SHAPE Society

1. Non-invasive Detection of
Vulnerable Plaque using
SPIO Enhanced MRI
Mitra Rajabi MD, Maamoun AbouQamar MD,
Michael Quast PhD, Jigna Wei MD, Daniel
Chan PhD, Mohammad Madjid MD, Khawar
Gul MD, Ponnada Narayana PhD, Ward
Casscells MD, James Willerson MD,
Morteza Naghavi MD
Texas Heart InstituteThe University of Texas-Houston

2. Everybody has
atherosclerosis, the
question is who has
Vulnerable Plaque

3. The Online Cardiovascular Research Community
www.VulnerablePlaque.org
All slides will be available on:

5. Vulnerable Plaque?
Atherosclerotic plaques
that cause sudden
luminal clot formation
and lead to heart attack
and stroke.

6. Different Types of Vulnerable Plaque
As underlying Cause of Acute Coronary Events
Normal
Rupture-prone
Fissured Eroded
Critical Stenosis Hemorrhage

7. Rupture-prone inflamed plaque
Vulnerable Plaque Type 1

8. Eroded Plaque with Exposed Proteoglycans Prone to Thrombosis
Vulnerable Plaque Type 2

9. Fissured Plaque with Old and Fresh Overlaying Thrombi
Vulnerable Plaque Type 3

10. Intra-Plaque Hemorrhage Prone to Thrombosis
Vulnerable Plaque Type 4

11. Vulnerable Plaque Type 5
Asymptomatic significantly stenotic plaque prone to occlusion

12. Further new types of Vulnerable
Plaque to be discovered in
future.

13. Less angiogenesis (?)Extensive angiogenesis
Low modified cholesterolHigh modified cholesterol
High collagen contentLow collagen content
Small or no lipid poolLarge lipid pool
Thick fibrous capThin fibrous cap
Low-Risk Plaque,
Hard Plaque
Unstable Plaque, High-Risk
Plaque, Soft Plaque
Structural or Morphologic Classification
Vulnerable Plaque Stable Plaque

14. Not exposed but may
contain as much
Exposed proteoglycans
(versican and hyaluronan)
Intact endothelial lawyerEndothelial denudation
High collagen contentOverlaying thrombosis
No thrombosisDisrupted / fissured cap
Concentric (negative
remodeling)
Eccentric (positive
remodeling)
Small or large plaque
volume
Small or large plaque
volume
Structural or Morphologic Classification
Vulnerable Plaque Stable Plaque
Cont…

15. Hemodynamically
significant
(>75% stenosis)
…
Hemodynamically
insignificant
(< 75% stenosis)
…
Low strain (stiff)High strain (elasticity)
More calcifiedLess calcified ?
Structural or Morphologic Classification
Stable PlaqueVulnerable Plaque
Cont…

16. Minimum apoptosis
…
Excessive apoptosis
…
Low oxidative stressHigh oxidative stress
(excessive oxygen and nitrogen free
radical formation)
Normal or high pH with
minimum pH heterogeneity
Acidic with high pH
heterogeneity
Normal temperature with
minimal heterogeneity
Hot with increased
temperature heterogeneity
Low traffic (monocyte and T
cell recruitment)
High traffic (monocyte and T
cell recruitment)
Quiescent Plaque
Low-Risk Plaque
Active Plaques
Unstable Plaque
High-Risk Plaque
Functional or Physiologic Classification
Vulnerable Plaque Stable Plaque

17. Plaque characterization
by MRI has been
introduced by Toussaint
and others to study
structural properties of
atherosclerotic plaque.
MRI and Plaque Characterization:

18. Carotid artery plaqueCCA
Carotid bifurcation
ICA stenosis & plaque
Courtesy of
Dr. Chun Yuan
University of
Washington,
Seattle

19. Question!
 Lets assume that we are in our
dreamland and non-invasive MR
imaging of coronary artery with
<100 micron resolution is easily
obtained, now the question is
whether we are able to accurately
detect all vulnerable plaques only
by studying their structural
properties or we need more?

20. Plaque Morphology
vs.
Plaque Activity
 Why do we need to go beyond
morphological assessment of
plaques? Why do we need both?
 The short answer is: because not all
plaques with similar morphology would
result in similar outcome.

21. Functional vs. Structural Imaging
Inactive and
non-inflamed
plaque
Active and
inflamed plaque
Different
Similar
IVUS OCT MRI
w/o CM
Structural:
Functional:
Thermography,
Spectroscopy, MRI w/ CM

22. Therefore,
We need a
combined method
to image both
morphology and
activity of plaques

23. We need MRI with vulnerable plaque
targeted contrast media that identifies:
1- Inflammation (macrophage infiltration),
2- Fissured/Permeable Cap,
3- Leaking Angiogenesis and
4- Intra-Plaque Hemorrhage
5- …

24. Willerson et al:
Study of fluorescent labeled
macrophage homing into
Apo E deficient mice
Circ 1998

25. SPIO
 Super
 Paramagnetic
 Iron
 Oxide
Colloidal coated nano-particles of iron oxide, e.g.
dextran coated SPIO
20-100 nanometer particle size
Phagocyted by, and accumulated in cells with
phagocytic activity
Shortening MR relaxation time, early T2 and late
T1 effect

26. USPIO
 Ultra
 Super
 Paramagnetic
 Iron
 Oxide
Smaller particle size which yields a
longer circulation time, yet less
phagocytosis and more uptake by non-
immune cells

27. Particle Core Size Particle Size Blood
(nm) (nm) Half-life
Combidex 5-6 20-30 8h
Feridex 4-6 35-50 2.4±0.2h
MION 4-6 17 varies
… … … ….
Examples of Commercially Available SPIO

28. Prior works done by
others for imaging
inflammation by MRI
and SPIO

29. Maamoun add Ref

30. Flash MR Image of a Rat Kidney With
Experimental Nephritic Syndrome, Before
(Right)and 24h After USPIO Injection(left)
Maamoun add Ref

31. Correlation between macrophage and MR
signal reduction in the kidney cortex
Maamoun add Ref

32. Cardiac Application
 Monitoring rejection of transplanted
heart and lungs following rat
allograft and homograft
transplantation, w/wo cyclosporin
Ho et al, ISMRM 2000

33. Old literature!
Iron particles observed
immediately under the
endothelium 5 hours after the
administration, in artery, in a
rat with 7 days hypertension
33 years ago !!!
Gordon et al, 1968
Maamoun add Ref

34. Our Hypothesis:
 Vulnerable atherosclerotic plaques
which have 1) active recruitment of
monocytes and T cells, 2)
extensive leaking angiogenesis 3)
fissured or permeable cap can be
detected by excessive uptake of
SPIO particles.

35. vasa vasorum
Over magnification is a major advantage of SPIO
Darkening property of SPIO in the white background of fat and
water of plaque is another advantage

36. Why negative enhancement?!!
Positive Contrast Negative ContrastV.S.
Gd-compounds SPIOs
+ -
Knowing that plaque has white background due to its fat and water

37. What we have done:
 - In vitro study of SPIO uptake by
macrophages using fluorescent
labeled home-made SPIO
 -In vitro study of SPIO uptake by
macrophages and its effect on T2
relaxation time
 -In vitro study of effect of SPIO on
macrophage biology and super
oxide production

38. What we have done:
 - In vivo study of bio distribution of
SPIO in Apo E deficient
atherosclerotic mice vs normal wild
type C57 black mice
 In vivo MRI study of aortic wall in
Apo E deficient mice vs. wild type
normal mice 4.7 T -in collaboration
with Dr Quast’s lab UTMB

39. Invitro Study of
Macrophage SPIO Uptake
 In a series of invitro studies we
have tested the rate of SPIO
uptake by human activated
monocytes in different conditions
regarding incubation time and
concentration of SPIO. All SPIO
were labeled by a fluorescent dye
(DCFA)

40. FL-labeled SPIO Incubated Macrophages 24hr

41. Mouse Peritoneal Macrophages Incubated with SPIO after 6hr

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

44. SPIO and T2 Effect
Invitro study to show the effect
of macrophage SPIO uptake on
their T2 relaxation time

45. Protocol:
 We used 8 flasks of CBM macrophages.
 After preparing the cells, Feridex was
added with the proper concentration to
each labeled tube.
 Incubation was done at 37 C.
 For each time, pellet the tubes at 1000
rpm’s for 5 min.
 Washed with 1X PBS for 5 min 3 times.
 Resuspended in 2% paraformaldehyde, to
fix the cells.

46. time
concentration
20
Min
1
Hour
6
Hours
24
Hours
50µl 100µl 250µl 500µl control control
Expected T2 Reduction Effect

49. Macrophage Uptake of Feridex After 20
Min Shown by T2 Reduction
0
10
20
30
40
50
60
70
80
90
50 100 250 500 control control
20 min
Concentration µl

50. 0
10
20
30
40
50
60
70
80
90
50 100 250 500 control control
60 min
Macrophage Uptake of Feridex After 60
Min Shown by T2 Reduction
Concentration µl

51. 0
10
20
30
40
50
60
70
80
90
50 100 250 500 control control
6 Hours
Macrophage Uptake of Feridex After 6hr
Shown by T2 Reduction
Concentration µl

52. 0
10
20
30
40
50
60
70
80
90
50 100 250 500 control control
24 Hours
Macrophage Uptake of Feridex After 24hr
Shown by T2 Reduction
Concentration µl

53. 0
10
20
30
40
50
60
70
80
90
50 100 250 500 control control
20 min
60 min
6 hours
24 hours
Macrophage Uptake of Feridex with Time
and Concentration Shown by T2
Reduction
Concentration µl

54. 0
10
20
30
40
50
60
70
80
90
20 Min 60 Min 6 Hours 24 Hours
50
100
250
500
control
control
Macrophage Uptake of Feridex with
Concentration and Time Shown by T2
Reduction
µl

55. Study of production of
Reactive Oxygen Species
by SPIO Incubated
Macrophages

56. Since the production of
reactive oxygen species
(ROS) in the plaque might
have unfavorable effects on
the biology of the plaque,
we have planned to check if
the SPIO would excessively
produce ROS.

57. Facts
 Any event of phagocytosis is immediately followed by
a transient release of super oxide due to the
assembly of the NADPH oxidase against the plasma
membrane. Subsequently the oxidase translocates
onto the phagosomes containing the SPIO to
produce intracellular ROS.
 Thus an early extra cellular secretion of super oxide
is detectable (using luminol) soon after phagocytosis
and a later event of intracellular secretion is
measurable using DCFDA dye .

58. Method
 · The suspension of SPIO (1.25-10 uL) was added to
macrophages (1x10*4/well in 96 well plates). Cells
were incubated for 1 h and washed to remove extra
cellular FDIO. For each dose three wells were tested.
Isoluminol substrate was added and super oxide
induced luminescence measured at 15, 30 and 45
min intervals using a luminometer.

59. Results
· SPIO was internalized by macrophages as
early as 15 min after addition.
· Uptake was followed by release of super oxide
for all four doses tested.
Super oxide was released by SPIO at all
doses tested (1.25-10 ul)

60. Dosage of SPIO: 1.25micL
0
500
1000
1500
2000
2500
3000
3500
15min 30min 45min NOSPIO
Sample1
Sample2
Sample3
ROS Production: Time VS SPIO Concentration

61. Dosage at 2.5micL
0
500
1000
1500
2000
2500
3000
3500
15min 30min 45min
Sample1
Sample2
Sample3
ROS Production: Time VS SPIO Concentration

62. Dosage at 5micL
0
500
1000
1500
2000
2500
3000
15min 30min 45min
Sample1
Sample2
Sample3
ROS Production: Time VS SPIO Concentration

63. Dosage at 10micL
0
200
400
600
800
1000
1200
1400
1600
1800
15min 30min 45min
Sample1
Sample2
Sample3
ROS Production: Time VS SPIO Concentration

64. SPIO biodistribution in
ApoE mice

65. Iron staining of mouse circulating
monocyte after 15 minutes

66. Iron staining of mouse circulating
monocyte after 30 minutes

67. Specimens were
taken at interval
of 3mm from
arotic root to the
renal aorta

68. SPIO Accumulation in
Atherosclerotic Plaque
Atherosclerotic plaque
in aortic root
Normal aortic segment
Iron staining of Apo E K/O Aorta, 24 hour after SPIO injection
Iron
particles

69. ApoE Mouse 3 Days After
Injection
H&E Pearl’s
Aorta-2
Atherosclerotic plaque in thoracic aorta

70. Aortic Root after 5 days
Dense infiltration of iron particles as
shown by light blue in Pearl’s staining

71. Aortic Plaque at Renal Level After 3 days

76. Control C57Black
No plaque, No Iron
Pearl’s staining

77. 0
5
10
15
Atherosclerotic
Aorta
Average
numberofiron
particlesper
sample
P<0.001
Comparison of the Number of the Iron Particles in Apo
E KO Mice Plaque vs. Normal Wall
Normal-Looking
Vessel Wall of Same
Apo E Mice

78. MRI SPIO study of
Apo E v.s. Normal Mice

81. TE: 12ms TR: 2500 FOV: 6x6
256x256
Only respiratory gating was done

82. Images of aorta from renal level

83. MR Image of Abdominal Aorta After
SPIO Injection in Apo E and Control Mice
Apo E
deficient
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 in pathology

84. MRI Imaging of
Atherosclerosis using SPIO
Studies done recently by others:
 1- Schmitz SA, Coupland SE, Gust R,
Winterhalter S, Wagner S, Kresse M,
Semmler W, Wolf KJ
Superparamagnetic iron oxide-enhanced
MRI of atherosclerotic plaques in
Watanabe hereditable hyperlipidemic
rabbits.
Invest Radiol. 2000 Aug;35(8):460-71.

85. Group I II III IV
USPIO 0 50µmol Fe/kg 50µmol 200µmol
Time - 8 hr 24 hr 48 hr
Schmitz et al J. Inv. Radiol. 2000

86. Control
SPIO
Injected
Schmitz et al J. Inv. Radiol. 2000

87. Schmitz et al J. Inv. Radiol. 2000

88. Schmitz et al
J. Inv. Radiol.
2000

90. Schmitz et al
J. Inv. Radiol. 2000

91. 2- Ruehm SG, Corot C, Vogt P, Kolb S,
Debatin JF.
Magnetic resonance imaging of atherosclerotic
plaque with ultrasmall superparamagnetic
particles of iron oxide in hyperlipidemic rabbits.
Circulation. 2001 Jan 23;103(3):415-22.
Studies done recently by others:
MRI Imaging of
Atherosclerosis using SPIO

92. A, Coronal MIP and (B) sagittal oblique and (C) coronal oblique
reformatted images of contrast-enhanced 3D MRA data set collected
after intravenous administration of Gd-DOTA displaying aorta of 7-
month-old hyperlipidemic rabbit. Aortic wall is smooth, without
evidence of luminal narrowing.
Reuhm et al,
Circulation
2001

93. A, Coronal MIP and (B) sagittal oblique and (C) coronal oblique
reformatted images of contrast-enhanced 3D MRA data sets of same
hyperlipidemic rabbit as depicted in Figure 1 obtained 5 days after
intravenous injection of USPIO agent Sinerem. Note susceptibility
effects originating within vessel wall and representing Fe uptake in
macrophages embedded in plaque.
Reuhm et al,
Circulation
2001

94. A, Intraluminal signal measured in single large ROI (9 mm2
) revealed significant
increase in SNR, with maximum reached at day 5 after contrast administration. These
changes reflect T2* effects, which decreased over time. B, SNR values based on 3
ROI measurements in aortic wall of each animal failed to reveal statistical difference
between precontrast and 5 days post-Sinerem image sets in normal control rabbits. In
hyperlipidemic animals, conversely, significant decrease in SNR corresponding to
select USPIO uptake in plaque formations containing MPS cells was evident.
Reuhm et al, Circulation 2001

95. Ex vivo imaging of contrast-filled aortic specimen of (A) hyperlipidemic rabbit 5 days
after administration of Sinerem, (B) normal control rabbit 5 days after administration of
Sinerem, and (C) hyperlipidemic rabbit that did not receive Sinerem. Marked
susceptibility artifacts are present in aortic wall of hyperlipidemic rabbit that had
received Sinerem (A). No such changes are visualized in other 2 rabbits (B, C).
Reuhm et al,
Circulation
2001

96. Cross-sectional histopathological sections with Prussian blue staining of aorta of same
hyperlipidemic rabbit as depicted in Figures 1 and 3, killed 5 days after administration
of USPIO agent Sinerem. Note thickening of intima with marked staining of Fe
particles embedded in atherosclerotic plaque formations.
Rheum et al,
Circulation
2001

97. Conclusion:
 Non-invasive MRI study of
atherosclerotic plaques using SPIO
(pre and post injection
comparison) may be a likely
method for detection of vulnerable
plaques
 Further studies particularly human
clinical trials are warranted

98. SPIO Clinical Trial:
- The first human clinical trial on
detection of carotid vulnerable plaque
using SPIO in patients undergoing
carotid endartherectomy
Baseline
SPIO
Injection
1hr post-
injection
5days
Scan
Surgery

99. Dr. Naghavi – The first
volunteer subject in his
Carotid MRI SPIO Study

104. Multi-Center Trial:
 The second site of the study is
going to be Univ. of Washington
Seattle directed by Dr. Yuan.
 The interim report of the trial will be
presented at AHA 2001 in
Anaheim

105. The Online Cardiovascular Research Community
www.VulnerablePlaque.org
All slides will be available on:

107. WWW.HotPlaque.Com