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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
Everybody has atherosclerosis, the question is who has Vulnerable Plaque
The Online Cardiovascular Research Community
www.VulnerablePlaque.org
All slides will be available on:
Vulnerable Plaque?Atherosclerotic plaques that cause sudden luminal clot formation and lead to heart attack and stroke.
Different Types of Vulnerable Plaque As underlying Cause of Acute Coronary Events
NormalRupture-prone
Fissured ErodedCritical Stenosis Hemorrhage
Rupture-prone inflamed plaque
Vulnerable Plaque Type 1
Eroded Plaque with Exposed Proteoglycans Prone to Thrombosis
Vulnerable Plaque Type 2
Fissured Plaque with Old and Fresh Overlaying Thrombi
Vulnerable Plaque Type 3
Intra-Plaque Hemorrhage Prone to Thrombosis
Vulnerable Plaque Type 4
Vulnerable Plaque Type 5
Asymptomatic significantly stenotic plaque prone to occlusion
Further new types of Vulnerable Plaque to be
discovered in future.
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 ClassificationVulnerable Plaque Stable Plaque
Not exposed but may contain as much
Exposed proteoglycans(versican and hyaluronan)
Intact endothelial lawyer Endothelial denudation
High collagen contentOverlaying thrombosis
No thrombosis Disrupted / fissured cap
Concentric (negative remodeling)
Eccentric (positive remodeling)
Small or large plaque volume
Small or large plaque volume
Structural or Morphologic ClassificationVulnerable Plaque Stable Plaque
Cont…
Hemodynamically significant(>75% stenosis)
…
Hemodynamically insignificant(< 75% stenosis)
…
Low strain (stiff)High strain (elasticity) More calcifiedLess calcified ?
Structural or Morphologic ClassificationStable PlaqueVulnerable Plaque
Cont…
Minimum apoptosis…
Excessive apoptosis …
Low oxidative stress High 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 PlaqueLow-Risk Plaque
Active PlaquesUnstable Plaque
High-Risk Plaque
Functional or Physiologic ClassificationVulnerable Plaque Stable Plaque
Plaque characterization by MRI has been introduced by Toussaint and others to study structural properties of atherosclerotic plaque.
MRI and Plaque Characterization:
Carotid artery plaqueCCA
Carotid bifurcation
ICA stenosis & plaque
Courtesy of
Dr. Chun Yuan University of Washington, Seattle
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?
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.
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
Therefore,
We need a combined method to image both morphology and activity of plaques
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- …
Willerson et al:
Study of fluorescent labeled macrophage homing into Apo E deficient mice
Circ 1998
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
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
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
Prior works done by others for imaging inflammation by MRI and SPIO
Maamoun add Ref
Flash MR Image of a Rat Kidney With Experimental Nephritic Syndrome, Before (Right)and 24h After USPIO Injection(left)
Maamoun add Ref
Correlation between macrophage and MR signal reduction in the kidney cortex
Maamoun add Ref
Cardiac Application Monitoring rejection of transplanted
heart and lungs following rat allograft and homograft transplantation, w/wo cyclosporin
Ho et al, ISMRM 2000
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
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.
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
Why negative enhancement?!!
Positive Contrast Negative ContrastV.S.
Gd-compounds SPIOs
+ -Knowing that plaque has white background due to its fat and water
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
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
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)
FL-labeled SPIO Incubated Macrophages 24hr
Mouse Peritoneal Macrophages Incubated with SPIO after 6hr
Double DAPI Staining with Fluorescence-labeled SPIO Macrophages after 24hr Incubation
SPIO and T2 Effect Invitro study to show the effect of macrophage SPIO uptake on their T2 relaxation time
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.
time
concentration
20 Min
1 Hour
6 Hours
24 Hours
50µl 100µl 250µl 500µl control control
Expected T2 Reduction Effect
Macrophage Uptake of Feridex After 20 Min Shown by T2 Reduction
0102030405060708090
50 100 250 500 control control
20 min
Concentration µl
0102030405060708090
50 100 250 500 control control
60 min
Macrophage Uptake of Feridex After 60 Min Shown by T2 Reduction
Concentration µl
0102030405060708090
50 100 250 500 control control
6 Hours
Macrophage Uptake of Feridex After 6hr Shown by T2 Reduction
Concentration µl
0102030405060708090
50 100 250 500 control control
24 Hours
Macrophage Uptake of Feridex After 24hr Shown by T2 Reduction
Concentration µl
0102030405060708090
50 100 250 500 control control
20 min60 min6 hours24 hours
Macrophage Uptake of Feridex with Time and Concentration Shown by T2 Reduction
Concentration µl
0102030405060708090
20 Min 60 Min 6 Hours 24 Hours
50100250500controlcontrol
Macrophage Uptake of Feridex with Concentration and Time Shown by T2 Reduction
µl
Study of production of Reactive Oxygen Species by SPIO Incubated Macrophages
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.
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 .
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.
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)
Dosage of SPIO: 1.25micL
0
500
1000
1500
2000
2500
3000
3500
15min 30min 45min NOSPIO
Sample1Sample2Sample3
ROS Production: Time VS SPIO Concentration
Dosage at 2.5micL
0
500
1000
1500
2000
2500
3000
3500
15min 30min 45min
Sample1Sample2Sample3
ROS Production: Time VS SPIO Concentration
Dosage at 5micL
0
500
1000
1500
2000
2500
3000
15min 30min 45min
Sample1Sample2Sample3
ROS Production: Time VS SPIO Concentration
Dosage at 10micL
0200400600800
10001200140016001800
15min 30min 45min
Sample1Sample2Sample3
ROS Production: Time VS SPIO Concentration
SPIO biodistribution in ApoE mice
Iron staining of mouse circulating monocyte after 15 minutes
Iron staining of mouse circulating monocyte after 30 minutes
Specimens were taken at interval of 3mm from arotic root to the renal aorta
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
ApoE Mouse 3 Days After Injection
H&E Pearl’s
Aorta-2
Atherosclerotic plaque in thoracic aorta
Aortic Root after 5 days
Dense infiltration of iron particles as shown by light blue in Pearl’s staining
Aortic Plaque at Renal Level After 3 days
Control C57BlackNo plaque, No Iron
Pearl’s staining
0
5
10
15
AtheroscleroticAorta
Averagenumber of ironparticles persample
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
MRI SPIO study of Apo E v.s. Normal Mice
TE: 12ms TR: 2500 FOV: 6x6
256x256
Only respiratory gating was done
Images of aorta from renal level
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
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.
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
Control
SPIO Injected
Schmitz et al J. Inv. Radiol. 2000
Schmitz et al J. Inv. Radiol. 2000
Schmitz et al
J. Inv. Radiol. 2000
Schmitz et al J. Inv. Radiol. 2000
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
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
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
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
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
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
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
SPIO Clinical Trial:- The first human clinical trial on
detection of carotid vulnerable plaque using SPIO in patients undergoing carotid endartherectomy
Baseline
Scan
SPIO Injection
1hr post-injection
5days
Scan
Surgery
Dr. Naghavi – The first volunteer subject in his Carotid MRI SPIO Study
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
The Online Cardiovascular Research Community
www.VulnerablePlaque.org
All slides will be available on:
WWW.HotPlaque.Com