Center for Vulnerable Plaque ResearchCVPR

UT- HoustonTexas Heart Institute

How the World Dies Today?YLLS: Years of Life Lost

Everybody has atherosclerosis, the question is who has vulnerable plaqueSudden Cardiac DeathAcute MIVulnerablePlaque(s)

Plaque Eruption (Volcano!)

Compensatory Enlargement of Human Atherosclerotic Coronary ArteriesN Engl J Med 1987 May 28;316(22):1371-5

How important is detection of vulnerable plaque?

In 50-70% of cases the first symptom of having a vulnerable plaque is sudden death out-of-hospital.

Kill It Before It Kills You!

Human Carotid PlaqueCourtesy of Dr. Chun Yuan University of Washington

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 Hemorrhage5- ?

Superparamagnetic and Ultra-superparamagnetic Iron OxideBlood pool magnetic resonance (MR) imaging contrast media with a central core of iron oxide generally coated by a polysaccharide layer Shortening MR relaxation timeEngulfed by and accumulated in cells with phagocytic activity

Particle Core Size Particle Size Blood (nm) (nm) Half-life

Combidex 5-6 20-30 8hFeridex 4-6 35-50 2.40.2hDDM 43/34/102 6.4 20-30 6h ClariscanMION 4-6 17 variesFeruglose --- --- --- --- Examples of commercially available SPIOs

Current applications of SPIO in MR imaging:

Detection of Hepatic Lesions

(primary and metastatic cancers)

Experimental Nephritic syndrome in Laboratory animals

Monitoring rejection of transplanted heart or kidney in

the animal model of allograft transplantation.

Experimental detection of CNS lesions in laboratory

animals.

USPIOs Enter the Atherosclerotic Plaque Through-Macrophages that engulfed them-Fissured or thin cap -Extensive angiogenesis -vasa vasorum leakage -Intra plaque hemorrhage

In-vitro Study of Macrophage SPIO Uptake In a series of in-vitro studies we have tested

the rate of SPIO uptake by human activated monocytes in different conditions regardingincubation time and concentration of SPIO.All SPIO were labeled by a fluorescent dye(DCFA).

FL-labeled SPIO Incubated Macrophages 24hr

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

HypothesisActive macrophages residing inside the inflamed vulnerable plaques can be visualized following injection of SPIO or USPIOs into the systemic circulation by virtue of a decrease in the magnetic resonance signal intensity of the plaque,and correlate with histopathologic characteristics of vulnerability to rupture.

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

SPIO and T2 EffectIn-vitro study to show the effect of macrophage SPIO uptake on their T2 relaxation time

Macrophage Uptake of Feridex with Time and Concentration Shown by T2 ReductionConcentration mol/ml

Histopathologic study of the Mouse injected With SPIO (Thoracic Aorta)ApoE KO mouse, Movat staining, proximal aortaCoronary Cross section

Atherosclerosisplaque

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

Histopathologic study of ApoE KO Mouse injected With SPIO (Abdominal Aorta)H&E stainingIron Staining

CD 68 stainingIron particles

Histopathologic study of wild type Mouse injected With SPIO (Thoracic Aorta) H&E stainingCD68 stainingIron staining

Comparison of the Number of the Iron Particles (per HPF) in ApoE KO Mice Plaque vs. Normal Wall

Iron StainingH&E StainingApo E-deficient mouse injected with SPIO Cytokines added

MR Image of Abdominal Aorta After SPIO Injection in ApoE and Control Mice

ApoE 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

Typical in vivo MR images of a live mouse at the heart (left) and renal level (right). Various vessels and aortic arch can easily e seen in these images. The slice thickness is 0.5 mm and the in-plane resolution is 50 mm (7.1 T MR system).

We chose Watanabe Hereditary Hypercholesterolemic rabbits (WHHR) and New Zealand White rabbits (NZW) for this study.

We injected them with SPIO (Feridex)1 mMol Fe/kg and obtained baseline as well as 5-day post-SPIO injection MR images of the aorta (1.5 TeslaMRI system at the University of Texas, MD Anderson,Houston,Texas).

Then we compared the images in hypercholesterolemic rabbits with the normal,wild type NZW rabbits.Rabbit ex-vivo MRI studies:After the in-vivo MR images, we sacrificed the animals and excised the aorta.Then we put the isolated aorta in a gel medium, clamped both ends and anyside branches and injected gadolinium inside the lumen.

We did the same procedure for all rabbits. We also used 2 more rabbits, one WHHR and one NZW that were not injected with SPIO, as control, in the ex-vivo MR study.SPIO-Enhanced MRI study in rabbits

Histopathologic studies of Thoracic aorta in WatanabeHereditary Hypercholesterolemic rabbit after SPIO injectionH&E stainingIron stainingIron staining

Histopathologic studies of Thoracic aorta in Watanabe Hereditary Hypercholesterolemic rabbit after SPIO injectionH&E stainingIron stainingIron stainingIron particles

R=0.956Correlation between Iron positive cells in Iron staining and cell density in H&E staining in rabbitatherosclerotic aorta.

Chart1

35

0

12

6

24

17

9

8

8

10

18

22

8

0

5

6

12

3

50

24

10

40

2

35

4

10

52

1

Cell Denity in H&E staining

SPIO positive cell -Iron staining

Plaque Cell Density vs SPIO

Sheet1

HPFsNumber of nucleated cells per HPF(H&E staining)0.956113827

13735

270

32012

4106

52824

62617

7129

8118

9128

101810

111818

123322

1388

1430

15115

16146

171412

1853

196050

201424

211410

226540

2332

245035

2554

261010

276452

2821

574431

574431

Sheet1

Cell Denity in H&E staining

SPIO positive cell -Iron staining

Plaque Cell Density vs SPIO

Sheet2

Sheet3

MR Angiography 3D with Gadolinium-DTPA in Watanabe RabbitBefore SPIO injectionAfter SPIO injection

Ex-vivo MR study of the thoracic aorta in Watanabe and Wild type rabbit after SPIO injection compared to control.3D MR Angiography with Gadolinium-DTPAWatanabe rabbitPost-SPIOWatanabe rabbitcontrolNZW rabbitcontrolNZW rabbitPost-SPIO

Ex-vivo MR study of the thoracic aorta in Watanabe and Wild type rabbit after SPIO injection compared to control.(Gradient echo) Watanabe rabbitPost-SPIOWatanabe rabbitcontrol

NZW rabbitPost-SPIONZW rabbitcontrol