SHAPE Society

1. Introducing a Novel Model for StudyingIntroducing a Novel Model for Studying
Macrophage Homing intoMacrophage Homing into
Atherosclerotic Plaque in Apo E-Atherosclerotic Plaque in Apo E-
Deficient MiceDeficient Mice
Silvio Litovsky, MD, Philip Wyde, MD, WardSilvio Litovsky, MD, Philip Wyde, MD, Ward
Casscells, MD, James Willerson, MDCasscells, MD, James Willerson, MD
Morteza Naghavi, MDMorteza Naghavi, MD
Texas Heart Institute and University ofTexas Heart Institute and University of
Texas-Houston, Houston, USATexas-Houston, Houston, USA
European Society of Cardiology CongressEuropean Society of Cardiology Congress
Berlin, Sep 2Berlin, Sep 2ndnd
, 2002, 2002

2. Why Study Monocyte Recruitment?Why Study Monocyte Recruitment?
 Atherosclerosis is anAtherosclerosis is an inflammatoryinflammatory
disease.disease.
 Inflamed foci are associated with heavyInflamed foci are associated with heavy
traffic of immune cellstraffic of immune cells mainly monocytes.mainly monocytes.
 Monocytes/Macrophages are key playersMonocytes/Macrophages are key players
not only in plaquenot only in plaque initiationinitiation but also inbut also in
plaqueplaque progression and complicationsprogression and complications
through several mechanisms.through several mechanisms.

3. MonocyteMonocyte
Recruitment intoRecruitment into
AtheroscleroticAtherosclerotic
PlaquesPlaques
Available QuantificationAvailable Quantification
MethodsMethods

4. Studies done by:Studies done by:
 GerrityGerrity et alet al
 SteinbergSteinberg et alet al
 WillersonWillerson et alet al
 The potential role of SPIOThe 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, the seems to be a traffic jam later.
Fibrous cap and increased apoptosis play a role.

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 and labeled with FITC (fluorocin
Isothiocyanate 1-hydrochloride). Labeled monocytes were
reinjected back into the animal.
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.

8. 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. Antibodies to ICAM-1, integrin
and E-selectin were injected 6-8 hours before
macrophage injection.
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.

9. Steinberg et al (ATVB 2000;20:1976-82) reported on a
new method of detecting monocytes in the plaque.
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.
They also injected cytokines to enhance plaque
Inflammation.

10. 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.

11. Bottom line:Bottom line:
 They are either quantitative orThey are either quantitative or
qualitative.qualitative.
 Although very informative, all theseAlthough very informative, all these
techniques are invasive andtechniques are invasive and
therefore cannot be usedtherefore cannot be used
sequentially in the samesequentially in the same
experimental animalexperimental animal..

12. What is SPIO?What is SPIO?
 Super Paramagnetic Iron Oxide in form ofSuper Paramagnetic Iron Oxide in form of
nano-particles (10-100nm).nano-particles (10-100nm).
 Cored by iron oxide coated by a variety ofCored by iron oxide coated by a variety of
coating materials mainly polysaccharide.coating materials mainly polysaccharide.
 SPIO nano-particles are avidly taken up bySPIO nano-particles are avidly taken up by
macrophages.macrophages.
 Strong contrast enhancement effect inStrong contrast enhancement effect in
MRI.MRI.

13. OBJECTIVEOBJECTIVE
 Work from our group and other laboratories hasWork from our group and other laboratories has
shown that the SPIO administered intravenouslyshown that the SPIO administered intravenously
5 to 7 days before magnetic resonance imaging5 to 7 days before magnetic resonance imaging
localizes to aortic atherosclerotic plaques of apoElocalizes to aortic atherosclerotic plaques of apoE
k/o mice in addition to the reticuloendothelialk/o mice in addition to the reticuloendothelial
system (RES).system (RES).
 In the present studyIn the present study
• 1) we propose SPIO as a tool to both1) we propose SPIO as a tool to both
quantitatively (iron mass spectrometry)quantitatively (iron mass spectrometry)
and qualitatively (iron staining) evaluateand qualitatively (iron staining) evaluate
macrophage homing into atheroscleroticmacrophage homing into atherosclerotic
plaque.plaque.
• 2) we study evaluate the effect of2) we study evaluate the effect of
cytokine on macrophage homing intocytokine on macrophage homing into

14. FL-labeled SPIO Incubated Macrophages 24hr

15. Double DAPI Staining with Fluorescence-labeled SPIO Macrophages
after 24hr Incubation

16. METHODSMETHODS
 Eleven apoE k/o retired breeders, 11-Eleven apoE k/o retired breeders, 11-
months old, were divided in 2 groups. Sixmonths old, were divided in 2 groups. Six
received TNF-received TNF-αα 0.20.2μμg, IL-1g, IL-1ββ 0.20.2μμg andg and
IFNIFNγγ 100U/g intraperitoneally, the latter100U/g intraperitoneally, the latter
for 5 days; the five control received 0.5for 5 days; the five control received 0.5
mL saline containing 1% BSA. Three hoursmL saline containing 1% BSA. Three hours
later, all the animals were injected withlater, all the animals were injected with
SPIO (Feridex) 1 mMol/kg of iron.SPIO (Feridex) 1 mMol/kg of iron.
 Seven days later, the recipients wereSeven days later, the recipients were
euthanized, the heart and aorta perfusedeuthanized, the heart and aorta perfused
under physiologic pressure.under physiologic pressure.

17. ApoE K/O mice withApoE K/O mice with
SPIO but withoutSPIO but without
CytokineCytokine
H&E Iron Stain

18. APOE K/O Mice with SPIO andAPOE K/O Mice with SPIO and
CytokinesCytokines
H&E Iron Stain

19. Iron Deposition on the Edges ofIron Deposition on the Edges of
Atherosclerotic PlaquesAtherosclerotic Plaques
H&E Iron Stain

20. Iron Deposition in Endothelial CellsIron Deposition in Endothelial Cells

21. IMMUNOHISTOCHEMISTRYIMMUNOHISTOCHEMISTRY
MAC Stain CD3 Stain

22. Histopathologic study of ApoE KO Mouse injected
With SPIO (Thoracic Aorta)
CD68 staining
(aortic plaque)
Iron Staining (aortic plaque) Iron Staining (coronary section)
Iron particles Iron particles

23. Histopathologic studies of Thoracic aorta in Watanabe
Hereditary Hypercholesterolemic rabbit after SPIO injection
H&E staining
Iron staining
Mac staining

24. Histopathologic studies of Thoracic aorta in Watanabe
Hereditary Hypercholesterolemic rabbit after SPIO injection
H&E staining
Iron staining Iron staining
Iron particles

25. Quantitative AssayQuantitative Assay
270
280
290
300
310
320
330
340
350
Cytokine
treated
aorta
Iron
Content
 Iron content wasIron content was
higher in cytokine-higher in cytokine-
treated mice thantreated mice than
in sham-treatedin sham-treated
apo E K/O mice:apo E K/O mice:
Mass Spectrometry

26. Conclusion:Conclusion:
 SPIO allows both quantitative (massSPIO allows both quantitative (mass
spectrometry) and qualitative (ironspectrometry) and qualitative (iron
and macrophage staining) detectionand macrophage staining) detection
of infiltration of iron-ladenof infiltration of iron-laden
macrophages in the aortic plaques ofmacrophages in the aortic plaques of
apo E-deficient mice.apo E-deficient mice.
 This effect is significantly enhancedThis effect is significantly enhanced
after injection of proinflammatoryafter injection of proinflammatory
cytokines.cytokines.

27. IMPLICATIONS:IMPLICATIONS:
 Since SPIO is a MRI contrast agent,Since SPIO is a MRI contrast agent,
SPIO-enhanced MRI may thereforeSPIO-enhanced MRI may therefore
be useful for noninvasive monitoringbe useful for noninvasive monitoring
of monocyte recruitment intoof monocyte recruitment into
atherosclerotic plaques.atherosclerotic plaques.

28. MR Image of Abdominal Aorta After SPIO
Injection in ApoE and Control Mice
ApoE
deficien
t mouse
C57B1
(control)
mouse
Before Injection After Injection (5 Days )
Dark (negatively enhanced) aortic wall, full of iron particles
Bright aortic lumen and wall without negative
enhancement and no significant number of iron particles