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