1. The study found that periadventitial fat tissue in atherosclerotic mouse models takes up more SPIO nanoparticles, a MRI contrast agent, than normal mice. There are also more macrophage-like cells in the fat of atherosclerotic mice. 2. Preliminary studies in rabbits and humans also showed greater uptake of iron and more CD68 positive macrophage-like cells in the periadventitial fat of atherosclerotic vessels compared to normal vessels. 3. These macrophage-like cells in the periadventitial fat have not been fully characterized but show similarities to cells in fat tissue known as the stroma-vascular fraction, and may play a role in atherosclerosis progression and complications. Further study

1. Periadventitial Fat May Play an
Important Role in Plaque
Inflammation;
Introducing Macrophage-Like Activity of
Adipocytes in Periadventitial Fat
Mouse – Rabbit - Human
Silvio Litovsky, MD, Mohammad Madjid, MD,
Alireza Zarrabi, MD, Ward Casscells, MD, James
T. Willerson, MD, Morteza Naghavi, MD
Center for Vulnerable Plaque Research,
Texas Heart Institute, and University of Texas-Houston
Houston, TX

2. Introduction
• Our group has long been interested in the use of
SPIO (superparamagnetic iron oxide)
nanoparticles as a contrast agent for magnetic
resonance imaging of atherosclerotic plaques.
• SPIO 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 iron was present not only inside
plaque macrophages, but also in the
periadventitial fat.

3. Method:
• Twenty two female apoE K/O mice, 12 months old, and eleven
C57BL/6 female mice, 6 months old, were injected intravenously
with SPIO (1 mmol/kg iron).
• Six days later, mice were euthanized, and their aortas perfusion-
fixed. The entire aorta was formalin or Bouin’s-fixed and serially
sectioned. Subcutaneous 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 entire available
periadventitial fat was analyzed from each section.
• The 6 day time point was chosen because work from our laboratory
has shown that the highest MRI resolution is achieved 5-7 days
after injection; corresponding histology also showed highest iron
uptake around this time.

4. Methods
Day 0 Day 6
SPIO Injection Sacrifice and
Pathology

5. SPIO Uptake in Periadventitial Fat
A B
A – Wild type mouse B – ApoE K/O mouse

6. A
MAC-2 Positive Cells in Periadventitial Fat
B
B
A – Wild type mouse B – ApoE K/O mouse

7. F4/80 Positive Cells In Periadventitial Fat of ApoE Deficient Mice
ApoE K/O Mouse

8. Iron Stained Area in the Periaortic Fat of
C57BL/6 and ApoE K/O Mice.
0
200
400
600
800
1000
1200
1400
1600
1800
2000
C57BL Apo E K/O
C57BL/6 ApoE K/O p value
Total Iron Area (µm2
) /
Total Fat Area (mm2
) 382 ± 291 1896 ± 3847 0.032

9. Iron area in the subcutaneous abdominal
fat of C57BL/6 and ApoE K/O mice.
0
50
100
150
200
250
300
350
400
450
C57BL Apo E K/O
C57BL/6 ApoE K/O p value
Total Iron Area (µm2
) /
Total Fat Area (mm2
) 293 ± 265 427 ± 366 NS*

10. Rabbit Studies

11. Iron Uptake In WHHL Rabbit 7 Days After Administration of SPIO
plaque
media
adventitia
Periadventitial fat
H&E
Iron
Stain

12. Iron Co-localizes With Subendothelial & Periadventitial Fat
RAM11 Positive Cells
A
B

13. Iron Uptake In NZW Rabbit 7 Days After Administration of SPIO
H&E Iron Stain

14. Human Studies

15. CD68 in Periadventitial Fat of a Coronary Artery With Intimal
Thickening
H&E
CD68

16. CD68 Positive Cells Are Numerous in the Plaque and
Periadventitial Fat
A B
H&E CD 68

17. The Overwhelming Majority of CD68 Positive Cells Are Not S100
Positive
S100CD68

18. Toluidine blueCD68
The Overwhelming Majority of CD68 Positive Cells in the
Periadventitial Fat Are Not Toluidine Blue Positive

19. Co-localization of CD68 and CD34 Positive Cells
CD34CD68

20. There is moderate co-localization of CD68 & CD34 positive cells
CD68 CD34

21. Conclusions
• Periaortic fat tissue in apoE K/O mice takes up SPIO
nanoparticles (a MRI contrast agent).
• The magnitude of the uptake in apoE K/O mice is much greater
than in C57BL/6 mice. Similarly, the number of
macrophage/macrophage-like cells in the fat of apoE K/O mice is
greater than in C57BL/6 mice.
• Preliminary data on WHHL and New Zealand White rabbits
indicate a larger uptake of iron following SPIO in the periaortic
fat of the hypercholesterolemic atherosclerotic rabbit.

22. Conclusions (…continued)
• Preliminary studies on human coronaries show a much
greater density of CD68 positive cells in the periarterial
fat of atherosclerotic vessels compared to normal.
• Immunohistochemistry indicates that these cells are not
dendritic cells or mast cells. Moderate co-localization
with endothelial cells was seen.
• In many cases, the border between adventitia and fat is
not well defined, especially in humans. The cellular
infiltrate in the periadventitial fat was similar to the
infiltrate in the adventitia in all cases, suggesting they
behave as a single physiologic unit.

23. Conclusions (…continued)
• The exact lineage of these cells remains to be
further elucidated. Fat tissue is known to
contain a stroma-vascular fraction (SVF) and
the cells described here in the periadventitial
fat of mouse and rabbit aorta and human
coronaries, show similarities with this cell type.
• The possible significance of these cells with
macrophagic properties in the progression of
atherosclerosis, its complications and
restenosis remains to be investigated.

24. Conclusions (…continued)
• Upon further confirmation, these findings may
introduce a new marker of plaque
vulnerability based on inflammation in
periadventitial fat.
• From the imaging standpoint, the large
periadventitial area may provide a new
opportunity in imaging vulnerable plaque
where the spatial resolution for imaging
fibrous cap remains a challenge.

25. Texas Heart Institute University of Texas-Houston
Center for Vulnerable Plaque Research