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(Journal of Leukocyte Biology. 2001;69:732-740.)
© 2001 by Society for Leukocyte Biology

Mixed allogeneic chimerism with wild-type strains ameliorates atherosclerosis in apolipoprotein E-deficient mice

Naoki Ishimori*,{dagger}, Kazuya Iwabuchi*, Satoshi Fujii{dagger}, Keiko Watano*,{dagger}, Chikako Iwabuchi*, Manabu Ato*, Hitoshi Chiba{ddagger}, Shinya Tanaka§, Akira Kitabatake{dagger} and Kazunori Onoé*

* Division of Immunobiology, Research Section of Pathophysiology, Institute for Genetic Medicine, and Departments of
{dagger} Cardiovascular Medicine,
{ddagger} Laboratory Medicine, and
§ Molecular and Cellular Pathology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan

Correspondence: Kazunori Onoé, Division of Immunobiology, Research Section of Pathophysiology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. E-mail: kazunori{at}imm.hokudai.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atherosclerosis involves inflammatory processes between vascular tissues and hematocytes with a hyperlipidemic background. To examine whether variations of hematocytes constitute one of the genetic components in atherosclerosis, irradiated apolipoprotein E (apoE)-deficient (apoE-/-) mice with hypercholesterolemia and preexisting atherosclerotic lesions were reconstituted with mixed bone marrow cells (BMC) from syngeneic and wild-type (apoE+/+; atherosclerosis-resistant SJL or -susceptible B10.S) mice. Stable mixed allogeneic chimeras with small amounts of serum apoE were established without any detrimental complications. Compared with untreated apoE-/- mice or apoE-/- mice transplanted with syngeneic BMC alone, significant reduction of the cholesterol level and significant lesion regression were observed in the mixed chimeras. Furthermore, mixed chimeras given SJL BMC showed marked reductions in numbers of lesions compared with those reconstituted with B10.S BMC. Cholesterol levels in the former SJL chimeras, however, were significantly higher than those in the latter B10.S chimeras. These findings indicate that the resistance of SJL to atherosclerosis resides in the bone marrow-derived cells.

Key Words: bone marrow transplantation • bone marrow-derived cell • gene transfer • hypercholesterolemia • inbred strains • macrophage


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atherosclerosis, a principal contributor to coronary heart disease (CHD) and stroke, is often associated with disordered lipid metabolism. Although substantial gains were achieved through control of established risk factors, some patients suffering a CHD event have no obvious risk factors, suggesting the presence of unidentified risk factors. Atherosclerosis involves complex inflammatory processes between hematocytes and vascular tissues with a hyperlipidemic background [1 , 2 ]. In the earliest lesion of atherosclerosis (fatty streak), lipid-rich macrophages and T lymphocytes are recognized. In the advanced atheroma (fibrous plaques), aggregation of activated monocyte-derived macrophages, smooth muscle cells, and T lymphocytes develops into complex occlusive lesions [3 , 4 ]. Inflammatory cells, primarily monocyte-derived macrophages, play a pivotal role in the lesion development. Macrophages are present in all stages of atherosclerotic lesions, and they remove oxidized low-density-lipoprotein cholesterol (LDL-Cho) and produce growth-regulating molecules and cytokines. Thus, elucidation of the precise role of macrophages in the development of atherosclerosis appears to provide insights for a novel therapy for CHD.

Apolipoprotein E (apoE)-deficient (apoE-/-) mice [5 , 6 ] show spontaneous elevation of total plasma cholesterol and develop prominent atherosclerotic lesions simulating those in humans in a time-dependent manner [3 , 4 ]. Use of the mouse as an experimental model of atherosclerosis has many advantages because the genetic profile of this animal has been well characterized [7 8 9 10 11 ].

In the present study, to examine whether variations of hematocytes constitute one of the genetic components in atherosclerosis, mixed-allogeneic-chimera mice were established using combinations of apoE-/- mice as recipients and two representative strains of wild-type mice as donors. The partial reconstitution of apoE-/- mice with wild-type (apoE+/+) bone marrow cells (BMC) normalized high-density-lipoprotein cholesterol (HDL-Cho) levels and markedly reduced the atherogenic non-HDL-Cho level and atheroma formation in comparison with those observed in untreated apoE-/- mice or apoE-/- mice reconstituted with syngeneic BMC alone. Furthermore, compared with the mixed chimeras reconstituted with atherosclerosis-susceptible B10.S BMC, lesion formation was remarkably reduced in apoE-/- mice reconstituted with atherosclerosis-resistant SJL BMC, even though the cholesterol levels were significantly higher in the latter than in the former. Our results demonstrate for the first time that BMC-derived cells are involved in the regression of advanced atherosclerosis independently of the serum lipoprotein metabolism and vascular cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
C57BL/6J apoE-deficient mice (apoE-/- H-2b) and wild-type SJL/J (SJL; H-2s) were purchased from The Jackson Laboratories (Bar Harbor, ME). Wild-type B10.S/SgSlc (B10.S; H-2s) mice were purchased from Japan SLC (Hamamatsu, Japan). All mice were weaned at 4 weeks of age, and then a standard rodent chow diet (4.3% fat; Nihon-nohsan, Yokohama, Japan) and autoclaved water were provided ad libitum. The animals were housed in a temperature-controlled facility maintained in a room illuminated from 8 AM to 9 PM under specific-pathogen-free conditions. The animal care and experimental procedures conformed to the regulations of Hokkaido University Animal Care and Use Committee. After overnight fasting, mice were anesthetized with diethyl ether prior to collection of blood samples by axillary artery incision, and serum was obtained by centrifuging the blood samples for 5 min at 5,000 g and 4°C.

Irradiation and bone marrow transplantation
As recipients, female 28- to 29-week-old apoE-/- mice were subjected to 11 Gy of total body irradiation. Bone marrow transplantation (BMT) was performed as previously described [12 ]. In brief, BMC were obtained from either SJL mice or B10.S mice and from syngeneic apoE-/- mice. To deplete mature T cells, the BMC were treated with anti-Thy1.2 monoclonal antibody (mAb) (F7D5; Serotec, Bicester, UK) plus rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada). Then, recipient mice were injected via the tail vein with SJL or B10.S BMC (1.5 x 107 each) plus BMC from syngeneic apoE-/- mice (0.5 x 107) [13 ]. For 4 weeks after BMC injection, 300 mg/L of Terramycin (Pfizer, Tokyo, Japan) were added to the drinking water. These mixed-bone-marrow-chimera mice were referred to as either [SJL + apoE-/-->apoE-/-] or [B10.S+apoE-/-->apoE-/-]. Untreated apoE-/- mice and apoE-/- mice irradiated and reconstituted with syngeneic T-cell-depleted BMC (2 x 107 each) served as controls (henceforth referred to as apoE-/-,NT and [apoE-/-->apoE-/-], respectively).

Serum cholesterol and lipoprotein analyses
Total cholesterol (T-Cho) and triglyceride (TG) in the serum were measured by enzymatic methods using commercial kits from Kyowa Medex (Tokyo, Japan) and Serotekku (Sapporo, Japan), respectively, according to the manufacturers’ protocols. For HDL-Cho determination, each serum sample was mixed with an equal volume of aqueous 13% (w/v) polyethylene glycol 6000 (Wako Pure Chemicals, Osaka, Japan) and centrifuged for 15 min at 2,000 g and room temperature, as described elsewhere [14 ]. T-Cho in each supernatant was measured as the HDL-Cho. We confirmed by agarose gel electrophoresis that mouse ß- and pre-ß-migrating serum lipoproteins (non -HDL lipoproteins) were completely precipitated by this procedure, whereas {alpha}-migrating lipoproteins (HDL lipoproteins) were completely recovered in the supernatant. The amount of non-HDL-Cho was determined by subtracting HDL-Cho from T-Cho. All assays were performed within the linear range of the standard curve.

Flow cytometry
Heparinized mouse blood (0.3 mL) was centrifuged at 1,400 g for 5 min at 4°C, and each blood sample was incubated with 3 mL of NH4Cl, pH 7.2, for 20 min at room temperature to lyse the red blood cells. The remaining leukocytes were washed with phosphate-buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin and 0.1% NaN3 (wash buffer). The cells were stained with a biotinylated H-2Kb-specific mAb (E121.46; PharMingen, San Diego, CA). Then the cells were washed, incubated with a fluorescein isothiocyanate-conjugated H-2Ks-specific mAb (KH49; PharMingen) and phycoerythrin-conjugated streptavidin (Biomeda, Foster City, CA), and analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using Cell Quest (Becton Dickinson) software [15 ]. The proportion of SJL- or B10.S -derived cells in the mixed chimera (henceforth referred to as the chimerism) was calculated by using the following formula: (number of H-2Ks-positive cells)/[(number of H-2Ks-positive cells) + (number of H-2Kb-positive cells)] x 100.

Preparation and examination of aortae for atherosclerosis
To qualitatively assess the extent of atherosclerosis, mice were sacrificed 10 weeks after BMT. Evaluation of aortic lesions was described in detail elsewhere [16 ]. Briefly, the remaining blood was removed by perfusing the left ventricle with PBS (pH 7.4). The heart, together with the thoracic aorta, was removed from each of the animals and placed in 0.9% saline for at least 0.5 h. The heart was fixed in 10% neutral buffered formaldehyde and embedded by using a JB-4 embedding kit (Polysciences, Warrington, PA). Ten-micrometer-thick cross-sections were made, beginning with the lower portions of atria and continuing toward the aortic arch, and then fixed on silane-coated microscope slides (Muto Pure Chemicals, Tokyo, Japan) as described previously [16 ]. All sections were examined by microscopy prior to staining. For evaluation of the lesions, five sections were obtained, at 80-µm intervals, between the end of the aortic sinus and the junction site of the sinus and ascending aorta, which were determined by the presence of three valve cusps, and a rounded aorta, where the lesions were reproducibly most prominent. Sections were allowed to dry for a few days at room temperature. Foam cell and lipid deposition in the extracellular matrix were stained with Oil Red O (Sigma Chemical Co., St. Louis, MO) and hematoxylin, whereas collagen fiber and elastic fiber were stained with Masson’s trichrome and resorcin-fuchsin, respectively. Lesions were quantified by using a computerized image analysis system (Olympus, Tokyo, Japan) equipped with Power Movie MP/V (Canopus, Kobe, Japan), PhotoShop 4.0 (Adobe, San Jose, CA), and Scion Image (Scion, Frederick, MD) software. The areas of all lesions in each section were added together to give a total lesion area (in mean square micrometers) per section. The mean lesion area per section per animal was also calculated for each group of animals. The lesion area was measured and evaluated in a blinded manner.

Western blotting for apoE
Serum levels of apoE were determined by protein immunoblotting with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Briefly, serum albumin was partially eliminated from the serum by using CENTRICON centrifugal filter devices with YM-100 membranes (Millipore, Bedford, MA). Serum sample aliquots of 2.5 µL were separated on a sodium dodecyl sulfate–12% polyacrylamide gel. After being blotted and then blocked with 5% skim milk, the filter was incubated with a polyclonal rabbit antiserum to rat apoE that cross-reacts strongly with mouse apoE [17 ]. The secondary antibody was a goat antiserum to rabbit immunoglobulin G. Detection of bands was achieved by chemiluminescence with an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ). Serum apoE levels in mixed chimeras were compared with those of diluted sera of various control mice by using PhotoShop and Scion Image software.

In vitro foam cell assay
Peritoneal resident cells were harvested by washing the peritoneal cavities of 7- to 10-week-old female wild-type (SJL or B10.S) mice with 15-mL volumes of cold Hanks’ balanced salt solution. Similar numbers of cells were obtained from the two strains of mice. Cells (6.7 x 105/mL) were cultured in RPMI 1640 supplemented with 10% fetal calf serum (culture medium) on sterile glass-based dishes (Iwaki, Funabashi, Japan) for 72 h, and nonadherent cells were removed by washing with RPMI 1640. Adherent macrophages were incubated for the various time periods in 0.2 mL of culture medium with 10 µg of DiI-acetyl LDL (Biomedical Technologies, Stoughton, MA)/mL at 37°C. At the end of the incubation, the cells were fixed in 3% paraformaldehyde for 20 min at room temperature, rinsed with PBS for 5 s, and covered with a drop of 90% glycerol–10% PBS. Confocal laser microscopy was performed to quantitate the uptake of DiI-acetyl LDL into each cell. The amount of lipid uptake per cell (henceforth referred to as the fluorescence index) was calculated for each group of macrophages. The fluorescence index was measured and evaluated in a blinded manner.

Statistics
Results are expressed as means ±SE. Student’s t-test and analysis of covariance (ANCOVA) were performed on data for the chimerism, serum lipids, apoE levels, and atherosclerotic lesions by using Statview software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment of mixed-allogeneic-chimera mice
After 31 apoE-/- mice were subjected to lethal levels of radiation (11 Gy), 9 mice received BMT from SJL mice and syngeneic apoE-/- ([SJL+apoE-/-->apoE-/-]) mice, 10 mice received BMT from B10.S and syngeneic apoE-/- mice ([B10.S+apoE-/-->apoE-/-]) mice, and 12 mice received BMT from syngeneic apoE-/- ([apoE-/-->apoE-/-]) mice alone. The mixed-allogeneic-chimera mice survived for 10 weeks after BMT without overt graft-versus-host disease. Thus, it seemed that stable tolerance was induced in the immunocompetent cells, as we had reported earlier [18 , 19 ].

Indeed, chimerism was demonstrated in the peripheral blood leukocytes of these mixed allogeneic chimeras. Figure 1 shows the results of a representative experiment in which H-2Ks and H-2Kb antigens on the leukocytes were stained with specific mAbs. The majority of leukocytes from the [SJL+apoE-/-->apoE-/-] chimera were stained with anti-H-2Ks mAb, and ~10% were stained with anti-H-2Kb antibody (Fig. 1B) . Similarly, the majority of leukocytes from the [B10.S+apoE-/-->apoE-/-] chimera were stained with the anti-H-2Ks mAb (Fig. 1C) . No H-2Ks-positive leukocytes were observed in apoE-/-,NT mice (Fig. 1A) or [apoE-/-->apoE-/-] syngeneic chimeras (data not shown). The mean proportions of chimerisms seen in peripheral blood leukocytes of [SJL+apoE-/-->apoE-/-] and [B10.S+apoE-/-->apoE-/-] were 77.5% ± 8.8% (n = 9) and 70.9% ± 6.5% (n = 10), respectively. There was no statistically significant difference between [SJL+apoE-/-->apoE-/-] and [B10.S+apoE-/-->apoE-/-] chimeras in terms of mean proportion of chimerisms.



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  		Figure 1. Representative FACS analysis of chimerism of peripheral blood leukocytes. (A) apoE-/-,NT control. (B) [SJL+apoE-/-->apoE-/-] allogeneic chimera. (C) [B10.S+apoE-/-->apoE-/-] allogeneic chimera. Isolated leukocytes were reacted with fluorescein isothiocyanate-conjugated H-2Ks-specific mAb or biotinylated H-2Kb-specific mAb and phycoerythrin-conjugated streptavidin and analyzed on a FACScan flow cytometer.

 
Effect of chimerism on lipids and lipoprotein profile in apoE-deficient mice
To analyze the influence of the partial replacement of hematopoietic cells with wild-type cells on serum lipid levels, serum T-Cho, HDL-Cho, and TG levels were determined in chimeric mice 10 weeks after reconstitution. As seen in Table 1 , serum T-Cho levels in [SJL+apoE-/-->apoE-/-] and [B10.S+apoE-/-->apoE-/-] mice were reduced to 39% and 22%, respectively, of those levels in apoE-/-,NT mice. The mean reduction of T-Cho level in [B10.S+apoE-/-->apoE-/-] chimeras was significantly greater than that in [SJL+apoE-/-->apoE-/-] chimeras. In contrast, almost the same levels of HDL-Cho were detected in sera of both kinds of chimeras, and these levels were significantly higher than those in apoE-/-,NT mice and [apoE-/-->apoE-/-] syngeneic chimeras. Accordingly, non-HDL-Cho levels in [SJL+apoE-/-->apoE-/-] and [B10.S+apoE-/-->apoE-/-] mice were reduced to 29% and 11%, respectively, of the levels in apoE-/-,NT mice,. The mean reduction was significantly greater in [B10.S+apoE-/-->apoE-/-] mice than that in [SJL+apoE-/-->apoE-/-] mice. Thus, mixed allogeneic chimeras reconstituted with BMC from atherosclerosis-susceptible strain B10.S showed a profound reduction of atherogenic lipoproteins in comparison with those reconstituted with BMC of atherosclerosis-resistant SJL (Table 1) . The serum lipid levels in [apoE-/-->apoE-/-] syngeneic chimeras were essentially the same as those in apoE-/-,NT mice, although the mean HDL-Cho level in syngeneic chimeras was higher than that in apoE-/-,NT mice. No significant effect of the chimerism on the serum baseline TG concentration was observed in either the syngeneic and the allogeneic chimera mice.


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  		Table 1. Effect of Chimerism on Serum Cholesterol and Triglyceride Concentrations

 
Relationship between allogeneic donor chimerism and HDL-Cho or non-HDL-Cho and serum apoE expression in chimeras
The relationship between the donor chimerism and HDL-Cho levels or non-HDL-Cho levels in mixed-chimera mice was analyzed. No correlation of the HDL-Cho level with the chimerism was detected in either group of chimeras (Fig. 2 ).



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  		Figure 2. Correlation between chimerism and serum HDL-Cho. Correla-tion between allogeneic chimerism and serum HDL-Cho levels was analyzed in [SJL+apoE-/-->apoE-/-] mixed chimeras (open circles) and [B10.S+apoE-/-->apoE-/-] mixed chimeras (closed circles).

 
We then examined apoE production in sera of mixed chimeras. No apoE protein was detected in the sera of untreated apoE-/-,NT or [apoE-/-->apoE-/-] mice (Fig. 3 ). In both mixed allogeneic chimeras, small amounts of serum apoE proteins were detected. The mean apoE levels calculated for [SJL+apoE-/-->apoE-/-] and [B10.S+apoE-/-->apoE-/-] sera were 3.3% ± 0.4% (n = 9) and 3.0% ± 0.6% (n = 10) of those in normal SJL and B10.S mice, respectively. There was no statistical difference in the mean serum apoE level between [SJL+apoE-/-->apoE-/-] and [B10.S+apoE-/-->apoE-/-]. Thus, it seemed that amounts of apoE protein produced by hematopoietic cells were small in comparison with those produced in other tissue, such as liver. Small difference in the mobility of serum apoE seen between chimeras and wild-type mice appeared to be attributable to the different source of the apoE proteins (hematopoietic cells vs. mainly hepatocytes). These findings are consistent with a previous study showing that the difference is due to different degrees of glycosylation [20 ].



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  		Figure 3. Immunoblot analysis of serum apoE in allogeneic and syngeneic chimera mice 10 weeks after BMT. Aliquots (2.5 µL) of serum samples were subjected to sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis. Lanes 1 and 8, 2.5 µL of diluted sera (1:10) of wild-type SJL and B10.S, respectively; lanes 2–4, 6, and 7, 2.5 µL of undiluted serum from each chimera 10 weeks post-BMT; lane 5, 2.5 µL of undiluted serum from unmanipulated apoE-/- mice.

 
Effect of mixed allogeneic chimerism on atherosclerosis in apoE-/- mice
The chimeric animals were maintained for 10 weeks on a normal laboratory chow diet and the histological analysis of atherosclerosis in the aortic sinus and proximal aorta was performed in Oil Red O- or Masson’s trichrome-stained sections. The advanced atherosclerotic lesions had already been detected in the recipient apoE-/- mice (28 to 29 weeks old) before BMT (data not shown) [3 , 4 ]. Ten weeks later, the lesions in the apoE-/-,NT mice were increased in size and complexity. Figures 4A and 4B show that the lesions are present in the proximal aorta, beginning at the base of the aortic sinus and extending distally in the aorta, and that they are raised and contain a fibrous cap overlying a lipid-rich core, with foam cells, areas of necrosis, cholesterol clefts, and deposition of extracellular matrix containing lipid deposits in the intima. These are concordant with data from previous studies [3 , 4 ]. The mean lesion areas ± SD quantitatively evaluated per Oil Red O-stained section were 650,019 ± 63,808 µm2/section in apoE-/-,NT mice (n = 11). The aortic sinus and proximal aorta of an [apoE-/-->apoE-/-] mouse showed almost the same degree of atherosclerosis as that in apoE-/-,NT mice (Fig. 4C and D ). No differences were observed in the mean lesion areas of apoE-/-,NT and [apoE-/-->apoE-/-] mice (data not shown).



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  		Figure 4. Cross-sections of the aortic sinuses of chimeras and an untreated control mouse on a normal chow diet. Sections of the left-hand side (A, C, E, and G) were stained with Oil Red O and hematoxylin, and those of the right-hand side (B, D, F, and H) were stained with Masson’s trichrome and resorcin-fuchsin. (A and B) A typical lesion found in an apoE-/-, NT mouse. Large, advanced lesions are present in the aortic sinus. (C and D) Tissue sections from an [apoE-/-->apoE-/-] syngeneic chimera. In comparison with an Oil Red O-stained section of an apoE-/-,NT mouse, no significant differences are observed. (E–H) Tissue sections from [SJL+apoE-/-->apoE-/-] (E and F) and from [B10.S+apoE-/-->apoE-/-] (G and H) mixed chimeras. Mixed allogeneic chimeras show only a small focal lesion, localized to the aortic valve stump, with an oligocellular hyperplastic matrix. Bar = 500 µm. _art>

 
In contrast, the atheromas were exclusively localized to the base of the aortic valves in mixed allogeneic chimeras (Fig. 4E 4F 4G 4H) . These lesions were in a very stable state with low cellularity, almost no detectable foam cells, and no fibrous cap. Although the extracellular matrix content was increased in these lesions, no lipid-rich particles were observed. The mean lesion areas per section for the total region examined in the mixed-allogeneic-chimera [SJL+apoE-/-->apoE-/-] and [B10.S+apoE-/-->apoE-/-] mice were 30,712 ± 15,046 gmm2/section (n = 9) and 57,364 ± 23,023 µm2/section (n = 10), respectively. The mean lesion areas in the [SJL+apoE-/-->apoE-/-] and [B10.S+apoE-/-->apoE-/-] mice were reduced to 4.7% (P < 0.0001) and to 8.8% (P < 0.0001), respectively, of that of the apoE-/-,NT mice. These findings on the whole demonstrate that partial reconstitution of apoE-/- mice with wild-type BMC regresses impressively the preexisting atherosclerosis and that the effect of irradiation on the regression appears negligible.

Correlation between mean lesion area and donor mouse strain of BMT
Next, the correlation between non -HDL-Cho levels and the mean lesion area per section was analyzed. Figure 5 shows that the non-HDL-Cho levels in [B10.S+apoE-/-->apoE-/-] chimeras were considerably lower than those in [SJL+apoE-/-->apoE-/-] mice. Nevertheless, the mean lesion areas in both mixed chimeras were markedly reduced. In addition, it appeared that there was a correlation between the non-HDL-Cho levels and the mean lesion area, although the correlation was regarded as not statistically significant. It should be noted that the extent of atherosclerosis in the [SJL+apoE-/-->apoE-/-] mice was considerably less than that in [B10.S+apoE-/-->apoE-/-] mice. No significant difference was observed in the antiatherogenic HDL-Cho levels of these two groups. Then, to examine whether the donor strain of the BMT indeed determines the degree of resistance to atherosclerosis, we performed an ANCOVA with the degrees of allogeneic chimerism, the levels of apoE expression, and non -HDL-Cho as covariates. Compared with that of [B10.S+apoE-/-->apoE-/-] mice, the mean lesion area was significantly reduced in [SJL+apoE->apoE-/-] mice (P < 0.02 by ANCOVA). Thus, it was demonstrated that BMC-derived cells are responsible for development and regulation of atherosclerosis, independent of the serum lipoprotein metabolism.



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  		Figure 5. Correlation between the extent of atherosclerosis and non -HDL-Cho levels. The correlation was analyzed in [SJL+apoE-/-->apoE-/-] (open circles) and [B10.S+apoE-/-->apoE-/-] (closed circles) mixed chimeras. In both groups, the greater the reduction of non-HDL-Cho levels, the more profoundly the mean lesion area values were reduced, although the correlation was not statistically significant. Compared to that in [B10.S+apoE-/-->apoE-/-] chimeras, the extent of atherosclerosis in [SJL+apoE-/-->apoE-/-] chimeras was significantly reduced (P < 0.02 by ANCOVA).

 
Indeed, when lipid uptake was evaluated with resident peritoneal macrophages, macrophages derived from SJL mice took up lipids less efficiently than those from B10.S mice (Fig. 6 ). This finding appears to be related to the difference in the mean lesion areas of [SJL+apoE-/-->apoE-/-] and [B10.S+apoE-/-->apoE-/-] mice.



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  		Figure 6. Comparison of in vitro foam cell development in peritoneal resident macrophages of SJL and B10.S mice. Resident peritoneal macrophages isolated from female wild-type SJL (A) and B10.S (B) mice were cultured in the presence of 10 µg/mL of DiI-acetyl LDL for 0.5 h. Confocal laser microscopy was performed to quantitate the uptake of DiI-acetyl-LDL into each cell. The fluorescence index for indicated culture times was calculated for foam cells isolated from SJL (open bars) and B10.S (closed bars) (C). The mean fluorescence indices ± SE are shown in Fig. 6C . _art>

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, stable apoE-/- chimeric mice were established by reconstitution with mixed BMC from syngeneic apoE-/- (H-2b) and allogeneic SJL (H-2s) or B10.S (H-2s) mice. These mixed chimeras produced apoE protein in the serum. We found amelioration of hypercholesterolemia and marked regression of preexisting aortic atherosclerosis. Thus, allogeneic BMT from individuals with intact apoE may promote lipoprotein clearance and repair atherosclerosis in apoE-deficient individuals.

For potential clinical use, it is important to establish stable allogeneic chimerism without life-threatening complications. Mixed allogeneic chimerism, defined as engraftment of allogeneic, hematopoietic stem cells in the presence of the recipient hematopoietic cells, induces donor- and recipient-specific tolerance in the immune system [13 , 21 22 23 24 ]. Antigen-presenting cells derived from both donor- and recipient-type BMC migrate to the recipient thymus and eliminate T cells reactive to donor or recipient antigens (negative selection) [18 , 25 ]. Indeed, mixed chimerism is applicable in the treatment of autoimmune [22 ] and nonmalignant hematological [23 ] diseases and in the induction of tolerance to allogeneic grafts [24 ]. Nonlethal regimens for BMT were reported to reduce the excessive toxicity of total myeloablative conditioning [26 ]. In the present study, we demonstrated that partial replacement of the hematopoietic system of apoE-/- mice with that of the wild-type donor was sufficient for the absorption and/or remodeling of the preexisting atheromas. If rejection of allogeneic stem cells is appropriately regulated, partially myeloablative conditioning strategies may be preferable to minimize the toxicity related to total myeloablative conditioning.

Cells of a monocyte/macrophage lineage represent major apoE producers among hematopoietic cells, and the serum apoE level of apoE-/- mice after full reconstitution with wild-type syngeneic BMC ranges from undetectable [27 ] to 3.8% of normal [28 ] to 10–12% of normal values [29 ]. We demonstrated herein that [SJL+apoE-/-->apoE-/-] and [B10.S+apoE->apoE-/-] chimeras expressed serum apoE at levels ranging, respectively, from 2.2–5.4%, and from 1.0–5.1% of that of normal donor mice. The minimum concentration of serum apoE affecting cholesterol levels was approximately 0.04 mg/dL (~1% of normal levels) [30 ]. Thus, it seems that the serum apoE levels in our mixed chimeras are sufficient to reduce serum cholesterol levels. Recently, Hasty et al. [31 ] reported that reconstitution of apoE-/- mice with syngeneic BMC which had introduced intact apoE genes reduced early foam cell lesion formation but had no effect on the preexisting atherosclerosis. In contrast, our mixed chimeras showed a prominent regression of highly advanced atheromas. The serum apoE levels in syngeneic chimeras of Hasty et al. were approximately 0.5–1% of the levels of normal mice and were apparently lower than those in our mixed chimeras. Thus, the difference in the antiatherogenic efficacies of BMT seen by us and by Hasty et al. can be at least partly attributed to the difference in the serum apoE concentrations. Spangenberg and Curtiss [20 ] reported an influence of macrophage-derived apoE on serum lipoprotein metabolism, such as the compositional changes of HDL in apoE-/- mice. It seems that secretion of apoE by BMC-derived macrophages accelerates lipoprotein clearance, which results in amelioration of lipid metabolism and the prominent regression of preexisting lesions in our chimeric mice.

A direct effect of delaying atherogenesis has been attributed to apoE, independent of its role in serum lipoprotein clearance [31 32 33 ]. ApoE mediates reverse cholesterol transport, contributing to removal of cholesterol from lipid-rich foam cells for delivery to the liver. Thus, macrophage-derived apoE in our mixed allogeneic chimeras might also directly protect against the formation of atherosclerotic lesions. ApoE secreted by macrophages can stimulate cholesterol efflux from cholesterol-enriched macrophages in vitro [34 ], facilitate reverse cholesterol transport from the arterial wall in the presence of HDL [35 ], and/or inhibit lymphocyte proliferation as an immunoregulatory monokine [36 , 37 ].

In the present study, BMT from atherosclerosis-resistant SJL mice showed a considerable effect in terms of protection against and prevention of atherosclerosis compared with BMT from atherosclerosis-susceptible B10.S mice, even though serum levels of T-Cho and non-HDL were significantly higher in the former than the latter. Thus, functions of BMC-derived cells might influence lesion development independent of serum lipoprotein metabolism and clearance. Monocyte-derived macrophages play a pivotal role in foam cell formation [38 39 40 ]. Considering that macrophages are present in all stages of the atherosclerotic lesion, the differences seen in antiatherosclerotic effect between SJL and B10.S cells may be attributed to the functional differences of macrophages. Indeed, we showed that macrophages derived from SJL mice took up lipids less efficiently than those from B10.S mice. Furthermore, we reported previously that macrophage functions were determined by the genotype of precursors, but not by the recipient environment, in allogeneic bone marrow chimeras [41 ]. Thus, macrophages derived from SJL BMC might function in the apoE-/- recipients as they do in a normal SJL environment. Targeted disruption of class A [42 ] or class B [43 ] scavenger receptors protected against atherosclerotic lesion development in apoE-/- mice. Thus, these molecules may be important components in determination of susceptibility to atherosclerosis. When the molecular basis underlying the resistance represented by BMC-derived cells of SJL mouse is elucidated in further investigations, other factors, such as lipid modification, monocyte/macrophage differentiation and activation, chemotactic factors, and growth-regulatory molecules, should be taken into consideration.

Susceptibility to atherosclerosis differs among inbred strains of mice fed an atherogenic diet [44 ]. C58, C57BR, C57L, and C57BL mice, which have been derived from a common ancestor, are susceptible, whereas several strains such as A, C3H,and SJL are resistant. In addition, even in highly susceptible apoE-/- congenic mice, the genetic background determined the progression of atherosclerosis [45 ]. Genetic analysis of various recombinant inbred strains of mice has revealed multiple loci, a series of genes named ath1 to ath8, involved in the development of atherosclerosis [10 ]. A/J and C3H/HeJ mice, which carry a resistance allele in the ath1 locus, show low levels of serum T-Cho, moderate levels of serum HDL-Cho, and resistance to atherosclerosis [7 ]. Recently, Shi et al. [46 , 47 ] demonstrated that endothelial cells accounted for the difference in susceptibility to atherosclerosis. The atherosclerosis-resistant strain SJL mouse carries the other resistance locus, named ath7 [10 ]. Although details of the ath7 gene remain to be elucidated, studies by Paigen [10 ] and by us suggest that the ath7 gene determines the nature of BMC-derived cells in SJL mice. Thus, apoE-/- mice backcrossed with SJL (congenic) mice may serve as an appropriate model for future genomic studies to identify the ath7 gene. Our present study is, to the best of our knowledge, the first to show that the resistance to atherosclerosis presented by SJL mice resides in the BMC-derived cells and is independent of lipid metabolism and vascular tissues. Mixed allogeneic chimerism may be applicable to cell-mediated gene therapy for atherosclerotic disease.


   ACKNOWLEDGEMENTS
 
This study was supported in part by a Grant-in-Aid for Scientific Research awarded by the Ministry of Education, Science, Culture and Sports of Japan; by a Grant-in-Aid for Research on Immunology, Allergy and Organ Transplantation awarded by the Ministry of Health and Welfare, of Japan; by the Takeda Medical Research Foundation; and by grants from the Hokkaido Foundation for the Promotion of Scientific and Industrial Technology, the Tomakomai East Hospital Foundation, and the Nishimura Aging Fund.

The authors thank Ms. Yukiko Fujii for advice on pathological sample preparation, Ms. Mizuho Kasai for technical assistance, Dr. Eri Tsukishima for statistical support, and Ms. Kaori Kohno and Ms. Ryoko Hosohata for manuscript preparation.

Received November 8, 2000; revised January 2, 2001; accepted January 4, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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