Interleukin 1, tissue necrosis factor-, and interferon- increase spio accumulation
• In the sham-treated apo E K/O controls, iron-positive
macrophages were deposited in the subendothelial surface of the
atherosclerotic plaques (Figure 1, A&B).
• In the cytokine-treated mice, however, iron-positive
macrophages were much more numerous and tended to localize in
2 places. In all 6 cases, the iron-laden macrophages localized in
the subendothelium of plaques where they exhibited large and
foamy cytoplasms. In one section of two animals, they also
penetrated deeper into the plaque overlying advanced and
calcified plaques (Figure 1, C&D).
• In three mice, iron-laden macrophages localized in a thin layer
over the internal elastic lamina, suggesting their recruitment into
areas previously free of macrophage infiltration at the edges of
atherosclerotic plaques (Figure 2A & B). This phenomenon was
seen in sham-treated apo E K/O controls in a single case.
• Although iron staining is useful, it was not uncommon to
observe numerous brown golden particles in foamy macrophages
on H&E staining (Figure 2A). No iron deposition was seen in the
aortas of the wild-type control mice even after injection of
• In a single cytokine-treated mouse, iron-laden macrophages
were also present in a single subendothelial layer in plaque-free
areas (Figure 2C).
• Iron-positive cellularity was greater in cytokine-treated mice
than in sham-treated controls (42±11.8 vs. 11.6±5.9 iron
particles/cross-section) (P< 0.0001).
• Immunohistochemistry showed that most cells in atherosclerotic
plaques were positive for the macrophage stain Mac-2 and a few
for the T lymphocyte marker CD3 (Figure 3).
• One cytokine-treated mouse exhibited a prominent mural
infiltration of an intramural (muscular) coronary artery by foamy
macrophages. The surrounding myocardium showed healing
injury with mononuclear infiltration and early fine fibrosis
• Iron concentrations were higher in cytokine-treated mice than in
sham-treated apo E K/O controls: 345 vs. 298 ìg/g in the aorta
and 11,818 vs. 7,174 ìg/g in the liver. (Figure 5).
•Electron micrograph studies have shown that iron is not
distributed homogeneously in atherosclerotic plaques, but mainly
subendothelially and on rare endothelial cells. (Figure 6).
Interleukin-1â, Tissue Necrosis Factor-á, and Interferon-ã Increase SPIO Accumulation in
Atherosclerotic Plaques of Apo E Deficient Mice
Silvio Litovsky, MD, Mohammad Madjid, MD, Ward Casscells, MD, James T. Willerson, MD, Morteza Naghavi, MD
Center for Vulnerable Plaque Research
Texas Heart Institute & University of Texas at Houston
Material and Methods
•Eleven female apo E knockout (K/O) mice, each 11 months old,
were divided into 2 groups. Six received tissue necrosis factor-á
(0.2 µg once), interleukin-1b (0.2 µg once), and interferon-ã (100
U/g/day for 5 days) intraperitoneally; 5 received 0.5 mL saline
containing 1% bovine serum albumin and served as sham-treated
•Two wild-type C57BL/6 mice served as sham-treated
•Three hours after initial cytokine or sham treatment, all mice
received SPIO by intravenous injection (Feridex; Berlex
Laboratories, Wayne, NJ) (1 mmol/kg iron, undiluted, injected
over 3 minutes).
•Six days after initial cytokine or sham treatment, recipient mice
were euthanized with CO2, and their hearts and aortas were
perfused under physiological pressure, and studied histologically.
•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
• Prussian blue and MAC 2 (Accurate Chemical, Westbury, NY)
stains were used for detection of iron particles and macrophages,
• Total iron concentration (ìg/g of dry weight) was determined by
acid digestion of plaque followed by inductively coupled
plasma/mass spectrometry using a Perkin-Elmer ELAN 6100
series instrument at the Mayo Clinic Laboratories in Rochester,
MN on the aortas and livers of 2 cytokine-treated and 2 sham-
treated Apo E K/O.
• Student’s t test was used to test for statistical significance of the
difference observed in number of iron-positive cells in cytokine
and sham treated animals.
• SPIO (superparamagnetic iron oxide) is an MRI contrast agent in
the form of nanoparticle. SPIO is avidly taken up by monocyte
/macrophages in the reticuloendothelial system.
•We have previously studied SPIO enhanced MR imaging of
inflammation in atherosclerotic plaques of mice and rabbits.
• Administration of pro-inflammatory cytokines has been shown to
enhance the recruitment of monocytes into atherosclerotic lesions.
• The purpose of this study was to compare the uptake of SPIO into
plaques of cytokine-treated Apo E knockout (K/O) mice and age-
matched, sham-treated controls in order to:
1) assess the effect of cytokines on monocyte recruitment, and
2) validate SPIO as a marker of monocyte recruitment into
• Iron particles were detected subendothelially in aortic specimens
from apo E K/O mice, 6 days following intravenous SPIO. This
effect was much greater in mice that received cytokines
• The iron was mostly present in atherosclerotic plaques, though
most of the plaque area was free of iron because of the iron’s
predominant superficial localization. There were, in addition, thin
areas of iron-laden macrophages lying just above the internal
elastic lamina, suggesting that these areas were free of monocyte
infiltration before the administration of the cytokines and SPIO.
Most of these zones were at the edge of atherosclerotic plaques.
•No iron deposition was seen in the aortas of wild-type
• Most of the iron-laden macrophages in the cytokine-treated mice
were newly recruited monocytes.
• SPIO also homes on new fatty streaks in the aorta of animals
pretreated with cytokines.
• The iron staining and mass spectrometry offer two qualitative and
quantitative method for studying plaque inflammation in animal
model of atherosclerosis, respectively.
• Since SPIO is a contrast agent used in MRI, this may prove
useful as a noninvasive method for studying monocyte recruitment
into the atherosclerotic plaque in vivo.
Figure 1. A. Two aortic atherosclerotic plaques in a control Apo E deficient mouse (H&E stain x40). Foamy
macrophages constitute the majority of the cell population (arrows). B. Iron staining shows both plaques with faint
iron particles (arrows, Prussian Blue x40). C. Advanced plaque, largely denuded of endothelium, with significant
fibrosis and calcification. The intima is mainly occupied by foamy macrophages containing brown particles (arrow,
H&E staining x40). D. Iron staining confirms the brown particles correspond to iron (arrow). Almost all the iron is
intracellular, suggesting active uptake and not just diffusion into permeable plaque and binding to matrix. Moreover,
endothelial cells contain very little stain compared to macrophages. Very little iron is present deeper in the plaque
even in macrophages. (Prussian Blue x40).
Figure 2. A. Thin layer of foamy cells occupies the intima on the edge of an atherosclerotic plaque (arrow, H&E
x40) B. Iron staining confirms the presence of abundant iron (arrows, Prussian Blue x40). C. Area without
atherosclerotic plaque shows a single layer of subendothelial iron-laden macrophages (arrow, Prussian Blue x40).
Iron appears also in endothelial cells.
Figure 3. A. Macrophage (MAC-2) staining confirms the phenotype of the cells in the subendothelium (arrows,
MAC-2 x40). Rare cells appear to be lymphocytes (arrow head). B. Although a clear minority, some of the plaque
cells were T lymphocytes as shown by the CD3 staining (arrows, CD3 x40).
Figure 4. A. Intramural coronary artery with slit-like lumen and significant wall inflammation, mostly present in
the media and adventitia (arrow, H&E x40). The intima appears edematous and no luminal thrombus is seen. B.
The myocardium surrounding the abnormal vessel shows an organizing myocardial infarction (arrows, H&E x40).
Figure 5. Comparison of iron
content in aorta (A) and liver (B)
(µg/g dry weight) in cytokine-treated
versus sham-treated (control) mice.
Figure 6. A. Electron micrograph of thoracic aorta of a WHHL rabbit injected with SPIO (1 mmol/kg iron)
5 days before sacrifice. Multiple iron particles are present in subendothelial foamy macrophages (arrows).
The iron particles are about 0.5-1 micron (x4000). B. High magnification of iron particles (arrow) in
macrophage lysosomes (x40,000).
Texas Heart Institute®