SHAPE Society

1. INTRODUCTION
Our group has long been interested in the use of SPIO (superparamagnetic
iron oxide) as a contrast agent for magnetic resonance imaging of
atherosclerotic plaques. This compound, that is FDA approved for
detection of liver metastases by MRI, is taken up by fixed macrophages of
the reticuloendothelial system (RES) and by plaque macrophages, mainly
subendothelial. In our MRI-histopathology correlation studies we noted
that in addition to iron being present inside plaque macrophages, iron was
also taken up by the periadventitial fat. Although largely ignored in the
atherosclerosis literature, macrophage-like activity of the adipose tissue
(mainly or exclusively in the stroma vascular fraction, SVF) is well
described in the obesity and immunity literature.
The purpose of this study is to investigate the uptake of SPIO by
periadventitial fat of apoE k/o mice, a well-characterized animal model of
atherosclerosis. We also show evidence that a similar phenomenon occurs
in the WHHL (Watanabe heritable hyperlipidemic) rabbit and in the
human periadventitial coronary fat.

2. MATERIAL & METHODS
Twenty two female apoE k/o mice, each approximately 12 months old and eleven
C57 female mice, approximately 6 months old were studied. All animals were
injected intravenously with SPIO (Feridex; Berlex Laboratories, Wayne, NJ) (1
mmol/kg iron).
All experimental procedures in these animals were performed in accordance with
protocols approved by the Institutional Animal Care and Research Advisory
Committee.
Histopathology and Immunohistochemistry
Six days later, recipient mice were euthanized with CO2, and their aortas were
perfused under physiological pressure. In each case, the entire aorta from the
sinuses of Valsalva up to the iliac bifurcation was formalin-fixed and serially
sectioned transversely every 3 mm and stained with hematoxylin and eosin (H&E).
Sucutaneous abdominal fat was also obtained from every animal. Prussian blue and
MAC 2 stains were used for detection of iron particles and macrophages,
respectively. The slides were analyzed on an Olympus BX61 microscope with the
Microsuite TM 33SV image analysis software (Soft Imaging System Corp.,
Lakewood, CO). The entire available periadventitial fat was analyzed from each
slide.
The 6 day time point was chosen because work from our laboratory has shown that
the highest MRI resolution is obtained 5-7 days after injection; corresponding
histology also showed highest iron uptake around this time period.

3. RESULTS
Periadventitial adipose tissue around the aorta of C57 BL mice shows some uptake of iron
(Figure 1A). In the apoE k/o mouse, the uptake was clearly greater (Figure 1B). The location
and characteristics of these cells are consistent with stroma-vascular fraction (SVF) cells. Mac 2
stain shows clearly more positivity in the periarterial fat of apoE K/O mouse (Figure 2B) than
the C57 mouse (Fig 2A).
Table 1 shows the iron particles/total fat area and iron area/total fat area in C57 (control) mice,
and apoE k/o mice. The increase in iron of apoE k/o animals was more prominent when
analyzed in terms of iron area than iron particles, indicating the size of the particles in the
apoE k/o mice are larger than in control (non-atherosclerotic mice). Although the difference is
clearly significant, there is a large standard deviation. Possible explanations for this finding are
discussed later.
Next, we compared the uptake of iron in the subcutaneous fat of C57 (Figure 3A) and apoE k/o
mice (Figure 3B). Although there was a trend toward greater iron uptake in apoE k/o mice, it
did not reach statistical significance (Table 2). In the C57 mouse, the periarterial fat iron is
similar to the subcutaneous iron.
Since the experimental studies on adipose tissue have been performed on mice fat, we also
evaluated the presence of iron in the periaortic fat of WHHL (Watanabe hypercholesterolemic)
rabbits. Figure 4 shows a representative example. Iron was clearly seen in the subendothelial
macrophages but also in the periaortic fat. (Fig 4A). The anti-macrophage antibody RAM-11
was focally positive in the periaortic fat as well.
Finally, we evaluated the presence of macrophages in human atherosclerotic human coronaries
obtained at autopsy. Figure 5 shows the presence of macrophages (as manifested by the CD68
positivity) in the pericoronary fat in addition to their presence in the plaque.

4. A
Figure 1. Periaortic iron in C57 Bl (A) and ApoE K/O (B) mice 6 days
following intravenous administration of SPIO (1 mmol/kg iron). Iron uptake
is much greater in the periaortic fat of ApoE K/O mice.
B

5. A
Figure 2. Periaortic Mac2 stain in C57 Bl (A) and ApoE K/O (B) mice. There
is greater positivity in the latter. Mac2 x10.
B

6. Table 1. Iron particles and area in the periaortic fat of C57 and ApoE K/O mice.
C57 Apo E K/O p value
Iron particles / Total Fat Area (mm2
) 80±52 210±401 0.100
Total Iron Area (µm2
) / Total Fat Area (mm2
) 382±291 1896±3847 0.032
Table 2. Iron particles and area in the subcutaneous abdominal fat of C57 and ApoE
K/O mice.
C57 Apo E K/O p value
Iron particles / Total Fat Area (mm2
) 49±54 129±137 NS*
Total Iron Area (µm2
) / Total Fat Area (mm2
) 293±265 427±366 NS*
*NS – Not significant.

7. A B
Figure 3. Subcutaneous tissue iron in C57 Bl (A) and ApoE K/O (B) 6 days
following the intravenous administration of SPIO (1 mmol/kg. iron). There
is no significant difference in iron uptake.

8. A
Figure 4. Subendothelial and periaortic fat iron in WHHL rabbit (A) 6 days
following intravenous administration of SPIO. In this case, fat uptake was
greater than subendothelial. RAM-11 staining (B) shows positivity in the
plaque and periaortic fat.
B

9. A
Figure 5. A. Human atherosclerotic coronary
artery with prominent necrotic core and
macrophage infiltration. x4 B. CD68
demonstrates heavy macrophage
concentration in the plaque but also in the
pericoronary fat. C. Inset shows in higher
magnification the vascular-stroma positive
population.
C
B

10. CONCLUSION
• Periaortic fat tissue in apoE k/o mice takes up iron injected in the form of SPIO (a
contrast agent in MRI).
• The magnitude of iron uptake in apoE k/o mice is much greater than in C57 mice.
Similarly, the number of macrophage or macrophage-like cells in the fat of apoE
k/o mice.
• Preliminary data on rabbits comparing WHHL and New Zealand White seems to
indicate a larger uptake of iron following SPIO in the periaortic fat of the former.
• The significance of the cells with macrophagic properties in the progression of
atherosclerosis, its complications and restenosis remains to be investigated.
• From the imaging standpoint, the “competition” for the nanotracer between the
plaque macrophage and periaortic macrophage suggests that a “smarter delivery
system” to the plaque macrophage would be necessary to improve the signal-to-
noise ratio.
• The significant standard deviation in apoE K/O mice, larger than in C57 animals,
suggests that additional factors (cytokines, leptin, etc) might play significant role in
the presence and/or function of macrophage-like cells in the periarterial fat.
• The striking macrophage-like activity of the periarterial fat may have wide-
reaching implications both in terms of development of contrast agents for imaging
atherosclerotic plaques and possibly shed light on the role of fat-based
macrophages in the mechanisms of atherosclerosis progression and restenosis.