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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
Disclosure Statement of Financial Interest
No conflicts of interests.
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.
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
An inflammatory response is also intrinsic to collateral
development
Human bone marrow
phage display cDNA
library
1 day and
4 days
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.
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.
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
Human bone marrow matrix is used as a
source of millions of cDNAs
BM & derived cells
cDNA library
displayed on
cDNA library
phage
mRNA
(1 human
protein/phage)
transfect
phage
From Wellstein-2003
With this technique we can, in one study, test millions of
different human molecules to see which ones bind to the
vulnerable plaque.
The phage fishing strategy
Inject millions of phage,
carrying millions of
different proteins on
their surface
Isolate and amplify these phage
Identifying ligands and the genes that encode them that
functionally relate to human vulnerable plaque.
harvest tissue
with retained
phage
expand phage
Identification of ligand and of gene
Ischemic hindlimb model: Homing to collaterals
Human bone marrow
phage display cDNA
library
1 day and
4 days
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 obstructionquiescent collaterals
Wellstein, Schmidt, Zbinden,
Burnett, and Epstein-2007
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
Activated arteriosclerotic plaque
( apoE-/- & lard )
bc
lca
lsc
harvest tissue
with retained
phage
expand phage
Inject phage
into new
mouse
Human bone
marrow phage
display cDNA
library
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.
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
Vulnerable Plaque Homing Ligand Project and Future Directions
Novel animal models
Disease
Biomarkers:
Genetic
Identify SNPs in genes
encoding these
ligands—which ones
are linked to AMI/CAD?
Genetic biomarkers
for AMI
Ligand
Discovery
Novel phage fishing strategy
Discovery
Program:
We found ligands that are
encoded by newly identified
genes; these ligands are
used by stem/progenitor
cells and circulating
leukocytes.
Targeted Delivery
Program:
Targeted non-invasive
molecular imaging
vulnerable plaque
Targeted drug delivery to:
developing collaterals
vulnerable plaque
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.
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.
Genetic Risk Score
VP-and collateral-binding SNPs and risk of AMI
1.93
p = 0.00072
1.78
p = 0.0043
2.88
p = 1 × 10-8
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.
?
?
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Contrasting Complementary Discovery Strategies
Functional Strategy
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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.”
Vulnerable Plaque Homing Ligand Project and Future Directions
Novel animal models
Disease
Biomarkers:
Genetic
Identify SNPs in genes
encoding these
ligands—which ones
are linked to AMI/CAD?
Genetic biomarkers
for AMI
Ligand
Discovery
Novel phage fishing strategy
Discovery
Program:
We found ligands that are
encoded by newly identified
genes; these ligands are
used by stem/progenitor
cells and circulating
leukocytes.
Targeted Delivery
Program:
Targeted non-invasive
molecular imaging
vulnerable plaque
Targeted drug delivery to:
developing collaterals
vulnerable plaque
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