This document discusses a non-invasive technique using superparamagnetic iron oxide (SPIO) enhanced MRI to detect vulnerable plaque. SPIO particles are phagocytosed by macrophages and can thus be used to image inflammation. Studies in mice showed SPIO accumulation in atherosclerotic plaques after intravenous injection, indicating macrophage infiltration. MRI of abdominal aortas of mice after SPIO injection demonstrated higher signal in plaques compared to normal vessel walls, corresponding to greater iron uptake in inflamed plaques. This suggests SPIO enhanced MRI may non-invasively detect vulnerable plaque by imaging macrophage-mediated inflammation.

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

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

3. Detection of
Inflammation (macrophage infiltration),
Fissured/Permeable Cap,
Leaking Angiogenesis,
and Intra-Plaque
Hemorrhage
Introducing a non-invasive and potentially screening
technique for

4. No film!
No picture!!
Not even notes!!!
PLEASE

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

7. What is what?
 Vulnerable Plaque ?
 MRI in atherosclerosis?
 SPIO ?
 SPIO + MRI 
Detection of
Vulnerable Plaque
?

8. What is
Vulnerable Plaque?
 Based on the pioneering works done by
Michael Davies, Erling Falk, Valentin
Fuster, Jim Willerson, Peter Liby, Jim
Muller, Renu Virmani, Al van der Wal,
PK Shah, and many other
investigators…*
* For a complete list of pioneers see “Who is who” at
www.VulnerablePlaque.org

9. Vulnerable Plaque is
currently called as rupture-prone plaque
that is hemo-dynamically insignificant
(not stenotic <75%).
These plaques rupture into blood and their
thrombogenic content cause acute
thrombosis.
Plaque rupture may or may not be clinically
apparent.

10. Characteristics of VP
 Thin fibrous cap
(perhaps <50-100 microns)
 Large lipid core
 Dense macrophage infiltration

11. Plaque Erosion
 Virmani, and recently Thiene and van der Wal
have emphasized that plaque rupture is not
the underlying cause of all coronary thrombotic
events.
 In 30-60% of cases depending on the age and
sex, sudden death due to acute coronary
thrombosis is resulted from non-ruptured but
superficially eroded plaques.
 Eroded plaques may or may not be inflamed
 They are like prone-ruptured plaques mostly
hemo-dynamically insignificant (<75%)

12. Therefore,
 A definition based on
clinical outcome may well
recognize eroded plaque
as vulnerable plaque,
since they both cause
acute coronary thrombotic
event and yet both are
hemo-dynamically
insignificant

13. Also,
We think there are at least two other types
of plaque pathology that could be
included in the list of vulnerable plaques:
 Plaques with fissured cap and mural
thrombosis
 Plaques with extensive angiogenesis and
intra-plaque hemorrhage
These plaque may or may not overlap with
the other two categories

14. Vulnerability
 To rupture?
Or
 To induce thrombosis?
The latter seems to be a broader and
more conclusive definition. It is
also clinically more relevant.

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

16. Rupture-prone inflamed plaque
Vulnerable Plaque Type 1
To be
discussed
&
approved
by the
Scientific
Advisory
Board of
VP.org

17. Fissured Plaque with Old and Fresh Overlaying Thrombi
Vulnerable Plaque Type 2

18. Eroded Plaque with Exposed Proteoglycans Prone to Thrombosis
Vulnerable Plaque Type 3

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

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

21. Emerging Diagnostic Techniques
Angioscopy
Intravascular Ultrasound (IVUS)
Intravascular Thermography
Intravascular Optical Coherence Tomography (OCT)
Intravascular Elastography
Intravascular and Transesophageal MRI
Intravascular Nuclear Imaging
Intravascular Electrical Impedance Imaging
Intravascular Tissue Doppler
Intravascular Shear Stress Imaging
Intravascular (Photonic) Spectroscopy
A. Invasive Techniques -Intravascular

22. - Raman Spectroscopy
- Near-Infrared Diffuse Reflectance Spectroscopy
-Fibrousis and lipid measurement
-pH and lactate measurement
- Fluorescence Emission Spectroscopy
- Spectroscopy with contrast media
… Invasive Techniques
Intravascular (Photonic) Spectroscopy
Intra-coronary assessment of endothelial function
Intra-coronary measurement of MMPs and cytokines

23. Emerging Diagnostic Techniques
B. Non-Invasive Techniques:
A. MRI
1- MRI without contrast media
2- MRI with contrast media: Gadolinium-DPTA
2- MR Imaging of Inflammation: Super Paramagnetic
Iron Oxide (SPIO and USPIO)
3- MR Imaging of Thrombosis using monoclonal Ab
B. Electron Beam Computed Tomography (EBCT)
C. Multi-Slice Fast Spiral / Helical CT
D. Nuclear Imaging (18-FDG, MCP-1, Annexin V)

24. Emerging Diagnostic Techniques
C. Endothelial function test
-Flow mediated dilatation of brachial artery
-ACh induced vasorectivity of coronary artery
D. Blood Tests and Serum Markers
- CRP
- Proinflamatory cytokines
- Lp-PLA2
- MMPs ?
- Anti-body against endothelial cells

25. We sought a
 non-invasive technique
that could be used to
characterize
vulnerability of
atherosclerotic plaque
according to our
proposed definitions

26. Biochem Biophys Res Commun 1987 Dec 16;149(2):437-
42
High-resolution proton NMR spectra of human arterial plaque.
Zajicek J, Pearlman JD, Merickel MB, Ayers CR,
Brookeman JR, Brown MF.
Well-resolved proton (1H) NMR spectra of solid human arterial plaque can be acquired. Studies have been
carried out of human fatty plaque obtained postmortem (ex vivo), the total lipids extracted from human
atheroma, and a model mixture of cholesteryl esters whose lipid composition resembles that of human
atheroma. In each case, well-resolved 1H NMR spectra were obtained at body temperature (37 degrees C),
with little or no underlying broad signal. Such sharp 1H NMR spectra are typical of isotropic fluids, whereas
solid and liquid-crystalline materials give rise to much broader spectral lines. The results suggest the sharp
1H NMR spectra of human atheromatous lesions at body temperature are due largely to the presence of
intracellular and extracellular droplets of cholesteryl esters in the isotropic liquid phase. These findings
provide a necessary basis for use of 1H NMR techniques to image quantitatively the lipid constituents of
human atheroma in vivo, and to study their chemical and physical properties.
Despite a series of pioneering works by Brown et al, and others

27. Persistency goes above
pioneering
 In Early 90, as the body of
knowledge of vulnerable plaque
rapidly grew up, it was
persistent works led by Fuster,
Touissant, Yuan, and Fayad, and
later by others that brought to our
attention the feasibility of non-
invasive structural
characterization of plaque using
magnetic resonance imaging of
aortic, carotid, and finally coronary
wall.

28. Fayad et al,
Circulation.
2000 May
30;101(21):
2503-9.
Thoracic Aorta

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

30. Fayad et al
Circulation
2000 Aug
1;102(5):506-10
Coronary Art.

31. Our Dream!

32. Structure or Morphology
vs.
Function or Physiology
of plaque
 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.
Remember Erling Falk’s landmark experiments of mice swimming Olympic !

33. 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

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

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

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

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

38. MRI Structural
Characterization of Plaque
 Obviously it provides invaluable
information about the anatomy of plaque:
cap thickness, plaque volume, remodeling,
 Using multi-spectral imaging techniques, it
may also provide information of great
significance for histo-chemical
characterization of plaque: collagen and
lipid content, calcification, hemorrhage
 But it does not image inflammation nor
plaque activity

39. Therefore,
We need a way to
non-invasively
image
inflammation in
the plaque

40. MRI as a Screening Test
 If the goal is to include MRI in a package
of population based screening tests for
early detection of patients at risk for acute
coronary event, we need to ask MRI for all
it has to offer.
 MRI has many potentials that need to be
explored for studying vulnerable plaque.
 In our approach we simply have tried to
learn from experiences learned over years
in cancer and other fields

41. SPIO
 Super
 Paramagnetic
 Iron
 Oxide
Colloidal coated such as dextran
150-100 nanometer particle size
Shortening MR relaxation time mainly R2
Phagocyted by and accumulated in
cells with phagocytic activity

42. USPIO
 Ultra
 Super
 Paramagnetic
 Iron
 Oxide
Smaller size which yields a longer
circulation time

43. Particle Core Size ParticleSize 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

45. SPIO Labeled (A, C, E) andSPIO Labeled (A, C, E) and
Unlabeled (B, D, F) T-cellUnlabeled (B, D, F) T-cell
SamplesSamples

46. Intravenously injected SPIO are
opsonized by plasma proteins and are then
efficiently internalized into macrophages
and other phagocytic cells
Cellular uptake into macrophages is through C3 receptors. Both
phagocytosis and pinocytosis are involved.
It has previously demonstrated that SPIO particles
extravasate into the intrstitium of some solid tumors and
that these particles accumulate within tumor cells located
adjacent to vessels.

47. Type Species Iron Uptake ± SD (ng/106
cells) Particles per Cell
Primary isolates
Peritoneal macrophages Mouse 970 ± 77 4,850,000
Lymph node lymphocytes Mouse 19.1 ± 0.3 95,500
Lymph node lymphocytes Rat 11.5 ± 4.3 57,500
Splenic lymphocytes Mouse 18.7 ± 0.1 93,500
Splenic lymphocytes Rat 19.8 ± 1.3 99,000
Blood lymphocytes Human 27.9 ± 3.6 139,500
Tumor cells
SVEC4-10 endothelial cells Mouse 118 ± 25 590,000
C6
glioma Rat 25.0 ± 3.0 125,000
9L gliosarcoma Rat 17.0 ± 2.0 85,000
J774 sarcoma (macrophage-like) Rat 310 ± 28 1,550,000
LX1 small cell lung cancer Human 29.2 ± 2.2 145,000
BT 20 breast adenocarcinoma Human 38.2 ± 4.0 190,000
MCF-7 breast adenocarcinoma Human 11.9 ± 1.6 60,000
HBL100 breast adenocarcinoma Human 33.2 ± 0.1 165,000
D4.475 breast adenocarcinoma Human 21.1 ± 0.8 105,000
LS174T colon adenocarcinoma Human 60.0 ± 1.5 300,000
U87 glioma Human 18.4 ± 0.5 90,000
Note.—Uptake was measured after 1 hour incubation of 100,000 cells in the presence of 100 µg of 125
I-labeled LCDIO.

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

49. Correlation between macrophage and MR
signal variation in the kidney cortex

50. Iron staining,shows particles in perivascular in
encephalitis and immunohistochemistry shows
positivity of macrophages
Encephalitis

51. Cardiac Application
 Monitoring rejection of transplanted
heart and lungs following rat
allograft and homograft
transplantation, w/wo cyclosporin
Hu et al

52. Old literature never ceases to amaze me!
Iron particles observed
immediately under the
endothelium 5 hours after the
administration, in artery, in a
rat with 7 days hypertension
33 years ago !!!

53. vasa vasorum
Over magnification is a major advantage of
SPIO

54. Iron staining of mouse circulating
monocyte after 15 minutes

55. Iron staining of mouse circulating
monocyte after 30 minutes

57. SPIO Accumulation in
Atherosclerotic Plaque
Atherosclerotic plaqueNormal aortic segment
Iron staining of Apo E K/O Aorta, 24 hour after SPIO injection
Iron
particles

58. ApoE Mouse 3 Days After
Injection
H&E Pearl’s
Aorta-2

59. Aortic Root after 5 days

60. C57Black
No plaque, No Iron
Pearl’s staining

61. 0
5
10
15
Atherosclerotic
Aorta
Average
number of iron
particles per
sample
P <0.001
Comparison of the Number of the Iron Particles in Apo
E KO Mice Plaque vs. Normal Wall
Normal
Vessel Wall

64. TE: 12ms TR: 2500 FOV: 6x6
256x256

66. MR Image of Abdominal Aorta After
SPIO Injection in Mouse
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

67. Figure 4. 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

68. Figure 3. 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

69. Figure 5. 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