1. Cardiovascular Research Institute Washington Hospital Center
A Novel Mouse Model Containing Most of the Elements of Human Vulnerable Plaque
Stephen E. Epstein
Cardiovascular Research Institute
Washington Hospital Center 1

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

3. The impetus for this project evolved from the VP Project, the purpose of which was to identify ligands binding to VP-like lesions in an animal model. 3

4. The phage fishing strategy
Inject millions of phage, carrying millions of different proteins on their surface
Isolate and amplify these phage 4

5. The problem was that a compelling animal model of activated or vulnerable plaques did not exist.5

6. The model we choose to test was a mixed strain Apo-E KO mouse (that had attributes we thought might produce the lesions we were looking for); we further subjected these mice to chronic cold stress (standing in 1cm ice water 1hr/day for 4 wks).

7. 16 wks 20 wks Age 0 6 wks 12 wks Start lard diet
Start daily cold stress
Start lard diet
Start daily cold stress
Age 0
6 wks
16 wks
20 wks
Age 0
6 wks
36 wks
40 wks
Start lard diet
Start daily cold stress

8. Class I
Lumen
Class 1: single layer of foam cells 8

9. Class 2
Lumen
Class 2: multiple layers of foam cells

10. Class 3
Lumen
Class 3: multiple layers of foam cells with free cholesterol and rare SMCs.

11. Class 4 L MФ D FC NC
Class 4 (“fibrous Cap atheroma): well-formed necrotic core (NC) and cholesterol clefts with overlying fibrous cap and some degree of macrophage (MФ) invasion.

12. Class 5 Lumen FC E NC MФ IPH
Class 5 (activated atheroma): All the class 4 features plus neovascularization and/or intraplaque hemorrhage (IPH).

13. Comparison of Brachiocephalic lesions of 16 week old stressed C57 and stressed mixed background.

14. Neovascularization and intraplaque hemorrhage in lesions of 20 week old stressed, mixed strain mice.B A A
Neovascularization (arrows)
CD 31 staining confirms the presence of neovascularization (arrow).

15. Neovascularization and intraplaque hemorrhage in lesions of 20 week old stressed, mixed strain mice.
Intraplaque hemorrhage in the shoulder area.

16. Neovascularization and intraplaque hemorrhage in lesions of 20 week old stressed mice.
TER 119 staining of the same lesion confirms the presence of RBCs.
CD 31 staining of the same lesion shows presence of ECs in the region of plaque hemorrhage.

17. Effects of stress on intraplaque hemorrhage in 20 week old mixed strain mice
100 80
P=0.03
Percent of lesions
with NV/IPH 60 40 20
no stress
stress
20 week old stressed mice have higher percentage of lesions with neovascularization / intraplaque hemorrhage

18. (Percent of lesion area)
Effects of stress on intraplaque hemorrhage in 20 week old mixed strain mice 20 16
P=0.02
(Percent of lesion area)
Macrophage content 12 8 4
No Stress
Stress
20 week old stressed mice have higher macrophage content.

19. Lesion phenotype in 40 week old stressed mouse.
Movat staining of a 40 week old stressed mouse.
Mac3 staining of a 40 week old plaque. There is no evidence of macrophages.

20. Differences in lesion “vulnerability” phenotype in 16 and 20 vs
Differences in lesion “vulnerability” phenotype in 16 and 20 vs. 40 week old mice, and differences in the response to stress.
No Stress
Stress *
Stress increases frequency of Class 5 atheromas at the age of 16 and 20 weeks. However, there are essentially no Class 5 lesions in the 40 week old mice, whether stressed or non-stressed. The lesions at this age appear “burned out”.

21. Indirect validation that this mouse model contains elements common to human vulnerable plaque.

22. 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 22

23. Human bone marrow phage display cDNA library
Ischemic hindlimb model: Homing to the proliferating ECs of developing collaterals
1 day and
4 days
Human bone marrow phage display cDNA library 23 23

24. harvest tissue with retained phage
Strategy for identifying ligands and the genes that encode them that functionally relate to development of human vulnerable plaque and plaque rupture.
harvest tissue with retained phage
Identification of ligand and its gene
expand phage 24

25. 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. 25 25

26. 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 26

27. Conclusions
We have developed a mouse model of atherosclerosis in which the plaques contain many of the attributes of human vulnerable plaque. The model has a well defined time course of development of lesions, and is highly reproducible.
Using this model:
We seem to have discovered a new group of homing molecules, probably used by stem/progenitor cells for homing to injured tissue.
This model may provide an opportunity to learn more about the evolution and biology of vulnerable plaque, as well as provide a model for screening new drugs being tested for plaque pacification properties—or for unanticipated plaque activation properties.

28. Georgetown University Zofia Zukowska
Investigators
CRI
Mary Susan Burnett
Amir Najafi
Nima Aghili
Lee Alderman
Jim Andrews
Jusin Tilan
XinZhi Peng
Stephen E. Epstein
Georgetown University
Zofia Zukowska
Cardiovascular Pathology Institute
Renu Virmani
Frank D. Kolodgie