* Division of Immunobiology, Research Section of Pathophysiology, Institute for Genetic Medicine, and Departments of
Cardiovascular Medicine,
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
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Key Words: bone marrow transplantation • bone marrow-derived cell • gene transfer • hypercholesterolemia • inbred strains • macrophage
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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.
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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
-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.
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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.
 View larger version (21K): [in a new window] |
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.
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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. |
View this table: [in a new window] |
Table 1. Effect of Chimerism on Serum Cholesterol and Triglyceride
Concentrations
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 View larger version (9K): [in a new window] |
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).
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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
].
 View larger version (31K): [in a new window] |
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.
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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).
 View larger version (80K): [in a new window] |
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>
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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+apoEapoE-/-] 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.
 View larger version (15K): [in a new window] |
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).
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apoE-/-] and
[B10.S+apoE-/-apoE-/-]
mice.
 View larger version (76K): [in a new window] |
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>
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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+apoEapoE-/-] 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.
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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ß+ thymocytes against a CD4+8+ thymocyte population associated with intact Fas antigen expression on the target J. Exp. Med. 180,423-432This article has been cited by other articles:
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  Y. Nakai, K. Iwabuchi, S. Fujii, N. Ishimori, N. Dashtsoodol, K. Watano, T. Mishima, C. Iwabuchi, S. Tanaka, J. S. Bezbradica, et al. Natural killer T cells accelerate atherogenesis in mice Blood, October 1, 2004; 104(7): 2051 - 2059. [Abstract] [Full Text] [PDF]
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