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.

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.

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.

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.

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

AA

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.

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.

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.

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.

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.