free Full Text]

13. Herijgers N, Van Eck M, Groot, PHE, Hoogerbrugge PM, Van Berkel TJC. Effect of bone marrow transplantation on lipoprotein metabolism and atherosclerosis in LDL receptor–knockout mice. Arterioscler Thromb Vasc Biol. 1997;17:1995–2003.[Abstract/Free Full Text]

14. Fazio S, Hasty AH, Carter KJ, Murray AB, Price JO, Linton MF. Leukocyte low density lipoprotein receptor (LDL-R) does not contribute to LDL clearance in vivo: bone marrow transplantation studies in the mouse. J Lipid Res. 1997;38:391–400.[Abstract]

15. Boisvert WA, Spangenberg J, Curtiss LK. Role of leukocyte-specific LDL receptors on plasma lipoprotein cholesterol and atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 1997;17:340–347.[Abstract/Free Full Text]

16. Nishina PM, Verstuyft J, Paigen B. Effects of dietary fats from animal and plant sources on diet-induced fatty streak lesions in C57BL/6J mice. J Lipid Res. 1992;31:859–869.[Abstract]

17. Groot PHE, van Vlijmen BJM, Benson GM, Hofker M, Schiffelers R, Vidgeon-Hart M, Havekes LM. Quantitative assessment of aortic atherosclerosis in apoE3 Leiden transgenic mice and its relationship to serum cholesterol exposure. Arterioscler Thromb Vasc Biol. 1996;16:926–933.[Abstract/Free Full Text]

18. Redgrave TG, Roberts DCK, Wert CE. Separation of plasma lipoproteins by density gradient ultracentrifugation. Anal Biochem. 1975;65:42–49.[Medline] [Order article via Infotrieve]

19. McFarlane AS. Efficient trace-labelling of proteins with iodine. Nature. 1958;182:53.[Medline] [Order article via Infotrieve]

20. van Tol A, van Gent T, van het Hooft FM, Vlaspolder F. High density lipoprotein catabolism before and after partial hepatectomy. Atherosclerosis. 1978;29:439–448.[Medline] [Order article via Infotrieve]

21. Basu SK, Goldstein JL, Anderson GW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976;72:3178–3182.

22. Lowry OH, Rosebrough NJ, Fair AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275.[Free Full Text]

23. Henriksen T, Mahoney EM, Steinberg D. Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells: recognition by receptors for acetylated low-density lipoprotein. Proc Natl Acad Sci U S A. 1981;78:6499–6503.[Abstract/Free Full Text]

24. Havekes LM, de Wit ECM, Princen HMG. Cellular free cholesterol in HepG2 cells is only partially available for down-regulation of the low density lipoprotein receptor. Biochem J. 1987;247:739–746.[Medline] [Order article via Infotrieve]

25. Breslow JL. Mouse models in atherosclerosis. Science. 1996;272:685–688.[Abstract]

26. Purcell-Huynh DA, Farese RV Jr, Johnson DF, Flynn LM, Pierotti V, Newland DL, Linton MF, Sanan DA, Young SG. Transgenic mice expressing high levels of human apolipoprotein B develop severe atherosclerotic lesions in response to a high-fat diet. J Clin Invest. 1995;95:2246–2257.

27. Cohen RD, Castellani, LW, Qiao JH, Van Lenten BJ, Lusis AJ, Reue K. Reduced aortic lesions and elevated high density lipoprotein levels in transgenic mice overexpressing mouse apolipoprotein A-IV. J Clin Invest. 1997;99:1906–1916.[Medline] [Order article via Infotrieve]

28. Arai T, Wang N, Bezouevski M, Welch C, Tall AR. Decreased atherosclerosis in heterozygous low density lipoprotein receptor-deficient mice expressing scavenger receptor BI transgene. J Biol Chem. 1999;274:2366–2371.[Abstract/Free Full Text]

29. Koo C, Wernette-Hammond ME, Innerarity TL. Uptake of canine ß-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor. J Biol Chem. 1986;261:11194–11201.[Abstract/Free Full Text]

30. Soutar AK, Knight BL. Structure and regulation of the LDL-receptor and its gene. Br Med Bull. 1990;46:891–916.[Abstract/Free Full Text]

31. Srivastava RAK, Jiao S, Tang J, Pfleger BA, Kitchens RT, Schonfeld G. In vivo regulation of low-density lipoprotein receptor and apolipoprotein B gene expressions by dietary fat and cholesterol in inbred strains of mice. Biochim Biophys Acta. 1991;1086:29–43.[Medline] [Order article via Infotrieve]

32. Goldstein JL, Ho YK, Brown MS, Innerarity TL, Mahley RW. Cholesteryl ester accumulation in macrophages resulting from receptor-mediated uptake and degradation of hypercholesterolemic canine ß-very low density lipoproteins. J Biol Chem. 1980;255:1839–1848.[Abstract/Free Full Text]

33. Linton MF, Babaev VR, Gleaves LA, Fazio S. A direct role for the macrophage low density lipoprotein receptor in atherosclerotic lesion formation. J Biol Chem. 1999;274:19204–19210.[Abstract/Free Full Text]

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