This document summarizes studies examining methods to track macrophage recruitment into atherosclerotic plaques. Initial EM studies in pigs found bi-directional movement of foam cells through the endothelium. Subsequent studies labeled monocytes with fluorescent dyes or microspheres and tracked their recruitment to plaques in pigs and mice. A newer method used PCR to detect transfusion of male monocytes into female mice. The presented study examined the effects of cytokines on SPIO uptake in plaques of ApoE-deficient mice, finding greater iron particle deposition in cytokine-treated mice, indicating increased monocyte recruitment. This suggests SPIO could non-invasively monitor monocyte recruitment during MRI.

1. Introducing a Novel Model for Studying
Macrophage Homing into Atherosclerotic
Plaque in APOE-Deficient Mice
• S. Litovsky, P. Wyde, M. Madjid, A. Akhtar, S.W.
Casscells, M. Naghavi
• Texas Heart Institute and University of Texas-
Houston, Houston, USA
European Atherosclerosis Society Meeting
Salzburg, July 7-10, 2002

2. Why Study Monocyte Recruitment?
• Monocytes/Macrophages are key players
not only in plaque initiation but also in
plaque progression and complications
through several mechanisms.

3. Monocyte Recruitment into
Atherosclerotic Plaques
Quantification Methods

4. Review of the studies done by:
• Gerrity et al
• Steinberg et al
• Willerson et al
• The potential role of SPIO

5. Initial EM (electron microscopy) observation by
Gerrity et all (Artery 1980;8:208-14), led to the
hypothesis of “macrophage assisted lipid clearance
mechanism”.
The swine model of hypercholesterolemia was
employed. Yorkshire pigs were fed high cholesterol
diets and sacrificed at the ages of 12, 15 and 30
weeks. Samples of aortic arch areas were examined
by electron microscopy.

6. EM studies indicated bi-directional movement of
foam cells through the endothelium overlying
the fatty streak lesion.
The above mentioned phenomenon may explain
the stability of fatty streaks for a period of time.
It also explains that macrophages may initially
serve as carriers of lipid material out of the plaque.
However, focal endothelial damage would result
from this process, which may contribute to later
lesion progression by introducing SMC
proliferating factors into the wall.

7. In a 2nd
study by Gerrity et al (Atherosclerosis 1988;
71:17-25) an additional method to trace macrophages
was used:
Monocytes were isolated from peripheral blood of pigs with
hypercholesterolemia.
Monocytes were labeled with FITC (fluorocin
Isothiocyanate 1-hydrochloride)
Labeled monocytes were reinjected back into the animal.
Labeled cells were found in the blood.
Animals were killed after 9 days, followed by histologic
examination.

8. Result
1)FITC-labeled monocytes were found adherent
to the thickened site of the intima but not to
normal areas.
2)Labelled cells were also found within the
atherosclerotic lesions.

9. In the study by S. Patel, James T. Willerson and
Edward Yeh, (Circulation 1998;97:75-81), wild-type
mice peritoneal macrophages were labeled with
fluorescent latex microspheres and IV injected into
ApoE deficient mice.
Animals were sacrificed after 48 hours, and tissues
obtained for histology.
A quantitative survey was done to detect the number
of labeled macrophages in the arterial wall.
Antibodies to ICAM-1, integrin and E-selectin were
injected 6-8 hours before macrophage injection.

10. Results
-Study was performed in 2 groups, (control and test) with
respect to the use of antibodies.
-After 48 hours macrophages were observed adhering to
all stages of plaques.
-The mean number of macrophages in the proximal 1mm
of aortic root was estimated to be 143+17.
-Antibodies against ICAM-1 and integrin significantly
reduced the number of macrophage homing.

11. Steinberg et al (ATVB 2000;20:1976-82) reported on a
new method of detecting monocytes in the plaque.
The basic idea was the introduction into a recipient
animal of leukocytes differing from those of the recipient
by virtue of one easily identified and quantified genetic
marker.
Monocytes were transfused from a male donor into
a female recipient and PCR was used to amplify
the Sry gene (testis-determining gene) on the Y
chromosome.

12. Method
The animal model was the LDL receptor deficient mice.
Males and females from the same highly inbred strain
were used as donors and recipients, so that no significant
immune response occurred.
Females who were to be recipients, were fed an atherogenic
diet for 3-6 months, beginning at 6 to 9 months of age.
All animals showed lesions in the aortic arch covering 13%
to 49% of the total surface area.

13. Methods
(cont.)
Monocytes collected from male mice were injected into 3
female mice that served as controls. A second group of
three female mice also received IP cytokine injection
(TNF-α, IL-1β).
Twenty four hours later the animals were euthanized and
aorta obtained for the study.

14. Results
Cytokine-treated mice showed 100% more recruitment
of monocytes in the aortic wall, compared to the control
group.
There was no cytokine effect in the animals with >40%
of the aortic arch covered by lesions.
The data in the control animals showed that monocyte
recruitment continued even when 30% to 50% of the
aortic surface was covered by lesions, but the rate of
recruitment was slower than it was in the earlier
stages of the disease.

15. • Although very informative, all these
techniques are invasive and
therefore cannot be used sequentially
in the same experimental animal.

16. OBJECTIVE
• Work from our group and other laboratories has
shown that the tracer Superparamagnetic Iron
Oxide (SPIO) administered intravenously 5 to 7
days before magnetic resonance imaging localizes
to aortic atherosclerotic plaques of apoE k/o mice
in addition to the reticuloendothelial system
(RES).
• In the present study we assessed by histopathology
the effect of several cytokines on monocytes
homing into aortas of apoE k/o mice.

17. METHODS
• Eleven apoE k/o retired breeders, 11-months old,
were divided in 2 groups. Six received TNF-α
0.2μg, IL-1β 0.2μg and IFNγ 100U/g
intraperitoneally, the latter for 5 days; the five
control received 0.5 mL saline containing 1%
BSA. Three hours later, all the animals were
injected with SPIO (Feridex) 1 mMol/kg of iron.
• Seven days later, the recipients were euthanized,
the heart and aorta perfused under physiologic
pressure and the entire aorta studied histologically.

18. ApoE K/O mice with SPIO but
without Cytokine
H&E Iron Stain

19. APOE K/O Mice with SPIO and
Cytokines
H&E Iron Stain

20. Iron Deposition on the Edges of
Atherosclerotic Plaques
H&E Iron Stain

21. Iron Deposition in Endothelial Cells

22. IMMUNOHISTOCHEMISTRY
MAC Stain CD3 Stain

23. Intramural Coronary Involvement
and Myocardial Injury

24. CONCLUSIONS
• Numerous iron particles are detected
subendothelially in the aorta specimens of
apoE k/o mice that received cytokines and
SPIO intravenously 7 days before sacrifice.
• Aortic iron particle levels are much lower in
mice not treated with cytokines.

25. IMPLICATIONS
• Since SPIO is a contrast agent used in
magnetic resonance imaging, this agent
offers great hope of studying monocyte
recruitment into the atherosclerotic plaque
non-invasively.