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

Disclosure Statement of Financial Interest No conflicts of interests. 2

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

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

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

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

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

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

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

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

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

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

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

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

Summary: collateral-binding ligands Collaterals in ischemic legs enriched for 14 different cDNAs -- 11 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

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

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

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

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

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

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

VP-and collateral-binding SNPs and risk of AMI Genetic Risk Score VP-and collateral-binding SNPs and risk of AMI p = 0.00072 p = 0.0043 p = 1 × 10-8 1.93 1.78 2.88 22

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

* * * * * * * * * * * * 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

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

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