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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 8, 2001 as doi:10.1096/fj.01-0017fje. |
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,
,¶,
,1,¶,,¶,¶,¶,
* Program of Developmental Cardiovascular Biology, Cardiovascular Division, and
Pulmonary and Critical Care Division, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA;
Department of Medicine, Harvard Medical School;
¶ Cardiovascular Biology Laboratory and
Division of Biological Sciences and Department of Nutrition, Harvard School of Public Health;
|| Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA; and
** Department of Nutritional Sciences, Rutgers University, New Brunswick, New Jersey 08901, USA
2Correspondence: Program of Developmental Cardiovascular Biology, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115, USA. E-mail: mperrella{at}rics.bwh.harvard.edu
SPECIFIC AIM
The present study was designed to elucidate a role for the adipocyte fatty acid binding protein AFABP or aP2 in the development of atherosclerosis. We evaluated the expression of aP2 in atherosclerotic lesions, assessed aP2 gene regulation in macrophages, and intercrossed aP2-/- mice with apolipoprotein E-deficient (ApoE-/-) mice to study the effect of aP2 deficiency on lesion formation in this hypercholesterolemic model of atherosclerosis.
PRINCIPAL FINDINGS
1. aP2 is expressed in atherosclerotic lesions from
ApoE-/- mice
Lesions from ApoE-/- mice were
evaluated because they developed severe hypercholesterolemia and
atherosclerotic lesions characteristic of human disease. aP2 mRNA was
not detectable in the arterial walls of wild-type mice except in
adipose tissue of the adventitia. However, an intense aP2 signal was
visible in the arterial walls of ApoE-/- mice.
The signal for aP2 was prominent in both the atherosclerotic lesion and
adipose tissue of the adventitia, with much less signal in the media.
2. aP2 is induced in mouse and human macrophages during
differentiation and stimulation with oxidized low density lipoprotein
(oxLDL).
Peritoneal macrophages were harvested from wild-type mice and the
level of aP2 mRNA was examined. aP2 mRNA was present in mouse
peritoneal macrophages and the level of aP2 message increased during
macrophage differentiation. aP2 message increased when the macrophages
were exposed to oxLDL but not upon exposure to nonoxidized LDL. We also
examined aP2 mRNA levels in human macrophages. The message for aP2
increased during monocyte to macrophage differentiation in human cells,
and this pattern of aP2 mRNA induction paralleled the increase in
scavenger receptor class A mRNA (a marker of monocyte to macrophage
differentiation). These data demonstrate that aP2 is expressed in both
mouse and human macrophages and that aP2 is specifically induced by
oxLDL, a stimulus for the development of atherosclerosis.
3. Plasma cholesterol and triglyceride levels are markedly
increased in both ApoE-/-aP2-/- and
ApoE-/- mice
To determine whether aP2 is important for the development of
atherosclerosis, we generated mice deficient in both ApoE and aP2.
Serum lipid profiles in the mice on a high-fat Western diet revealed an
overall reduction (P<0.05) in total circulating cholesterol
levels in ApoE-/-aP2-/-
mice (735±58 mg/dl, n=15) compared with
ApoE-/- mice (1192±137 mg/dl,
n=10). However, total cholesterol levels remained extremely
high in both groups. Total circulating triglyceride levels did not
differ in ApoE-/-aP2-/-
and ApoE-/- mice. We also characterized the
distribution of cholesterol in various lipoprotein fractions from the
two groups by fast-phase liquid chromatography. Unlike the plasma of
wild-type mice in which high density lipoprotein (HDL) predominates as
the major cholesterol-carrying lipoprotein, the plasma of
ApoE-/-aP2-/- and
ApoE-/- mice showed a predominance of lower
density lipoproteins.
4. Vascular lipid accumulation and lesion size is markedly reduced
in ApoE-/-aP2-/- mice
We next investigated atherosclerotic lesion formation in the
aortic arch and its branches (Fig. 1A
) in animals from the two groups on a Western diet. Sudan IV
staining revealed a marked decrease in lipid accumulation in
ApoE-/-aP2-/- mice vs.
ApoE-/- mice (Fig. 1B
, red). We also
measured the cross-sectional area of lesions in the proximal and distal
portions of the right brachiocephalic artery (Fig. 1C
).
Large occlusive lesions were present in the proximal
(71±12x103 µm2,
n=7) and distal (39±4x103
µm2, n=7) brachiocephalic arteries
of ApoE-/- mice. These lesions occluded 80 ± 5% of the proximal and 55 ± 7% of the distal arteries. In
contrast to the ApoE-/- mice, lesions in
ApoE-/-aP2-/-
mice were small and nonocclusive in both proximal
(7±2x103 µm2,
n=12) and distal (0.5±0.3x103
µm2, n=12) brachiocephalic arteries.
In fact, 5 of 12
ApoE-/-aP2-/- mice did
not develop any detectable atherosclerotic lesions after 12 wk on a
Western diet.
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5. Collagen accumulation and macrophage infiltration is reduced in
atherosclerotic lesions of ApoE-/-aP2-/-
mice
Atherosclerotic lesions from ApoE-/- mice
were complex, with fibrous caps (Fig. 2A
, arrow), and contained large amounts of collagen
(Fig. 2C
, blue). The clear areas within these advanced,
complicated lesions (marked by an asterisk in Fig. 2A
) are
areas of lipid accumulation.
ApoE-/-aP2-/- mice, in
contrast, had very small, uncomplicated lesions that lacked fibrous
caps and deposition of excess extracellular matrix (Fig. 2B
,
D
, arrows). In ApoE-/- mice, 45 ± 5% (n=4) of the lesion was composed of collagen, whereas
only 13 ± 4% (n=4) of the lesion was composed
of collagen in
ApoE-/-aP2-/- mice. We
also assessed macrophage accumulation in the two groups by
immunostaining the arteries for MOMA-2 (a marker for macrophages). The
percentage of lesions staining positive for MOMA-2 (brown) in the
proximal arteries was 30-fold higher in ApoE-/-
mice (Fig. 2E
) than in
ApoE-/-aP2-/- mice
(Fig. 2F
).
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6. Influence of aP2 on lesion formation is related to
hypercholesterolemia
To determine whether aP2 deficiency prevented lesion
formation in another type of occlusive vascular disease, we assessed
lesion size and macrophage accumulation in a model of
transplant-associated arteriosclerosis that does not depend on
hypercholesterolemia. We transplanted carotid arteries from mice of a
different genetic background into wild-type or
aP2-/- mice. We found no difference in lesion
size and no decrease in macrophage accumulation in donor carotid
arteries that were transplanted into wild-type (Fig. 2G
) or
aP2-/- (Fig. 2H
) mice.
CONCLUSIONS AND SIGNIFICANCE
Our experiments implicate aP2 as an important mediator in the development of diet-induced atherosclerosis. Atherosclerotic lesions from ApoE-/- mice, but not arterial walls from normal mice, contain high levels of aP2 mRNA. Moreover, aP2 is present in isolated mouse and human macrophages, and oxLDL is a stimulus for aP2 induction in macrophages. Compared with ApoE-/- mice, ApoE-/-a2-/- mice on a Western diet developed trivial lesions that were markedly smaller, less complex, and less macrophage-rich even though the ApoE-/-aP2-/- mice remained hypercholesterolemic. In contrast, the absence of aP2 did not prevent lesion formation and macrophage accumula-tion in transplant-associated arteriosclerosis, which does not depend on elevated levels of cholesterol. These results indicate a critical role for aP2 in the development of hypercholesterolemia-induced atherosclerosis.
The dramatic decrease in atherosclerotic lesions in ApoE-/- aP2-/- mice is not likely secondary to the modest reduction in plasma cholesterol levels in ApoE-/- aP2-/- mice compared with ApoE-/- mice. Indeed, Zhang and colleagues have shown that similar reductions in plasma cholesterol levels of ApoE-/- mice, caused by hypolipidemic drugs, did not reduce atherosclerotic lesion formation in ApoE-/- mice. It is noteworthy that plasma cholesterol levels of ApoE-/- aP2-/- mice remained fivefold higher than values reported for wild-type mice. Thus, although we cannot exclude the possibility that the 38% decrease in plasma cholesterol levels may have contributed to the decrease in atherosclerotic lesion formation, it is likely that the effects are not a direct result of lowering the plasma cholesterol levels, but due to the absence of aP2.
Macrophages play a critical role in the development of atherosclerosis.
Since adipocyte FABP is expressed in macrophages and in inflammatory
cells of atherosclerotic lesions, we focused our attention on
macrophage aP2 in atherosclerosis. In ApoE-/-
aP2-/- mice there was a marked decrease in
lesion macrophage accumulation that occurred only in the setting of
hypercholesterolemia. These data suggest that aP2 is an important
mediator in the development of atherosclerosis induced by
hypercholesterolemia and that a lack of aP2 leads to a significant
decrease in macrophage accumulation and complex lesion formation
(Fig. 3
). The discovery of aP2 as a critical component of atherosclerosis opens
up previously unrecognized avenues for novel therapeutic targets for
this disease.
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Although the precise biological functions and mechanisms of action of aP2 are not yet completely defined, the collective results suggest that adipocyte FABP may play a role in modulating systemic lipid and glucose metabolism and fatty acid flux. However, the relationship, if any, between such metabolic alterations and the anti-atherosclerotic effects observed in aP2 deficiency in the present study is not known. Future studies will explore how alterations in hypercholesterolemia-induced inflammatory responses, lipid and glucose metabolism, and fatty acid flux contribute to the decrease in atherosclerotic lesion formation in the absence of adipocyte FABP.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0017fje ; to cite this article, use FASEB J. (June 8, 2001) 10.1096/fj.01-0017fje 
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; E, F, brown). MOMA-2
staining for macrophages (M