1. The Future of Non-Invasive Molecular Imaging of the Vulnerable Plaque
Stephen E. Epstein, MD
Cardiovascular Research Institute
Washington Hospital Center

2. Stephen E. Epstein, MD DISCLOSURES Royalty Consulting Fees
Receipt of Intellectual Property Rights/Patent Holder Patent No. 5,244,460 (issued), entitled, "Method To Foster Myocardial Blood Vessel Growth And Improve Blood Flow To The Heart." Inventors: E. Unger and S. Epstein
Holder of a patent entitled, “Injection of Autologous Bone Marrow into Ischemic Tissue as a Novel strategy to Enhance Collateral Blood Vessel Formation and Tissue Perfusion. Inventors: R. Kornowski, S Fuchs, M Leon, and S. Epstein.
Consulting Fees
Athersys, Inc
Ownership Interest (Stocks, Stock Options or Other Ownership Interest)
Myocardial Therapeutics Inc.

3. Multiple invasive imaging modalites are being tested to identify vulnerable plaques and attempt to link them to future events during follow-up.
Imaging modalities being evaluated:
IVUS
virtual histology
palpography
thermography
angioscopy
Near infrared spectroscopy
OCT
Problem:
Because these are all invasive (intracoronary) strategies, they therefore apply to a very limited patient cohort.

4. The critical clinical question:
How can vulnerable plaques be detected, not only in symptomatic patients, but in the much larger cohort of individuals who are asymptomatic and appear normal, but are in fact at high risk?

5. Plaque activation, rupture and thrombosis
Question: What elements of the vulnerable plaque can be effectively targeted—for targeted delivery of imaging agents (and of therapeutic agents)?
Hansson GK, Libby P. Nat Rev Immunol. 2006;6:508-19

6. Some of the “usual suspects” residing within vulnerable plaques
Ligands
CXCLl (growth- regulated oncogene-alpha);
CXCL5 (ENA-78);
CXCL8 (JL-S);
CXCL9 (Mig);
CXCLlO (IP-10);
CXCLI l (I-TAC);
CXCL12 (SDF-I);
CCL2 (MCP-I);
CCL3 (MIP-lalpha);
CCL5 (RANTES);
CCL7 (MCP-3);
CCL17 (TARC);
CCL22 (MDC);
fractalkine (FKN);
HSP60,
(S)-2-amino-4-phosphonobutanoic acid
(S)-3,4-dicarboxyphenylglycine;
IL-6;
IL-8;
IFN-γ;
TNF-α.
VCAM-I
ICAM-I .
Receptors of:
CXCLl (growth-regulated
oncogene-alpha);
CXCL5 (ENA-7S);
CXCL8 QL-S);
CXCL9 (Mig);
CXCLlO (IP-IO);
CXCLH (I-TAC);
CXCL12 (SDF-I);
CCL2 (MCP-I);
CCL3 (MIP-I alpha);
CCL5 (RANTES);
CCL7 (MCP-3);
CCLl 7 (TARC);
CCL22 (MDC);
fractalkine (FKN);
HSP60,
(S)-2-amino-4-ρhosphonobutanoic acid
(S)-3,4- dicarboxyphenylglycine);
IL-6,
IL-8,
IFN-γ
TNF-α.
Other Receptors
CXCR3,
CXCR4,
CCR4,
CX3CR1,
toll-like receptors,
metabotropic glutamate receptors (mGluRs),
αvβ3-integrins

7. POTENTIAL “USUAL SUSPECT” TARGETS
Molecules upregulated during endothelial proliferation
αvβ3-integrins
Lipid-laden plaques
Image oxidized low-density lipoprotein in atheromatous plaques with MRI
Gd3-loaded micelles targeted
to the macrophage scavenger receptor
Inflammation
Increased VCAM-1 present in aortic plaques of apoE/ mice imaged by using a dual contrast agent detectable by both MRI and fluorescence imaging.
Labeling of monocytes
In vivo labeling
Ultrasmall long-circulating superparamagnetic iron-oxide nanoparticles are engulfed by macrophages in vivo. These macrophages alter MRI signal in proportion to the degree of atherosclerotic plaque inflammation.
In rabbits, specific uptake of an iodine-containing contrast agent by macrophages allows inflamed atherosclerotic lesions to be detected with CT.
Ex-vivo labeling
Ex-vivo labeling of monocytes and then injection into intact animal/human homing and detection

8. We are using a very different strategy, based on the fact that we don’t really know what the best targeting molecules are—we therefore determine, without prior assumptions, what molecules actually bind preferentially to vulnerable plaques.

9. Our approach attempts to mimic how stem/inflammatory/immune cells normally home to injured vessels.
Homing occurs by receptor/ligand interactions between molecules expressed by endothelial cells and by circulating stem/inflammatory/immune cells.
Hansson GK, Libby P. Nat Rev Immunol. 2006;6:508-19

10. To identify ligands that bind to vulnerable plaques, we’ve used the “phage fishing” technique, which provides a means to test millions of molecules to determine which ones actually bind.
A phage (bacteriophage) is a virus that infects only bacteria.

11. Phage have hundreds of molecules attached to its surface, which are critical to its function.
We employ molecular techniques that cause the phage to produce one human molecule on its surface .
From Wellstein-2003

12. Human bone marrow matrix is used as a source of millions of cDNAs
BM & derived cells
cDNA library
displayed on phage
(1 human protein/phage)
cDNA library
mRNA
transfect
phage
From Wellstein-2003

13. With this technique we can, in one study, test millions of different human molecules to see which ones bind to the vulnerable plaque.

14. The phage fishing strategy
Inject millions of phage, carrying millions of different proteins on their surface
Isolate and amplify these phage

15. Mouse Model of “Activated” Plaque
Genetically-based; mice physically stressed
and fed a high cholesterol/lard diet
Lesions have many of the components present in human vulnerable plaque

16. Mice sacrificed at 5 months of age
Left common carotid artery
Flow-limiting lesions
Brachiocephalic
artery
Left subclavian artery
Necrotic
core
Cholesterol clefts
Thin cap
Vasa vasorum
Vasa vasorum
Burnett, Kolodgie, Virmani, Epstein

17. Negative control
Movat
Anti-glycophorin A
Anti-CD31

18. Activated arteriosclerotic plaque
( apoE-/- & lard ) bc
lca
harvest tissue with retained phage
lsc
expand phage
Inject phage into new mouse
Human bone marrow phage display cDNA library

19. Interestingly, these ligands are:
Using this approach, we have recently identified 11 human ligands that appear to preferentially bind to plaques in mice.
Interestingly, these ligands are:
encoded by genes not previously recognized as being genes.
expressed by BM stem and progenitor cells and different types of circulating leukocytes.
Reiter, Burnett, Schmidt, Wellstein, and Epstein-2009

20. To confirm that these ligands actually do bind to lesions present in our mouse model, we labeled the ligands (using Qdots), injected them into mice with atherosclerotic lesions, and determined whether the lesions could be imaged.
Reiter, Burnett, Schmidt, Wellstein, and Epstein-2009

21. Plaque-binding ligands
Aorta, selected for λ605
(control)
Aorta, selected for λ655
(VP binding ligands 1-11)
Higher power image of aorta, selected for λ605
Control protein
Higher power image of aorta, selected for λ655
Plaque-binding ligands
Reiter, Burnett, Schmidt, Wellstein, and Epstein-2009

22. Normal Leukocyte Homing Mechanisms
We have apparently discovered a new group of homing molecules, expressed by bone marrow precursor cells as well as different types of circulating leukocytes.
Gordon & Taylor Nat Rev Immunol 2005

23. Since these ligands are homing molecules and appear to be used by stem/progenitor cells and circulating leukocytes, we thought they might play a role in vascular injury.
We therefore hypothesized that polymorphisms in the genes encoding these ligands have an impact on vulnerable plaque development—ie, they are linked to AMI.

24. To test this hypothesis, we analyzed the relationship of SNPs present in these 11 ligands to the risk of AMI in 3 cohorts of patients in whom GWAS studies had been performed:
Medstar
PennCATH
Ottawa Heart

25. Ligand Homing to:
Region N
Meta analysis p-value
Odds ratio (95% CI)
Atherosclerotic plaque 9q
>3000
0.03
0.67 ( ) 8p
0.003
0.53 ( )
12q
0.031
1.21 ( )
21q
0.026
1.17 ( )
0.018
1.26 ( )
0.0061
1.29 ( )
0.037
1.16 ( )

26. We are in the process of adding additional cohorts to determine whether these results can be confirmed in a still larger group of patients.

27. Conclusions
We have developed a mouse model of atherosclerosis in which the plaques contain many of the attributes of human vulnerable plaque. Using this model:
We have discovered a new group of homing molecules, probably used by stem/progenitor cells for homing to injured tissue.
These novel ligands appear to play a critically important physiological role, since polymorphisms in proximity to the genes encoding them predict increased or decreased risk of AMI.
These polymorphisms may constitute a new class of genetic biomarkers, predicting either increased or decreased risk of AMI.

28. The Future
There is great need for non-invasive methods to identify individuals at risk of myocardial infarction and stroke.
Enormous experimental progress has been made employing targeted imaging using the candidate gene/”usual suspect” approach.
An alternative approach using non-biased strategies to identify targeting ligands is promising.
It remains to be determined whether these approaches can identify ligands that bind preferentially to vulnerable plaques in patients and lead to clinically “actionable” information.

29. Georgetown University Anton Wellstein Marcel Schmidt
Investigators
CRI
Stephen E. Epstein
Mary Susan Burnett
Ron Reiter
Amir Najafi
Stephan Zbinden
Jinsong Wang
Remi Adenika
David Hellinga
Richard Baffour
Ron Waksman
Georgetown University
Anton Wellstein
Marcel Schmidt
Cardiovascular Pathology Institute
Renu Virmani
Frank D. Kolodgie