1. Cardiovascular Research Institute Washington Hospital Center
Genetic Variations of Human Proteins Binding to Mouse Vulnerable Plaque-like Lesions Identify Individuals at High Risk of Myocardial Infarction
Stephen E. Epstein
Cardiovascular Research Institute
Washington Hospital Center 1

2. Disclosure Statement of Financial Interest
No conflicts of interests. 2

3. Vascular wall “injury” activates endothelial cells
Hypothesis:
Vascular wall “injury” activates endothelial cells
Causing them to express pro-inflammatory and other as yet unknown receptors nature normally uses to evoke and augment the healing responses to injury.
Based on Hansson GK, Libby P. Nat Rev Immunol. 2006;6:508-19 3 3

4. The resulting inflammatory/injury response leads to plaque progression and predisposes to plaque rupture.
Hansson GK, Libby P. Nat Rev Immunol. 2006;6:508-19 4

5. An inflammatory response is also intrinsic to collateral development
Human bone marrow phage display cDNA library
1 day and
4 days 5 5

6. Inflammatory and stem/progenitor cell responses critically influence both vulnerable plaque and collateral development.
Homing mechanisms designed to deliver these cells to injured tissue depend on the interaction between ligands expressed by the cells, and the receptors expressed by the endothelial cells lining the vessels supplying the injured tissue. 6

7. To identify ligands that bind to vulnerable plaques, we’ve used a modified “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. 7

8. 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 8

9. 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 9

10. With this technique we can, in one study, test millions of different human molecules to see which ones bind to the vulnerable plaque. 10

11. The phage fishing strategy
Inject millions of phage, carrying millions of different proteins on their surface
Isolate and amplify these phage 11

12. harvest tissue with retained phage
Identifying ligands and the genes that encode them that functionally relate to human vulnerable plaque.
harvest tissue with retained phage
Identification of ligand and of gene
expand phage

13. Human bone marrow phage display cDNA library
Ischemic hindlimb model: Homing to collaterals
1 day and
4 days
Human bone marrow phage display cDNA library 13 13

14. Binding of phage to developing collaterals vs
Binding of phage to developing collaterals vs. normal quiescent collaterals
130-fold greater binding
Phage(pfu/ml)
10,000,000
5,000,000
Developing collaterals
due to obstructed artery
No obstruction-quiescent collaterals
Wellstein, Schmidt, Zbinden,
Burnett, and Epstein-2007 14

15. Summary: collateral-binding ligands
Collaterals in ischemic legs enriched for 14 different cDNAs unknown genes, 3 new exons
The selected mRNAs are
- highly expressed in bone marrow precursor or stem cells
- differentially expressed in distinct leukocyte populations 15

16. 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 16

17. Using this approach, we have identified 11 human ligands that appear to preferentially bind to plaques in mice in a model in which the lesions have many phenotypic features of human vulnerable plaque. 17

18. Normal Leukocyte Homing Mechanisms
What we have apparently discovered are a new group of homing molecules, expressed by bone marrow precursor cells as well as different types of circulating leukocytes, the apparent function of which is to deliver these cells to sites of vascular or tissue injury.
Gordon & Taylor Nat Rev Immunol 2005 18

19. Targeted Delivery Program:
Vulnerable Plaque Homing Ligand Project and Future Directions
Novel animal models
Novel phage fishing strategy
Ligand
Discovery
Disease
Biomarkers:
Discovery Program:
Targeted Delivery Program:
Genetic
We found ligands that are encoded by newly identified genes; these ligands are used by stem/progenitor cells and circulating leukocytes.
Targeted non-invasive molecular imaging
Targeted drug delivery to:
developing collaterals
vulnerable plaque
Identify SNPs in genes encoding these ligands—which ones are linked to AMI/CAD?
Genetic biomarkers
for AMI 19 19

20. Hypothesis
If these homing molecules are involved in delivering cells to the injured vessel as part of a “healing” response, then it is possible that genetic variation in and surrounding the genes encoding these ligands will be associated with altered risk of plaque rupture. 20

21. Performed Genetic Risk Score Analysis using these 56 SNPs.
Isolated genetic variation +/- 100kb from 23 binding sequences (n = 1784 SNPs; Affy 6.0)
Discovered 56 variants with p<0.05 in the WHC population (500 CAD-MI; 500 CAD no MI)
Performed Genetic Risk Score Analysis using these 56 SNPs. 21 21

22. VP-and collateral-binding SNPs and risk of AMI
Genetic Risk Score
VP-and collateral-binding SNPs and risk of AMI
p =
p =
p = 1 × 10-8
1.93
1.78
2.88 22

23. Hundreds to thousands of SNPs identified…
Contrasting Complementary Discovery Strategies
GWAS Strategy
Hundreds to thousands of SNPs identified…
…Most with unknown function, unknown mechanistic relation to disease, and each, if relevant to disease, probably playing a small role. ? * ? 23

24. * * * * * * * * * * * * Contrasting Complementary Discovery Strategies
Functional Strategy * * * * * * * * * * * *
Discover ligands binding to receptors expressed uniquely by “activated” ECs; these identify final common effects of multiple pathways each involving multiple molecules—it is the aggregate total of polymorphisms that influence the response of the vessel wall to “injury.” 24

25. Targeted Delivery Program:
Vulnerable Plaque Homing Ligand Project and Future Directions
Novel animal models
Novel phage fishing strategy
Ligand
Discovery
Disease
Biomarkers:
Discovery Program:
Targeted Delivery Program:
Identify SNPs in genes encoding these ligands—which ones are linked to AMI/CAD?
Genetic
We found ligands that are encoded by newly identified genes; these ligands are used by stem/progenitor cells and circulating leukocytes.
Targeted non-invasive molecular imaging
Targeted drug delivery to:
developing collaterals
vulnerable plaque
Genetic biomarkers
for AMI 25

26. Georgetown University Anton Wellstein Marcel Schmidt
Investigators
CRI
Stephen E. Epstein
Mary Susan Burnett
Ron Reiter
Amir Najafi
Stephan Zbinden
Jinsong Wang
Remi Adenika
Georgetown University
Anton Wellstein
Marcel Schmidt
Cardiovascular Pathology Institute
Renu Virmani
Frank D. Kolodgie
Intermountain Medical Center
Benjamin Horne