Effect of macrophage-derived apolipoprotein E on hyperlipidemia and atherosclerosis of LDLR-deficient mice

Effect of Macrophage-Derived Apolipoprotein E onEstablished Atherosclerosis in

Apolipoprotein E–Deficient MiceWeibin Shi, Xuping Wang, Nicholas J. Wang, William H. McBride, Aldons J. Lusis

Abstract—Apolipoprotein E–deficient (apoE2/2) mice have hyperlipidemia and develop spontaneous atherosclerosis in atime-dependent manner. Although macrophage-derived apoE has been shown to prevent the development ofatherosclerosis in apoE2/2 mice, whether it would induce regression of established atherosclerosis is unknown. Todetermine this, 8-week-old apoE2/2 mice were transplanted with apoE1/1 bone marrow. Four weeks after transplanta-tion, when plasma cholesterol levels had reached normal levels, a group of mice (n512) were killed and their aorticlesions were measured and used as a baseline to judge regression. Twelve and 20 weeks after transplantation, aorticlesion areas of the mice were 934062184mm2 (mean6SEM, n58) and 12 21161433mm2 (n59), respectively, valuesnot significantly different from the lesion areas of the baseline mice (12 34762487mm2; n512, P.0.05). In contrast,apoE2/2 mice reconstituted with apoE2/2 bone marrow developed severe atherosclerotic lesions (453 036629 767mm2,n57) 20 weeks after transplantation. These data suggest that macrophage-derived apoE was insufficient to inducesignificant regression of established atherosclerotic lesions in apoE2/2 mice, although it was sufficient to eliminatehypercholesterolemia and prevent progression of aortic lesions.(Arterioscler Thromb Vasc Biol. 2000;20:2261-2266.)

Key Words: atherosclerosisn macrophagesn apolipoprotein En regressionn bone marrow transplantation

Previous studies have provided evidence that atheroscle-rosis can undergo regression in both humans and animal

models,1–4 but results have varied greatly, depending on theanimal models used or the population investigated.5–7 Miceare increasingly used in the study of atherosclerosis, andcertain mice such as apoE-deficient (apoE2/2) mice developall phases of lesions found in humans.8,9 Recently, severalstudies have reported that atherosclerotic lesions of mice canundergo considerable regression.10–16

ApoE is a 34-kD glycoprotein that plays an important rolein lipoprotein metabolism.17 It mediates uptake and degrada-tion of chylomicron and VLDL remnants by acting as aligand for the LDL receptor and the LDL receptor–relatedprotein.17,18 Although the vast majority of plasma apoE isderived from the liver,19–22 apoE is also synthesized bymacrophages in various organs.23,24 ApoE deficiency resultsin severe hypercholesterolemia and diffuse atheroscleroticdisease in humans25 and in gene-targeted mice.8,9 Targetedmice develop foam cell–rich fatty streaks in the aortic sinusand proximal aorta by the age of 3 months, and after 5months, fibrous lesions are present.26,27 Because apoE issynthesized by monocytes/macrophages23,24 but not by gran-ulocytes and lymphocytes,28 bone marrow transplantation(BMT) has been used to examine the role of macrophage-de-rived apoE in atherosclerosis in vivo. Recent BMT studies

have shown that macrophage-derived apoE results in thenormalization of serum cholesterol levels and prevents thedevelopment of atherosclerosis in apoE2/2 mice.29–31 How-ever, it is unknown whether macrophage-derived apoE caninduce regression of established atherosclerotic lesions. Thepurpose of the present study was to determine the effect ofmacrophage-derived apoE on established atherosclerosis inapoE2/2 mice by BMT.

MethodsMiceApoE2/2 C57BL/6J mice and wild-type C57BL/6J mice were pur-chased from the Jackson Laboratory, Bar Harbor, Me. Mice were fedwith a regular chow diet and maintained in a temperature-controlledroom with a 12-hour light/dark cycle. All procedures were inaccordance with current National Institutes of Health guidelines andwere approved by the UCLA Animal Research Committee.

Bone Marrow TransplantationFemale recipient apoE2/2 mice were lethally irradiated with a dose of1000 rads from a cobalt source. Bone marrow cells were harvestedby flushing the femurs and tibias of male donor mice with Dulbec-co’s modified Eagle’s medium containing 10% fetal bovine serumand 5 U/mL heparin. Red blood cells were lysed with ACK buffer(150 mmol/L NH4Cl, 61 mmol/L KHCO3, and 1 mmol/L Na2EDTA,pH 7.3). The remaining cells were washed and suspended inDulbecco’s modified Eagle’s medium with 1% bovine albumin.

Received June 8, 2000; revision accepted July 17, 2000.From the Department of Medicine (W.S., X.W., N.J.W., A.J.L.), Department of Microbiology and Molecular Genetics, and the Department of Radiation

Oncology (W.H.M.), School of Medicine, University of California, Los Angeles.Correspondence to Aldons J. Lusis, Department of Medicine, UCLA School of Medicine, 47-123 CHS, Los Angeles, CA 90095-1679. E-mail

[email protected]© 2000 American Heart Association, Inc.

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Each recipient mouse was injected with 107 bone marrow cells in 0.3mL through the tail vein.

Experimental ProtocolsAt 8 weeks of age, female apoE2/2 mice (n529) were reconstitutedwith bone marrow cells from wild-type male C57BL/6J mice(apoE1/13apoE2/2) and maintained on a chow diet. Four weeks aftertransplantation, a group of mice (n512) were killed and served as thebaseline group by which to judge regression of atheroscleroticlesions. The remaining mice were killed at 12 (n58) or 20 (n59)weeks after transplantation for lesion analyses. In addition, a groupof age-matched, female apoE2/2 mice were transplanted with bonemarrow from male apoE2/2 mice (apoE2/23apoE2/2) and main-tained on a chow diet for 20 weeks (n57).

Western Blot Analysis for ApoEThe presence of apoE in plasma was determined by Western blotanalysis. In brief, 1mL of plasma was separated by electrophoresison 12% SDS polyacrylamide gels and electrophoretically transferredto nitrocellulose membranes. The membranes were incubated with apolyclonal rabbit anti-mouse apoE antibody (BioDesign Interna-tional) for 1 hour and then incubated for 0.5 hour with a horseradishperoxidase–conjugated anti-rabbit secondary antibody. The signalswere detected by the enhanced chemiluminescent detection methodaccording to the manufacturer’s instructions (ECL Western blotting,Amersham).

DNA Preparation and PCR ofMale-Specific SequencesOvernight-fasted mice were bled from the retro-orbital vein underisoflurane anesthesia. After centrifugation, the plasma was collectedand used for lipid analyses as indicated below. The blood cell pelletwas lysed in ACK buffer to remove red blood cells. DNA wasprepared by adding polymerase chain reaction (PCR) solution(10 mmol/L Tris, pH 8.0; 2.5 mmol/L MgCl2; 1% Tween-20; and 0.4mg/mL proteinase K) to each leukocyte pellet and incubating theresulting mixture at 60°C for 2 hours followed by a 95°C incubationfor 20 minutes. PCR was performed to amplify a 250-bp sequence ofthe Y chromosome.32 The upstream primer was 59-GAG GGC CATGTC AAG CGC CCC ATG AATG-39and the downstream primerwas 59-AGA CAC TGT GAA ATC GGG AGG CT-39. The cyclingconditions were denaturing for 1 minute at 94°C, annealing for 1minute at 62°C, and extension for 1 minute at 72°C.

Aortic Lesion AnalysisMethods for the quantification of atherosclerotic lesions in the aortawere done as previously reported.33 In brief, the heart and proximalaorta were excised and embedded in OCT compound. Serial 10-mm-thick cryosections from the middle portion of the ventricle to theaortic arch were collected and mounted on poly-D-lysine–coatedslides. In the region from the appearance to the disappearance of theaortic valves, every other section was collected. In all other regions,every fifth section was collected. Sections were stained with oil redO and hematoxylin, counterstained with fast green, and examined bylight microscopy.

Immunohistochemical Analyses ofAtherosclerotic LesionsImmunohistochemical analyses of atherosclerotic lesions in theaortic root were performed as previously described.33 In brief,10-mm-thick cryosections were fixed in acetone and incubated witha rabbit polyclonal antibody to mouse apoE or a rat monoclonalantibody to mouse macrophages, MOMA-2 (Accurate Chemicals),followed by incubation with biotinylated anti-rabbit or anti-ratsecondary antibodies. Signals were detected with peroxidase chro-mogen kits (Vector Laboratories). We used an FITC-labeled poly-clonal antibody to human smooth muscle cell actin (Sigma) to detectsmooth muscle cells in the lesions.

Plasma Lipid MeasurementsEnzymatic assays for total cholesterol, HDL cholesterol, and triglyc-eride were performed in 96-well plates on a Biomek 2000 automatedlaboratory workstation (Beckman Instruments, Inc) as described.34

Measurements on plasma samples were performed in triplicate withknown control samples on each plate to ensure accuracy.

Statistical AnalysisPlasma lipid levels were expressed as mean6SEM, with n indicatingthe number of mice. Atherosclerotic lesion areas were expressed asvalues of individual mice. ANOVA was used to compare differencesin atherosclerotic lesions and lipid levels among different groups ofmice over time. Differences were considered statistically significantat P,0.05.

ResultsReconstitution of Recipient Bone MarrowApoE was detected by Western blot analysis in the plasma ofapoE2/2 mice reconstituted with wild-type bone marrow asearly as 2 weeks after transplantation (Figure 1A). In contrast,apoE was absent in apoE2/2 mice reconstituted with apoE2/2

bone marrow. Moreover, because male mice containing XYchromosomes were used as donors for the female recipients,we designed primers to genotype a segment of the Ychromosome. As shown in Figure 1B, 2 weeks after trans-plantation, the Y chromosome was detected by PCR inperipheral leukocytes of recipient mice.

Effect of BMT on Plasma LipidsReconstitution of apoE2/2 mice with wild-type bone marrowresulted in dramatic changes in plasma cholesterol andtriglyceride levels (Figure 2). Two weeks after transplanta-tion, plasma total cholesterol and triglyceride levels weresignificantly reduced and HDL cholesterol levels increased(P,0.05). By 4 weeks after transplantation, plasma totalcholesterol, triglyceride, and HDL cholesterol levels hadreached normal levels. In contrast, in apoE2/23apoE2/2

mice, plasma total cholesterol levels gradually increased,from 334611 mg/L (mean6SEM) before transplantation to556624 mg/L 20 weeks after transplantation. There was asignificant decrease in triglyceride levels 2 weeks after

Figure 1. A, Western blot analysis of plasma apoE in apoE2/2

mice 2 weeks after BMT. One microliter of undiluted plasmawas electrophoresed on 12% SDS gels, transferred to nitrocel-lulose membranes, and probed with a polyclonal antibody tomouse apoE. Lanes 1 through 6, mice transplanted with wild-type bone marrow; lanes 7 through 9, mice transplanted withapoE2/2 bone marrow; lane 10, positive control (plasma fromwild-type mice). B, PCR of DNA extracted from the peripheralblood of female apoE2/2 mice 2 weeks after transplantation toamplify a 250-bp sequence of the Y chromosome. Lane 1,100-bp ladder; lanes 2 through 8, samples from transplantedmice; lane 9, negative control (female mouse DNA); lane 10,positive control (male mouse DNA).

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transplantation (P,0.05). Plasma HDL cholesterol levelswere not significantly altered after transplantation.

Effect on Aortic Atherosclerotic Lesion AreasThe size of atherosclerotic lesions at the aortic root wasquantified by light microscopy. The apoE1/13apoE2/2 micethat were killed 4 weeks after transplantation had an averagelesion area per section of 12 34762487mm2 (mean6SEM,n512; Figure 3). Twelve and 20 weeks after transplantation,the average lesion areas of apoE1/13apoE2/2 mice were934062185mm2 (n58) and 12 21161433mm2 (n59), re-spectively. Compared with mice that were killed 4 weeksafter transplantation, aortic lesion areas did not show asignificant increase or decrease in mice that were killed at 12and 20 weeks after transplantation (P.0.05). In contrast,apoE2/23apoE2/2 mice developed severe atherosclerosis 20weeks after transplantation, with an average lesion area of453 036629 767mm2 per section (n57).

Effect on Morphology of Atherosclerotic LesionsIn mice that were killed 4 weeks after transplantation, aorticlesions consisted primarily of macrophage-derived foam cells(Figure 4). Smooth muscle cells were undetectable in the

lesions, and there were no fibrous caps. In mice that werekilled 12 weeks and 20 weeks after transplantation, aorticlesions were flatter and had developed thin, fibrous caps inabout half of the mice. Macrophage-derived foam cells werestill the main cellular component of the lesions. Smoothmuscle cells were observed in the fibrous caps. In contrast,mice reconstituted with apoE2/2 bone marrow developedadvanced lesions containing numerous smooth muscle cells,calcification, and necrotic areas.

ApoE Expression in Atherosclerotic LesionsImmunohistochemistry analysis showed that apoE was abun-dantly expressed in atherosclerotic lesions of the apoE2/2

mice reconstituted with wild-type bone marrow (Figure 5). Incontrast, apoE was not detected in lesions of those apoE2/2

mice reconstituted with apoE2/2 bone marrow.

DiscussionThe principal aim of the present study was to evaluatewhether macrophage-derived apoE was sufficient to inducesignificant regression of established atherosclerotic lesions inapoE-deficient mice by BMT. Wild-type and apoE-deficientmice were used as bone marrow donors for apoE-deficientmice. We found that repopulation of cells in BMT-treatedmice with normal hematopoietic cells eliminated hypercho-lesterolemia and prevented progression of the lesions but didnot induce significant regression of established atherosclero-sis in apoE-deficient mice.

BMT leads to the replacement of recipient tissue macro-phages by macrophages of donor origin.35 Bone marrow cellsaccumulate in the liver, spleen, and bone marrow severalhours after injection.36,37 By 4 weeks after transplantation,.95% of macrophages in the bone marrow were of donor

Figure 2. Effects of BMT on plasma total cholesterol (A), HDLcholesterol (B), and triglyceride (C) levels in apoE2/2 recipientmice. Plasma lipid levels were measured before and 2, 4, 12,and 20 weeks after transplantation. ApoE2/2 mice were trans-planted with bone marrow from either apoE1/1 or apoE2/2 mice.Values are mean6SE of 7 to 12 mice. *P,0.05 vsapoE2/23apoE2/2.

Figure 3. Atherosclerotic lesion areas in cross sections of aorticroot from apoE2/2 mice transplanted with apoE1/1 or apoE2/2

bone marrow. Each point represents a mean lesion area persection from 1 mouse. Mice were fed a chow diet during theexperiment and were killed at 4, 12, and 20 weeks after trans-plantation. ApoE2/232/2 represents mice transplanted withapoE2/2 bone marrow and killed 20 weeks after transplantation.

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origin.29 In the present study, we observed that apoE waspresent in the peripheral circulation as early as 2 weeks aftertransplantation, and by 4 weeks, macrophage-derived apoEwas sufficient to normalize plasma lipid levels of apoE-deficient recipient mice. As in previous studies,38,39 we usedmale mice as bone marrow donors for female recipients sothat engraftment of bone marrow could be verified bygenotyping male-specific makers of the Y chromosome.Because donor and recipient mice were derived from thesame inbred strain, graft-versus-host disease would not occur.Moreover, all recipient mice were healthy throughout theexperiment.

Although macrophages produce only a small percentage('10%) of plasma apoE, this amount is sufficient to reversehypercholesterolemia and elevate plasma HDL levels.29,30

Our present findings are consistent with this observation. Thefinding that BMT induced a decrease of plasma triglyceridelevels in apoE1/13apoE2/2 mice is consistent with that of aprevious study.31 The temporary decrease at 2 weeks wasprobably caused by the transplantation procedure, because itwas also observed in apoE2/23apoE2/2 mice.

Previous studies29–31 and our present study have indicatedthat after transplantation, a period of 4 weeks is required formacrophage-derived apoE to achieve its full therapeutic

effect in apoE-deficient recipients. During this period, asperipheral tissues are reconstituted with apoE-expressingmacrophages, plasma cholesterol levels gradually fall butatherosclerotic lesions continue to progress. We observed asignificant increase in atherosclerotic lesion size, from26136781 mm2 at the time of transplantation to12 34762487mm2 4 weeks after transplantation. Therefore,selecting atherosclerotic lesions at 4 weeks after transplanta-tion as the baseline seems an appropriate standard by whichto judge regression in apoE-deficient mice. In the presentexperiment, apoE-deficient mice received transplantation at 8weeks of age, and by the time their bone marrow wasreplaced with donor marrow, they were 12 weeks of age.Nakashima et al27 reported that apoE-deficient mice of thisage develop fatty streak lesions. Indeed, we observed thatatherosclerotic lesions of these mice consisted primarily ofmacrophage-derived foam cells and that smooth muscle cellsand fibrous caps were absent in the lesions.

One important finding of the present study is that 16 weeksafter plasma lipid levels were normalized, the size of theatherosclerotic lesions was not significantly reduced in apoE-deficient mice. This finding is consistent with the notion thatthe regression of atherosclerotic lesions is a slow process.Indeed, in the rhesus monkey model, Tucker et al5 did not

Figure 4. Representative light photomi-crographs of aortic atherosclerotic lesionsin apoE2/2 mice transplanted withapoE1/1 or apoE2/2 bone marrow. Sec-tions were stained with oil red O andhematoxylin (A, B, C), an anti-mousemacrophage antibody MOMA-2 (D, E, F),or an FITC-labeled antibody to smoothmuscle cell actin (G, H, I). The first andsecond columns represent lesions frommice transplanted with apoE1/1 bonemarrow and killed at 4 and 20 weeks,respectively, after transplantation. Thethird column represents lesions from micetransplanted with apoE2/2 bone marrowand killed at 20 weeks after transplanta-tion. Original magnification, 325.

Figure 5. Immunohistochemical analysis ofapoE expression in aortic atheroscleroticlesions of apoE2/2 mice transplanted withapoE2/2 (A) or wild-type (B) bone marrow.Sections were stained with a polyclonal rabbitanti-mouse apo E antibody. Original magnifi-cation, 340.

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find definite evidence of regression after 4 months on alow-fat diet. Kokatnur et al40 reported that experimentalatherosclerosis in rhesus monkeys showed evidence of re-gression only after treatment with a low-fat diet for 64 weeks.However, Tsukamoto et al16 recently reported that liver-directed gene transfer and hepatic expression of humanapoE3 in chow-fed, apoE-deficient mice resulted in an almostcomplete regression of fatty streaks within 6 weeks, whereasexpression of human apoE4 reduced cholesterol levels to thesame extent as apoE3 but did not induce significant regres-sion. That study suggests that effects beyond the reduction ofplasma cholesterol levels are required to induce regression.Mouse apoE is similar to human apoE3 in term of the 2polymorphic amino acids.41 The reasons for the discrepancybetween the data of Tsukamoto et al16 and ours are unclear.One possible explanation is that gene transfer resulted inmore apoE production than did BMT. Indeed, Desurmont etal11 observed that regression of fatty streak lesions in apoE2/2

mice 6 months after injection of the adenovirus encodinghuman apoE cDNA was dependent on plasma apoEconcentration.

Hyperlipidemia plays an important role in the progressionof atherosclerosis. However, elimination of hyperlipidemiaalone seems insufficient to induce regression of atheroscle-rosis. Indeed, our failure to observe a significant reduction ofatherosclerotic lesions 16 weeks after normalization ofplasma lipid levels suggests that treatments other than nor-malizing plasma lipid levels are necessary to induce signifi-cant regression of atherosclerosis.

AcknowledgmentsThis work was supported by National Institutes of Health grantHL-30568. The authors thank Yishou Shi for technical help.

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Weibin Shi, Xuping Wang, Nicholas J. Wang, William H. McBride and Aldons J. LusisDeficient Mice−Apolipoprotein E

Effect of Macrophage-Derived Apolipoprotein E on Established Atherosclerosis in

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