This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marathe, S. Articles by Tabas, I. Search for Related Content PubMed PubMed Citation Articles by Marathe, S. Articles by Tabas, I. Related Collections Animal models of human disease Cell biology/structural biology Lipid and lipoprotein metabolism Other Vascular biology

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2607.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Sphingomyelinase Converts Lipoproteins From Apolipoprotein E Knockout Mice Into Potent Inducers of Macrophage Foam Cell Formation

Sudhir Marathe; Yunsook Choi; Andrew R. Leventhal; Ira Tabas

From the Departments of Medicine (S.M., Y.C., A.R.L., I.T.) and Anatomy and Cell Biology (I.T.), Columbia University, New York, NY.

Correspondence to Ira Tabas, MD, PhD, Department of Medicine, Columbia University, 630 West 168th St, New York, NY 10032. E-mail iat1{at}columbia.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—The apoE knockout (E0) mouse is one of the most widely used animal models of atherosclerosis, and there may be similarities to chylomicron remnant–induced atherosclerosis in humans. Although the lesions of these mice contain large numbers of cholesteryl ester (CE)-laden macrophages (foam cells), E0 plasma lipoproteins are relatively weak inducers of cholesterol esterification in macrophages. Previous in vivo work has suggested that arterial wall sphingomyelinase (SMase) may promote atherogenesis in the E0 mouse, perhaps by inducing subendothelial lipoprotein aggregation and subsequent foam cell formation. The goal of the present study was to test the hypothesis that the modification of E0 lipoproteins by SMase converts these lipoproteins into potent inducers of macrophage foam cell formation. When d<1.063 E0 lipoproteins were pretreated with SMase and then incubated with E0 macrophages, cellular CE mass and stimulation of the cholesterol esterification pathway were increased 5-fold compared with untreated lipoproteins. SMase-treated E0 lipoproteins were more potent stimulators of cholesterol esterification than either E0 lipoproteins in the presence of lipoprotein lipases or oxidized E0 lipoproteins. The uptake and degradation of SMase-treated E0 lipoproteins by macrophages were saturable and specific and substantially inhibited by partial proteolysis of cell-surface proteins. Uptake and degradation were diminished by an anti-apoB antibody and by competition with human S_f 100-400 hypertriglyceridemic VLDL, raising the possibility that a receptor that recognizes apoB-48 might be involved. In conclusion, SMase-modification of E0 lipoproteins, a process previously shown to occur in lesions, may be an important mechanism for foam cell formation in this widely studied model of atherosclerosis. Moreover, the findings in this report may provide important clues regarding the atherogenicity of chylomicron remnants in humans.

Key Words: sphingomyelinase • lipoproteins • macrophages • foam cells • apolipoprotein E knockout mice

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The apoE knockout (E0) mouse develops extensive atherosclerosis and has become one of the most widely used animal models to explore atherogenesis in vivo.^{1 2 3} It is presumed that the remnant-like particles that accumulate in the plasma of these mice are atherogenic and induce cholesteryl ester (CE)-laden macrophages (foam cells) formation,^{1 2} yet previous studies have shown that these plasma lipoproteins can induce only modest CE accumulation in cultured macrophages.^{4 5 6} The answer to this dilemma undoubtedly lies in the overall hypothesis that these lipoproteins become modified in the arterial wall to a form with increased ability to induce macrophage foam cell formation. Previous work has shown that the addition of 5 µg/mL lipoprotein lipase (LpL) or the oxidation of E0 lipoproteins can increase the ability of the particles to induce foam cell formation in cultured macrophages.^{4 5} Whether there is sufficient LpL or sufficiently extensive lipoprotein oxidation in E0 lesions to facilitate these processes in vivo remains to be determined.

A lipoprotein modification that is known to occur in lesions and that greatly enhances the ability of LDL to induce CE accumulation in macrophages is lipoprotein aggregation.^{7 8 9 10 11 12} One possible inducer of subendothelial lipoprotein aggregation is arterial wall sphingomyelinase (SMase).^{13 14 15 16 17 18 19} SMase treatment of lipoproteins results in aggregation and fusion of the particles, and aggregated LDL isolated from human lesions, but not monomeric lesional LDL or native plasma LDL, is enriched in ceramide, a marker of SMase action on the lipoproteins.¹⁴ A particular species of SMase called secretory SMase (S-SMase) is the best candidate for this enzyme because it is the only known extracellular SMase in mammals, it is secreted by cultured arterial wall cells, and it is found in normal endothelium and especially in atherosclerotic intima.¹⁷ The role of S-SMase in the E0 mouse may be particularly important for the following reasons: (1) lipoproteins from the lesions of E0 mice are aggregated (Maor et al²⁰ and unpublished data from our researchers); (2) lipoproteins isolated from the lesions of E0 mice are enriched in ceramide¹⁴ ; (3) E0 lesions stain abundantly for immunoreactive SMase¹⁶ ; (4) E0 lipoproteins are among the most susceptible lipoproteins to the action of S-SMase, probably because these lipoproteins uniquely have a high sphingomyelin-to-phospholipid ratio^{15 21} ; and, most important, (5) E0 mice on a SMase knockout background have smaller lesions than E0 mice with normal SMase.¹⁸

In light of these findings, we hypothesize that the hydrolysis of subendothelial E0 lipoproteins by arterial wall SMase converts these particles to a form that can induce macrophage foam cell formation. To test this hypothesis, we compared the ability of native and SMase-treated E0 lipoproteins to induce CE accumulation and to stimulate the cholesterol esterification pathway in macrophages. Our results show that the SMase-aggregated E0 lipoproteins are indeed potent inducers of macrophage foam cell formation. Moreover, our results indicate that a substantial portion of the cellular uptake of these aggregated particles involves the interaction of apoB with a cell-surface receptor that is uniquely competed by human hypertriglyceridemic VLDL.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
The Falcon tissue culture plasticware used in these studies was purchased from Fisher Scientific Co. Tissue culture media and other tissue culture reagents were obtained from GIBCO BRL. FBS was obtained from Hyclone Laboratories. LpL was purified from bovine milk as previously described²² and provided by Kirsten D. Mazany and Dr Kevin J. Williams. The source of S-SMase was serum-free conditioned medium harvested from DG44 Chinese hamster ovary cells stably transfected with human acid SMase cDNA.^{13 23} Bacterial SMase from Bacillus cereus, chondroitin ABC lyase from Proteus vulgaris, heparitinase from Flavobacterium heparinum, and trypsin from porcine pancreas (type IX, 13 000 U/mg) were purchased from Sigma Chemical Co. Goat anti-murine apoB was prepared and characterized as described previously²⁴ and provided by Drs Kevin J. Williams and Daniel Levine. Receptor-associated protein (RAP) was provided by Dr Dudley Strickland. Mouse IgG_2a and anti-mouse CD12/CD32 Fc receptor antibody were obtained from Chemicon and PharMingen, respectively. All other chemicals and reagents were obtained from Sigma Chemical Co, and all organic solvents were obtained from Fisher Scientific Co.

Native and Modified Lipoproteins
The VLDL and LDL fraction from the plasma of chow-fed 4- to 8-month-old female or male E0 mice was prepared through preparative ultracentrifugation (d<1.063 g/mL).¹⁵ S-SMase treatment of the lipoproteins was carried through the incubation of 50 µL (5 µg) lipoproteins in 100 mmol/L HEPES, pH 7.2, 100 µmol/L ZnCl₂ with 85 µL DG44 conditioned media for 16 hours at 37°C.¹⁵ For treatment with bacterial SMase, 1 mg/mL lipoproteins was incubated with 50 mU/mL B cereus SMase for 4 hours at 37°C in PBS containing 2 mmol/L MgCl₂.¹² LDL (d=1.020 to 1.063 g/mL) was isolated from fresh human plasma through preparative ultracentrifugation and acetylated as described previously.^{25 26} Oxidation was carried out by incubation of the E0 lipoproteins (1 mg/mL) with 5 µmol/L CuSO₄ for 18 hours at 37°C, followed by the addition of 1 mmol/L EDTA and dialysis against 150 mmol/L NaCl, 0.3 mmol/L EDTA. The lipoproteins were labeled with ¹²⁵I with the use of Iodogen-coated tubes (Pierce) and Na[¹²⁵I] (NEN Life Science Products)²⁷ ; the labeled lipoproteins had a specific activity of 250 to 400 cpm/ng protein and were used within 3 weeks of iodination. S_f 100-400 VLDL from a man with a plasma triglyceride level of 1320 mg/dL was obtained through preparative NaBr ultracentrifugation (d<1.006 g/mL), followed by discontinuous NaCl gradient centrifugation.²⁸ Trypsinized hypertriglyceridemic VLDL, which was generously provided by Drs Sandra Gianturco and William Bradley, was prepared through the incubation of hypertriglyceridemic VLDL with 10 mg/mL trypsin (Worthington 3x crystallized) for 2 hours at 37°C, followed by reisolation through ultracentrifugation.²⁹

Cells
J774.A1 macrophages (American Type Culture Collection) were maintained in spinner culture in DMEM, 10% (v/v) FBS containing 50 U/mL penicillin, 50 U/mL streptomycin, and 2 mmol/L glutamine. The medium was replaced with fresh medium each day. Mouse peritoneal macrophages from 25- to 35-g female C57BL6/J mice and from various gene-targeted mice were harvested from the peritoneum 3 days after the intraperitoneal injection of 40 µg concanavalin A in 0.5 mL PBS.³⁰ ApoE, LDLR, and class A scavenger receptor knockout mice were obtained from Jackson Laboratories, and CD36 knockout mice were kindly provided by Drs Maria Febbraio and Roy Silverstein (Weill Medical College of Cornell University, New York, NY). The cells were plated onto 22-mm dishes in DMEM containing 10% (v/v) FBS, 20% (v/v) L-cell conditioned medium (LCM), 100 U/mL penicillin, 100 µg/mL streptomycin, and 292 µg/mL glutamine and were used within 3 days.

Cellular Assays
Lipid extracts of the cells were assayed for total and free cholesterol mass with gas-liquid chromatography.¹² Cellular cholesterol esterification was assayed by measuring the incorporation of [¹⁴C]oleate into cellular cholesteryl [¹⁴C]oleate.²² Degradation of the ¹²⁵I-labeled lipoproteins was determined from the [¹²⁵I] cpm of TCA-soluble, non–chloroform-extractable material in the medium.²² Unless indicated otherwise, results are given as mean±SEM (n=3).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Treatment of d<1.063 Lipoproteins From E0 Mice With SMase Markedly Increases Their Ability to Induce Foam Cell Formation
To address the hypothesis that SMase-induced aggregation may contribute to foam cell formation in E0 mice, we incubated J774 macrophages with d<1.063 lipoproteins isolated directly from the plasma of E0 mice or with these lipoproteins after treatment with S-SMase, the enzyme proposed to induce lipoprotein aggregation in atherosclerotic lesions.¹⁷ Note that J774 macrophages do not synthesize apoE³¹ and thus provide a model for foam cell formation in apoE-deficient macrophages. In confirmation of previous results,^{15 21} S-SMase treatment led to visible lipoprotein aggregation. As shown in Figure 1A, the untreated, monomeric plasma lipoproteins caused some degree of CE accumulation in the macrophages, but the loading induced by S-SMase–aggregated lipoproteins was 5-fold higher. S-SMase alone had no effect, indicating that it was the aggregated lipoproteins and not residual SMase activity that caused the CE loading.

View larger version (17K): [in this window] [in a new window]   Figure 1. S-SMase–treated d<1.063 lipoproteins from E0 mice are potent inducers of cholesterol esterification in macrophages. A, J774 macrophages were incubated in medium alone (No LPs) or in medium containing 50 µg/mL untreated d<1.063 lipoproteins from E0 mice (E0 LPs), E0 lipoproteins treated with S-SMase, or the same solution containing S-SMase but no lipoproteins. After a 24-hour incubation, the cells were assayed for CE mass. B, Peritoneal macrophages from C57 mice were incubated in medium alone (No LPs) or in medium containing 50 µg/mL untreated d<1.063 lipoproteins from E0 mice (E0 LPs), E0 lipoproteins treated with bacterial SMase, oxidized E0 lipoproteins, or E0 lipoproteins in the presence of 5 µg/mL LpL (E0 LPs/LpL). After incubation for 18 hours, the cells were assayed for the incorporation of [14C]oleate into cholesteryl [14C]oleate.

To determine whether the increase in CE accumulation involved stimulation of the acyl-CoA:cholesterol acyltransferase pathway, mouse peritoneal macrophages were incubated with monomeric or SMase-aggregated d<1.063 E0 lipoproteins in the presence of [¹⁴C]oleate; for this and the following experiments, soluble bacterial SMase was used, which has the same effect on lipoproteins as human S-SMase.^{12 15} As shown in Figure 1B (first 3 columns), the SMase-aggregated lipoproteins led to a 5- to 6-fold higher increase in incorporation of the labeled oleate into cholesteryl [¹⁴C]oleate, indicating acyl-CoA:cholesterol acyltransferase–mediated cholesterol esterification and not simply cellular accumulation of undegraded lipoprotein CE. Importantly, SMase-aggregated d<1.063 E0 lipoproteins were also excellent inducers of cholesterol esterification in peritoneal macrophages from E0 mice (143±1.2 nmol cholesteryl [¹⁴C]oleate · mg^–1 · 18 h^–1 versus 107±1.0 in macrophages from wild-type mice). In addition, the uptake and degradation of SMase-treated ¹²⁵I-E0 lipoproteins were similar in macrophages from wild-type versus E0 mice (1050±39 versus 1012±25 ng degraded · mg^–1 · 5 h^–1). Thus, apoE secretion by macrophages is not necessary for the uptake and processing of SMase-modified E0 lipoproteins.

Others have proposed that E0 lipoprotein oxidation or LpL, acting as a bridging molecule, may facilitate E0 lipoprotein uptake by macrophages.^{4 5} As shown in Figure 1B, both of these perturbations indeed increased cholesterol esterification compared with untreated lipoproteins, but neither was as potent as SMase-induced aggregation. The addition of LpL to SMase-aggregated E0 lipoproteins, or SMase aggregation of oxidized E0 lipoproteins, did not increase cholesterol esterification above that seen with SMase-aggregated lipoproteins (data not shown).

The Uptake and Degradation of d<1.063 E0 Lipoproteins by Macrophages Involve an Interaction Between One or More Cell Surface Proteins and ApoB
The uptake and degradation of SMase-treated d<1.063 E0 lipoproteins showed evidence of saturability, as demonstrated by the concentration curve in Figure 2. Moreover, uptake and degradation were competed 78% by unlabeled SMase-treated E0 lipoproteins (data not displayed). These data are consistent with the notion that uptake of the lipoproteins is a receptor-mediated process. To determine whether a cell-surface protein was involved, cells were treated with a limiting concentration of trypsin to effect partial hydrolysis of cell-surface proteins without otherwise damaging the cells; cycloheximide was added to prevent synthesis on new receptor protein. As shown in Figure 3A, trypsin treatment resulted in 50% inhibition of the association of SMase-treated E0 lipoproteins with macrophages. In a parallel experiment, trypsin treatment of macrophages inhibited the cell association of acetyl-LDL, a lipoprotein known to interact with cell-surface receptor proteins,³² by 60% (data not shown). Thus, the lack of complete inhibition of SMase-treated E0 lipoprotein uptake by trypsin most likely represents incomplete hydrolysis of cell-surface proteins under the conditions of this experiment.

View larger version (13K): [in this window] [in a new window]   Figure 2. The uptake and degradation of SMase-treated d<1.063 lipoproteins from E0 mice represent a saturable, specific process.125I-labeled d<1.063 lipoproteins from E0 mice were treated with bacterial SMase and then incubated with mouse peritoneal macrophages at the indicated concentrations for 5 hours. The media were then assayed for 125I-lipoprotein degradation.

View larger version (19K): [in this window] [in a new window]   Figure 3. . The interaction of macrophages with SMase-treated d<1.063 E0 lipoproteins involves the interaction of a cell-surface protein with apoB and is competed by htgVLDL. A, Control mouse peritoneal macrophages were incubated in medium containing 50 µg/mL 125I-labeled E0 d<1.063 lipoproteins for 2 hours and then assayed for 125I-lipoprotein cell association. To effect partial proteolysis of cell-surface proteins, macrophages were incubated for 15 minutes at 37°C with 250 µg/mL trypsin and then rinsed with PBS and incubated with 250 µg/mL soybean trypsin/chymotrypsin inhibitor plus 2 µmol/L cycloheximide for 5 minutes at 37°C. The cells were then rinsed again with PBS and incubated with medium containing 50 µg/mL soybean trypsin/chymotrypsin inhibitor, 2 µmol/L cycloheximide, and 50 µg/mL 125I-labeled d<1.063 lipoproteins from E0 mice. After a 2-hour incubation, the cells were assayed for 125I-lipoprotein cell association. B, Macrophages were incubated with 50 µg/mL 125I-labeled E0 d<1.063 lipoproteins for 5 hours in the presence of either 0.4 µg/mL nonimmune goat IgG or goat anti-murine apoB IgG plus blockers of macrophage Fc receptors (25 µg/mL concentration of anti-mouse CD12/CD32 Fc receptor antibody and 50 µg/mL concentration of mouse IgG2a). After 5 hours of incubation, 125I-lipoprotein degradation was assayed. C, Peritoneal macrophages were incubated with 10 µg/mL SMase-treated 125I-lipoproteins from E0 mice alone or in the presence of 100 µg/mL unlabeled LDL, oxidized LDL, or human htgVLDL. After 5 hours of incubation, 125I-lipoprotein degradation was assayed.

The major protein on E0 lipoproteins is apoB-48.^{1 2} Figure 3B shows the results of an experiment in which a polyclonal antibody that recognizes murine apoB (both B-100 and B-48) was tested for its ability to inhibit the uptake and degradation of SMase-treated E0 lipoproteins. As shown, the anti-apoB antibody inhibited uptake by 40% compared with a similar incubation with a nonimmune IgG. The lack of complete inhibition could be due to the roles of other proteins on the aggregated E0 lipoproteins, partial uptake by Fc receptors despite our attempts to block these receptors (see Methods), or less-than-complete antibody binding to the apoB on the lipoproteins. In summary, at least a substantial portion of the interaction of macrophages with SMase-treated E0 lipoproteins involves the interaction of apoB on the lipoproteins with a cell-surface receptor on the macrophages.

Preliminary Investigation Into Possible Receptors That Mediate the Uptake of SMase-Treated E0 Lipoproteins by Macrophages
Although the LDL receptor would not be expected to mediate the uptake of SMase-treated E0 lipoproteins, we tested this possibility by comparing the degradation of SMase-treated ¹²⁵I-E0 lipoproteins in macrophages from wild-type versus LDL receptor knockout mice. As predicted, the degradation values were similar: 1050±39 versus 999±33 ng·mg^–1·5 h^–1. We next considered the idea that the particles might become modified (eg, by oxidation) to a form recognized by macrophage class A or B scavenger receptors. However, the degradation of SMase-treated E0 lipoproteins was similar in macrophages from wild-type mice (1496±99 ng · mg^–1 · 5 h^–1), scavenger receptor A knockout mice (1583±77 ng · mg^–1 · 5 h^–1), and CD36 knockout mice (1582±124 ng · mg^–1 · 5 h^–1). Finally, the treatment of macrophages with chondroitin ABC lyase plus heparitinase had no effect on lipoprotein uptake (data not shown; see Tabas et al²² ). Thus, neither members of the LDL receptor family, members of the scavenger receptor family, nor cell-surface glycosaminoglycans appear to mediate the interaction of macrophages with SMase-treated E0 lipoproteins.

Gianturco et al²⁸ and Bradley et al³³ demonstrated that a cell-surface protein on macrophages recognizes apoB-48 on S_f 100-400 VLDL from hypertriglyceridemic subjects (htgVLDL). Although the physiological role of this cell-surface protein is not yet known, we considered the possibility that it or another receptor for apoB-48–rich lipoproteins may also recognize the apoB-48 on SMase-treated E0 lipoproteins. Figure 3C shows a competitive inhibition experiment in which the abilities were compared of unlabeled LDL, oxidized LDL, and S_f 100-400 htgVLDL to block the uptake and degradation of SMase-treated ¹²⁵I-E0 lipoproteins. In corroboration of the data above, neither LDL nor oxidized LDL was a competitor. htgVLDL, however, was a potent competitor, and similar results were obtained with trypsinized htgVLDL (data not shown), which is a particularly potent ligand for the macrophage protein that recognizes apoB-48.²⁸ htgVLDL also inhibited the uptake of SMase-aggregated E0 lipoproteins by macrophages from apoE knockout mice (60% inhibition) and from LDL receptor knockout mice (70% inhibition), indicating that the portion of the interaction that was inhibitable by htgVLDL involved neither apoE nor the LDL receptor. Importantly, htgVLDL was a relatively poor competitor of degradation of monomeric ¹²⁵I-labeled E0 lipoproteins (only 12.6% inhibition) and of ¹²⁵I-acetyl-LDL (no inhibition), indicating a lack of toxic effects of htgVLDL and specificity of competition for SMase-aggregated E0 lipoproteins. Thus, the macrophage receptor activity that mediates the uptake of SMase-treated E0 lipoproteins is uniquely competed by S_f 100-400 human htgVLDL.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major objective of the present study was to suggest a new hypothesis as to how macrophage foam cells form in the widely studied E0 mouse model of atherosclerosis. Lipoproteins isolated directly from the plasma of these mice induce only a small amount of CE accumulation in macrophages compared with modified lipoproteins,^{4 5 6} and therefore these particles in their native state are unlikely to induce the massive CE accumulation seen in actual atherosclerotic lesions from these mice. In fact, the answer to this important dilemma is undoubtedly multifactorial. In addition to SMase-induced aggregation, others have shown, and we have verified, that the addition of LpL or oxidation of E0 particles can increase cholesterol esterification in macrophages. SMase was the most potent inducer (Figure 1B), and the addition of LpL to SMase-induced aggregates or the oxidation of E0 lipoproteins before SMase aggregation did not further enhance cholesterol esterification. Moreover, preliminary in vivo studies that show E0 mice without SMase have smaller lesions support a role for SMase in the promotion of atherogenesis in E0 mice.¹⁸ Nevertheless, the lesions of E0 mice probably contain a variety of modified particles, and each may contribute to macrophage foam cell formation.

Regarding other possible mechanisms of foam cell formation in E0 mice, Hayek et al³⁴ proposed that HDL from E0 mice is a relatively poor inducer of cholesterol efflux from macrophages, so it is possible that this factor also contributes to macrophage CE accumulation in this model. The role of macrophage-secreted apoE is uncertain. The hypothesis that macrophage-secreted apoE promotes cholesterol efflux has been supported by the results of three in vivo studies: transplantation of E0 bone marrow into wild-type mice led to increased lesion development compared with transplantation of wild-type marrow into these mice,³⁵ and macrophage-targeted expression of apoE in E0 mice, or transplantation of apoE-expressing bone marrow into E0 mice, led to a reduction in early lesion size.^{36 37} In another study, however, the hypothesis that macrophage-secreted apoE might contribute to particle uptake was supported by the finding that the transplantation of E0 bone marrow into wild-type mice (the same strategy of the first study mentioned above) led to a reduction in early lesion size.³⁸ In the present study, the internalization of E0 particles and the stimulation of cholesterol esterification were similar in macrophages from E0 mice versus those from wild-type mice.

We also presented some initial findings related to how SMase-aggregated E0 lipoproteins are recognized by macrophages. The data in Figures 2 and 3A strongly suggest that a cell-surface protein mediates at least a substantial portion of this interaction. A substantial portion of lipoprotein uptake could be inhibited by an antibody that recognizes murine apoB, and because most of the apoB on E0 lipoproteins is apoB-48, these data suggest an important role for this protein. The ability of native and trypsinized S_f 100-400 htgVLDL to inhibit the uptake of SMase-aggregated E0 lipoproteins is consistent with this idea, because a macrophage cell-surface protein that recognizes these VLDLs has been shown to bind apoB-48.^{28 33} The actual physiological function of this particular cell-surface protein, however, has not yet been elucidated, and its possible role in foam cell formation in E0 mice must await gene knockout studies.

There are several fundamental aspects of atherogenesis that are addressed by the findings in this report. First, the E0 mouse has been one of the most widely used models of atherosclerosis, and the presence of large numbers of macrophages with massive CE accumulation in the lesions of these mice is one of the most important characteristics of the model. Thus, the knowledge of how foam cells form in this model is critical for studies that address mechanisms of atherogenesis in E0 mice as well as for those that explore genetic, pharmacological, and dietary interventions to prevent lesions or to reduce lesion size. Second, the principle that modification of plasma lipoproteins in the subendothelium of developing lesions is necessary for foam cell formation is directly applicable to LDL-induced atherosclerosis in animals and humans.^{9 39 40} In particular, native plasma LDL is a weak inducer of CE loading in macrophages, but aggregated LDL, which is prominently found in atherosclerotic lesions, is a potent inducer of cholesterol esterification in macrophages.^{10 11 12} Because aggregated LDL from human lesions shows evidence of SMase hydrolysis,¹⁴ the findings in the present report suggest that the E0 model may be a reasonable model with which to explore the role of SMase in human atherogenesis. Third, although apoE deficiency is an extremely rare cause of human atherosclerosis, functional mutations in this protein can lead to a more common disease, namely, familial dysbetalipoproteinemia (type III hyperlipoproteinemia).^{41 42} Type III VLDL from most patients is only a modest inducer of CE accumulation in macrophages,^{43 44} so the mechanisms of foam cell formation in the lesions of these subjects may share characteristics with foam cell formation in E0 mice. Finally, the particles that accumulate in E0 mice share some properties with those of chylomicron remnants in humans,^{1 2} which are thought to be atherogenic and which may be quite abundant in the postprandial state.⁴⁵ Interestingly, human chylomicron remnants, unlike those that accumulated in fat-fed rabbits or dogs,⁴⁶ are rather weak inducers of cholesterol esterification in cultured macrophages.^{47 48} Thus, modification in the arterial wall by SMase may represent one mechanism that links these particles to atherosclerosis and heart disease in humans.

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-56984 (Dr Tabas) and a research grant from Berlex Biosciences (Dr Tabas). We thank Kirsten D. Mazany and Dr Kevin J. Williams for providing the LpL, Drs Kevin Williams and Daniel Levine for providing the anti-apoB antibody, Drs Sandra Gianturco and William Bradley for providing the trypsinized hypertriglyceridemic VLDL, Dr Ed Schuchman for providing the DG44 cells, Dr Dudley Strickland for providing the RAP, and Drs Maria Febbraio and Roy Silverstein for providing the CD36 knockout mice.

Received May 22, 2000; accepted September 1, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 1992;71:343–353.[Medline] [Order article via Infotrieve]

2. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992;258:468–471.[Abstract/Free Full Text]

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

4. Hendriks WL, van der Sman-de Beer F, van Vlijmen BJ, van Vark LC, Hofker MH, Havekes LM. Uptake by J774 macrophages of very-low-density lipoproteins isolated from apoE-deficient mice is mediated by a distinct receptor and stimulated by lipoprotein lipase. Arterioscler Thromb Vasc Biol. 1997;17:498–504.[Abstract/Free Full Text]

5. Whitman SC, Hazen SL, Miller DB, Hegele RA, Heinecke JW, Huff MW. Modification of type III VLDL, their remnants, and VLDL from apoE-knockout mice by p-hydroxyphenylacetaldehyde, a product of myeloperoxidase activity, causes marked cholesteryl ester accumulation in macrophages. Arterioscler Thromb Vasc Biol. 1999;19:1238–1249.[Abstract/Free Full Text]

6. Hakamata H, Sakaguchi H, Zhang C, Sakashita N, Suzuki H, Miyazaki A, Takeya M, Takahashi K, Kitamura N, Horiuchi S. The very low- and intermediate-density lipoprotein fraction isolated from apolipoprotein E-knockout mice transforms macrophages to foam cells through an apolipoprotein E-independent pathway. Biochemistry. 1998;37:13720–13727.[Medline] [Order article via Infotrieve]

7. Nievelstein PFEM, Fogelman AM, Mottino G, Frank JS. Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein. Arterioscler Thromb. 1991;11:1795–1805.[Abstract/Free Full Text]

8. Hoff HF, Morton RE. Lipoproteins containing apo B extracted from human aortas: structure and function. Ann N Y Acad Sci. 1985;454:183–194.

9. Tabas I. Nonoxidative modifications of lipoproteins in atherogenesis. Annu Rev Nutr. 1999;19:123–139.[Medline] [Order article via Infotrieve]

10. Khoo JC, Miller E, McLoughlin P, Steinberg D. Enhanced macrophage uptake of low density lipoprotein after self-aggregation. Arteriosclerosis. 1988;8:348–358.[Abstract/Free Full Text]

11. Suits AG, Chait A, Aviram M, Heinecke JW. Phagocytosis of aggregated lipoprotein by macrophages: low density lipoprotein receptor-dependent foam-cell formation. Proc Natl Acad Sci U S A. 1989;86:2713–2717.[Abstract/Free Full Text]

12. Xu X, Tabas I. Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages. J Biol Chem. 1991;266:24849–24858.[Abstract/Free Full Text]

13. Schissel SL, Schuchman EH, Williams KJ, Tabas I. Zn²⁺-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene. J Biol Chem. 1996;271:18431–18436.[Abstract/Free Full Text]

14. Schissel SL, Tweedie-Hardman J, Rapp JH, Graham G, Williams KJ, Tabas I. Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein: proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins. J Clin Invest. 1996;98:1455–1464.[Medline] [Order article via Infotrieve]

15. Schissel SL, Jiang XC, Tweedie-Hardman J, Jeong TS, Camejo EH, Najib J, Rapp JH, Williams KJ, Tabas I. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH: implications for atherosclerotic lesion development. J Biol Chem. 1998;273:2738–2746.[Abstract/Free Full Text]

16. Marathe S, Kuriakose G, Williams KJ, Tabas I. Sphingomyelinase, an enzyme implicated in atherogenesis, is present in atherosclerotic lesions and binds to extracellular matrix. Arterioscler Thromb Vasc Biol. 1999;19:2648–2658.[Abstract/Free Full Text]

17. Tabas I. Secretory sphingomyelinase. Chem Phys Lipids. 1999;102:131–139.[Medline] [Order article via Infotrieve]

18. Marathe S, Tribble DL, Kuriakose G, La Belle M, Chu BM, Gong EL, Johns A, Williams KJ, Tabas I. Sphingomyelinase transgenic and knockout mice: direct evidence that SMase is atherogenic in vivo.Circulation. 1999;100(suppl I):I-695. Abstract.

19. Pentikainen MO, Lehtonen EMP, Kovanen PT. Aggregation and fusion of modified low density lipoprotein. J Lipid Res. 1996;37:2638–2649.[Abstract]

20. Maor I, Hayek T, Hirsh M, Iancu TC, Aviram M. Macrophage-released proteoglycans enhance LDL aggregation: studies in aorta from apolipoprotein E–deficient mice. Atherosclerosis. 2000;150:91–101.[Medline] [Order article via Infotrieve]

21. Jeong TS, Schissel SL, Tabas I, Pownall HJ, Tall AR, Jiang XC. Increased sphingomyelin content of plasma lipoproteins in apolipoprotein E knockout mice reflects combined production and catabolic defects and enhances reactivity with mammalian sphingomyelinase. J Clin Invest. 1998;101:905–912.[Medline] [Order article via Infotrieve]

22. Tabas I, Li Y, Brocia RW, Wu SW, Swenson TL, Williams KJ. Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix: a possible mechanism for low density lipoprotein and lipoprotein(a) retention and macrophage foam cell formation. J Biol Chem. 1993;268:20419–20432.[Abstract/Free Full Text]

23. He X, Miranda SRP, Dagan A, Gatt S, Schuchman EH. Overexpression of human acid sphingomyelinase in Chinese hamster ovary cells: purification and characterization of the recombinant enzyme. Biochim Biophys Acta. 1999;1432:251–264.[Medline] [Order article via Infotrieve]

24. Levine DM, Williams KJ. Automated measurement of mouse apolipoprotein B: convenient screening tool for mouse models of atherosclerosis. Clin Chem. 1997;43:669–674.[Abstract/Free Full Text]

25. Havel RJ, Eder H, Bragdon J. The distribution and chemical composition of ultracentrifugally reported lipoproteins in human serum. J Clin Invest. 1955;34:1345–1353.

26. Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein producing massive cholesterol deposition. Proc Natl Acad Sci U S A. 1979;76:333–337.[Abstract/Free Full Text]

27. Buton X, Mamdouh Z, Ghosh R, Du H, Kuriakose G, Beatini N, Grabowski GA, Maxfield FR, Tabas I. Unique cellular events occurring during the initial interaction of macrophages with matrix-retained or methylated aggregated low density lipoprotein (LDL): prolonged cell-surface contact during which LDL-cholesteryl ester hydrolysis exceeds LDL-protein degradation. J Biol Chem. 1999;274:32112–32121.[Abstract/Free Full Text]

28. Gianturco SH, Ramprasad MP, Song R, Li R, Brown ML, Bradley WA. Apolipoprotein B-48 or its apolipoprotein B-100 equivalent mediates the binding of triglyceride-rich lipoproteins to their unique human monocyte-macrophage receptor. Arterioscler Thromb Vasc Biol. 1998;18:968–976.[Abstract/Free Full Text]

29. Gianturco SH, Bradley WA. The role of apolipoprotein processing in receptor recognition of VLDL. Methods Enzymol. 1986;129:319–344.[Medline] [Order article via Infotrieve]

30. Tang W, Walsh A, Tabas I, Macrophage-targeted CTP. phosphocholine cytidylyltransferase (1–314) transgenic mice. Biochim Biophys Acta. 1999;1437:301–316.[Medline] [Order article via Infotrieve]

31. Mazzone T, Reardon C. Expression of heterologous human apolipoprotein E by J774 macrophages enhances cholesterol efflux to HDL₃. J Lipid Res. 1994;35:1345–1353.[Abstract]

32. Krieger M, Herz J. Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem. 1994;63:601–637.[Medline] [Order article via Infotrieve]

33. Brown ML, Ramprasad MP, Umeda PK, Tanaka A, Kobayashi Y, Watanabe T, Shimoyamada H, Kuo WL, Li R, Song R, Bradley WA, Gianturco SH. A macrophage receptor for apolipoprotein B48: cloning, expression, and atherosclerosis. Proc Natl Acad Sci U S A. 2000;97:7488–7493.[Abstract/Free Full Text]

34. Hayek T, Oiknine J, Brook JG, Aviram M. Role of HDL apolipoprotein E in cellular cholesterol efflux: studies in apo E knockout transgenic mice. Biochem Biophys Res Comm. 1994;205:1072–1078.[Medline] [Order article via Infotrieve]

35. Fazio S, Babaev VR, Murray AB, Hasty AH, Carter KJ, Gleaves LA, Atkinson JB, Linton MF. Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages. Proc Natl Acad Sci U S A. 1997;94:4647–4652.[Abstract/Free Full Text]

36. Bellosta S, Mahley RW, Sanan DA, Murata J, Newland DL, Taylor JM, Pitas RE. Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice. J Clin Invest. 1995;96:2170–2179.

37. Hasty AH, Linton MF, Brandt SJ, Babaev VR, Gleaves LA, Fazio S. Retroviral gene therapy in ApoE-deficient mice: apoE expression in the artery wall reduces early foam cell lesion formation. Circulation. 1999;99:2571–2576.[Abstract/Free Full Text]

38. Boisvert WA, Curtiss LK. Elimination of macrophage-specific apolipoprotein E reduces diet-induced atherosclerosis in C57BL/6J male mice. J Lipid Res. 1999;40:806–813.[Abstract/Free Full Text]

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

40. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915–924.[Medline] [Order article via Infotrieve]

41. Havel RJ. Familial dysbetalipoproteinemia: new aspects of pathogenesis and diagnosis. Med Clin North Amer. 1982;66:441–454.

42. Mahley RW, Angelin B, Type III hyperlipoproteinemia: recent insights into the genetic defect of familial dysbetalipoproteinemia. Adv Internal Med. 1984;29:385–411.[Medline] [Order article via Infotrieve]

43. Fainaru M, Mahley RW, Hamilton RL, Innerarity TL. Structural and metabolic heterogeneity of ß-very low density lipoproteins from cholesterol-fed dogs and from humans with type III hyperlipoproteinemia. J Lipid Res. 1982;23:702–714.[Abstract]

44. Huff MW, Evans AJ, Sawyez CG, Wolfe BM, Nestel PJ. Cholesterol accumulation in J774 macrophages induced by triglyceride-rich lipoproteins: comparison of very low density lipoprotein from subjects with type III, IV, and V hyperlipoproteinemias. Arterioscler Thromb. 1991;11:221–233.[Abstract/Free Full Text]

45. Cohn JS. Postprandial lipemia: emerging evidence for atherogenicity of remnant lipoproteins. Can J Cardiol. 1998;14:18B–27B.

46. Mahley RW, Innerarity TL, Brown MS, Ho YK, Goldstein JL. Cholesterol ester synthesis in macrophages: stimulation by ß-very low density lipoproteins from cholesterol-fed animals of several species. J Lipid Res. 1980;21:970–980.[Abstract]

47. Yu KC, Mamo JC. Regulation of cholesterol synthesis and esterification in primary cultures of macrophages following uptake of chylomicron remnants. Biochem Mol Biol Int. 1997;41:33–39.[Medline] [Order article via Infotrieve]

48. Ellsworth JL, Cooper AD, Kraemer FB. Evidence that chylomicron remnants and beta-VLDL are transported by the same receptor pathway in J774 murine macrophage-derived cells. J Lipid Res. 1986;27:1062–1072.[Abstract]

This article has been cited by other articles:

				C. M. Devlin, A. R. Leventhal, G. Kuriakose, E. H. Schuchman, K. J. Williams, and I. Tabas Acid Sphingomyelinase Promotes Lipoprotein Retention Within Early Atheromata and Accelerates Lesion Progression Arterioscler. Thromb. Vasc. Biol., October 1, 2008; 28(10): 1723 - 1730. [Abstract] [Full Text] [PDF]

				C. Shah, G. Yang, I. Lee, J. Bielawski, Y. A. Hannun, and F. Samad Protection from High Fat Diet-induced Increase in Ceramide in Mice Lacking Plasminogen Activator Inhibitor 1 J. Biol. Chem., May 16, 2008; 283(20): 13538 - 13548. [Abstract] [Full Text] [PDF]

				P. G. Yancey, W. G. Jerome, H. Yu, E. E. Griffin, B. E. Cox, V. R. Babaev, S. Fazio, and M. F. Linton Severely altered cholesterol homeostasis in macrophages lacking apoE and SR-BI J. Lipid Res., May 1, 2007; 48(5): 1140 - 1149. [Abstract] [Full Text] [PDF]

				F. Samad, K. D. Hester, G. Yang, Y. A. Hannun, and J. Bielawski Altered Adipose and Plasma Sphingolipid Metabolism in Obesity: A Potential Mechanism for Cardiovascular and Metabolic Risk Diabetes, September 1, 2006; 55(9): 2579 - 2587. [Abstract] [Full Text] [PDF]

				A. Nilsson and R.-D. Duan Absorption and lipoprotein transport of sphingomyelin J. Lipid Res., January 1, 2006; 47(1): 154 - 171. [Abstract] [Full Text] [PDF]

				H. Yu, W. Zhang, P. G. Yancey, M. J. Koury, Y. Zhang, S. Fazio, and M. F. Linton Macrophage Apolipoprotein E Reduces Atherosclerosis and Prevents Premature Death in Apolipoprotein E and Scavenger Receptor-Class BI Double-Knockout Mice Arterioscler. Thromb. Vasc. Biol., January 1, 2006; 26(1): 150 - 156. [Abstract] [Full Text] [PDF]

				K. J. Williams and I. Tabas Lipoprotein Retention--and Clues for Atheroma Regression Arterioscler. Thromb. Vasc. Biol., August 1, 2005; 25(8): 1536 - 1540. [Abstract] [Full Text] [PDF]

				Z. Zhao, M. C. de Beer, L. Cai, R. Asmis, F. C. de Beer, W. J.S. de Villiers, and D. R. van der Westhuyzen Low-Density Lipoprotein From Apolipoprotein E-Deficient Mice Induces Macrophage Lipid Accumulation in a CD36 and Scavenger Receptor Class A-Dependent Manner Arterioscler. Thromb. Vasc. Biol., January 1, 2005; 25(1): 168 - 173. [Abstract] [Full Text] [PDF]

				S.-y. Morita, M. Kawabe, A. Sakurai, K. Okuhira, A. Vertut-Doi, M. Nakano, and T. Handa Ceramide in Lipid Particles Enhances Heparan Sulfate Proteoglycan and Low Density Lipoprotein Receptor-related Protein-mediated Uptake by Macrophages J. Biol. Chem., June 4, 2004; 279(23): 24355 - 24361. [Abstract] [Full Text] [PDF]

				F. B. Kraemer and J. Suzuki Lipoprotein Receptors, Macrophages, and Sphingomyelinase Arterioscler. Thromb. Vasc. Biol., December 1, 2000; 20(12): 2509 - 2510. [Full Text] [PDF]

				S. W. Sakr, R. J. Eddy, H. Barth, F. Wang, S. Greenberg, F. R. Maxfield, and I. Tabas The Uptake and Degradation of Matrix-bound Lipoproteins by Macrophages Require an Intact Actin Cytoskeleton, Rho Family GTPases, and Myosin ATPase Activity J. Biol. Chem., September 28, 2001; 276(40): 37649 - 37658. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marathe, S. Articles by Tabas, I. Search for Related Content PubMed PubMed Citation Articles by Marathe, S. Articles by Tabas, I. Related Collections Animal models of human disease Cell biology/structural biology Lipid and lipoprotein metabolism Other Vascular biology