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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 15, 2001 as doi:10.1096/fj.01-0374fje. |
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,
,¶
,2
* Program of Developmental Cardiovascular Biology, Cardiovascular Division, and
Pulmonary and Critical Care Division, Brigham and Women’s Hospital;
Department of Cancer Biology, Dana Farber Cancer Institute; Departments of
Medicine and
¶ Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA;
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: Pulmonary and Critical Care Division, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115, USA. E-mail: mperrella{at}rics.bwh.harvard.edu
SPECIFIC AIMS
The present study was designed to elucidate the mechanism(s) responsible for the prevention of advanced atherosclerotic lesion formation in hypercholesterolemic mice lacking aP2. We wanted to determine whether the reduction in lesion formation in the absence of aP2 was related to an alteration in bone marrow-derived macrophages (function or inflammatory/chemoattractant cytokine expression), cholesterol levels, glucose, or insulin levels.
PRINCIPAL FINDINGS
1. Bone marrow cells from ApoE-/-aP2-/- mice transplanted into ApoE-/- mice promotes a marked reduction in atherosclerotic lesion formation
We transplanted bone marrow cells from ApoE-/-aP2-/- mice into ApoE-/- mice; as a control, bone marrow cells from ApoE-/- mice were transplanted into ApoE-/- mice. Mice were placed on a high-fat Western diet for 12 wk and the right brachiocephalic arteries were analyzed. Representative vessels from ApoE-/- mice receiving marrow cells from ApoE-/- donor mice are depicted in Fig. 1
A, B. Large atheromas were present in the brachiocephalic arteries from these mice, producing lesions occluding the vessel by 42 ± 8% (Fig. 2
A, black bar, n=6). Vessels from ApoE-/- mice receiving cells from ApoE-/-aP2-/- donor mice contained significantly smaller lesions (Fig. 1
C, D, P=0.0006) occluding the brachiocephalic arteries by only 9 ± 2% (Fig. 2A
, white bar, n=9). Lesions in the brachiocephalic arteries of ApoE-/- mice receiving ApoE-/-aP2-/- marrow cells were 5.5-fold smaller by area (P=0.0012) than ApoE-/- mice receiving cells from ApoE-/- mice (Fig. 2B
). These results strongly suggest that bone marrow-derived cells are the primary mediators of the anti-atherosclerotic effects in ApoE-/-aP2-/- mice we demonstrated recently.
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2. No difference in plasma cholesterol levels of ApoE-/- mice receiving bone marrow cells from ApoE-/-aP2-/- or ApoE-/- donors
We measured total plasma cholesterol levels in ApoE-/- mice receiving donor bone marrow cells from either ApoE-/- or ApoE-/-aP2-/- mice. There was no difference (P=0.95) in total cholesterol levels between mice receiving marrow cells from ApoE-/- donors (1291±157 mg/dl, n=6) and mice receiving marrow cells from ApoE-/- aP2-/- donors (1280±85 mg/dl, n=11). These data confirm that an alteration in circulating cholesterol levels cannot account for the decrease in lesion size previously found in ApoE-/- aP2-/- mice.
3. No differences in cholesterol esterification in macrophages harvested from aP2-/- mice
Peritoneal macrophages were harvested from wild-type (aP2+/+) and aP2-/- mice, then stimulated with oxLDL. OxLDL caused a significant increase in cholesterol ester accumulation (P<0.05, 0 µg/ml vs. 50 µg/ml oxLDL) in peritoneal macrophages from both aP2+/+ and aP2-/- mice. After 24 h of oxLDL exposure, no difference in the formation of cholesterol esters was noted in macrophages from aP2+/+ and aP2-/- mice. These data demonstrate no difference in cholesterol uptake by aP2-deficient macrophages that could account for the decreased lesion formation.
4. No difference in plasma glucose or insulin levels of ApoE-/- mice in the presence or absence of aP2
Fasting glucose or insulin levels in ApoE-/- and ApoE-/-aP2-/- mice that had been on a high-fat Western diet for 12 wk were not different. Moreover, ApoE-/- mice receiving bone marrow cells from ApoE-/- or ApoE-/-aP2-/- mice showed no difference in fasting glucose or insulin levels on the same diet. Taken together, these data indicate that there are no obvious differences in systemic glucose metabolism that could explain the decrease in atherosclerotic lesion formation in ApoE-/-aP2-/- mice.
5. Decrease in expression of inflammatory cytokines and chemokines, but no alteration in NO production, in aP2-deficient macrophages
Peritoneal macrophages were harvested from ApoE-/- and ApoE-/-aP2-/- mice exposed to vehicle or oxLDL for 48 h. RNase protection assays revealed that levels of the proinflammatory cytokine tumor necrosis factor 
(TNF-) and macrophage inflammatory protein 1 (MIP-1, and ß) were decreased (58±5%, 45±9%, and 46±11%, respectively) in ApoE-/-aP2-/- cells vs. ApoE-/- cells. Levels of MCP-1, particularly in the presence of oxLDL, were considerably decreased (62±7%) in ApoE-/-aP2-/- cells. Nitrite levels were induced to a similar level by LPS in wild-type and ap2-/- macrophages. Macrophages from ApoE-/- mice also deficient in aP2 (ApoE-/-aP2-/-) showed nitrite inducibility that was not different from wild-type or aP2-/- macrophages. These data suggest that macrophages deficient in aP2 are fully activated by inflammatory stimuli yet show a decreased message for inflammatory cytokines and chemokines.
CONCLUSIONS AND SIGNIFICANCE
Bone marrow cells transplanted from ApoE-/-aP2-/- mice into ApoE-/- mice were able to reduce brachiocephalic lesion size in mice fed a high-fat Western diet to a similar degree found previously for ApoE -/- mice entirely deficient in aP2. Thus, even in the presence of adipocyte aP2 expression in other cell types, the absence of macrophage expression reproduced the phenotype observed in the ApoE-/-aP2-/- mice. These data establish the importance of aP2 from bone marrow-derived cells in mediating atherosclerotic lesion formation. Moreover, since monocytes and macrophages constitute 
80% of inflammatory cells in atherosclerotic lesions, and aP2 is highly expressed in isolated macrophages and atherosclerotic lesions, these data convincingly support our hypothesis regarding the importance of macrophage aP2 in the development of atherosclerosis.
ApoE-/- mice hematologically reconstituted with bone marrow cells from ApoE-/-aP2-/- mice had small, trivial lesions even in the setting of a very high plasma cholesterol levels. In fact, plasma cholesterol levels for these mice were almost identical to ApoE-/- mice transplanted with syngeneic ApoE-/- cells, which had dramatically larger lesions. These data suggest that the beneficial effects of aP2 deficiency in preventing atherosclerotic lesion formation are not a direct result of lowering the plasma cholesterol levels, but are mediated by bone marrow-derived cells.
In the development of atherosclerosis, circulating monocytes enter the arterial wall, differentiate into macrophages, and then scavenge cholesteryl esters that originate from plasma lipoproteins to become lipid-filled foam cells. When these foam cells accumulate within the intima, the first identifiable lesion of atherosclerosis develops. Our experiments revealed no marked difference in cholesterol ester accumulation between aP2-/- and aP2+/+ macrophages that could account for the dramatic decrease in lesion formation.
Diabetes is an important risk factor in the development of atherosclerosis. We therefore investigated whether an alteration in fasting plasma glucose and insulin levels may contribute to the smaller lesion size in ApoE-/-aP2-/- mice that were fed a high-fat Western diet. Plasma glucose and insulin levels were not different between ApoE-/-aP2-/-and ApoE-/- mice or between ApoE-/- mice receiving bone marrow cells from ApoE-/-aP2-/- or ApoE-/- donors.
We wanted to determine whether the expression of inflammatory factors known to modulate atherosclerotic lesion formation—proinflammatory and chemoattractant cytokines—is altered in the absence of aP2. Indeed, mRNA levels for TNF- (inflammatory cytokine) and MIP-1, MIP-1ß, and MCP-1 (chemokines) were reduced in macrophages harvested from ApoE-/-aP2-/- mice compared with macrophages from ApoE-/- mice. Moreover, the reduction in inflammatory and chemoattractant cytokines was present when macrophages were stimulated with oxLDL. These data are consistent with studies showing that hypercholesterolemic mice deficient in MCP-1 or its receptor CCR2 developed smaller and fewer macrophage-rich lesions. Our data suggest that decreased expression of inflammatory and chemoattractant cytokines in macrophages from ApoE-/-aP2-/- mice may contribute to the decrease in atherosclerotic lesion formation rather than circulating insulin, glucose, or cholesterol levels (Fig. 3
). Future studies will explore how alterations in cytokine expression in macrophages in the presence and absence of aP2 contribute to the development of atherosclerotic lesions.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0374fje; to cite this article, use FASEB J. (October 15, 2001) 10.1096/fj.01-0374fje 
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) in plasma cholesterol, insulin, or glucose levels. The formation of cholesterol esters was not significantly different (
) in the absence of aP2, indicating a potential mechanism for the altered severity of the atherosclerotic lesions.