1. Role of Inflammation and Immune Responses
Robert L Wilensky, MD
Professor of Medicine
Hospital of the University of Pennsylvania

2. Robert L. Wilensky, MD Grants/Contracted Research GlaxoSmithKline
Ownership Interest (Stocks, Stock Options or Ownership Interest)
Johnson and Johnson
I intend to reference off label or unapproved uses of drugs or devices in my presentation.
I intend to discuss darapladib which is in Phase III clinical trial evaluation

3. Wilensky, Hamamdzic Curr Opin Cardiol 2007;22:545
Immunomodulatory cells and development of unstable coronary artery disease
Wilensky, Hamamdzic Curr Opin Cardiol 2007;22:545

4. Recent data on the role of inflammation-immunomodulation: DM/HC porcine studies
Increase in inflammatory gene expression in animals with complex disease.
Selective inhibition of Lp-PLA2 reduces necrotic core size by effecting macrophage & T-lymphocyte recruitment and functioning.
IVUS/NIRS predicts vulnerable plaque development and local vascular inflammation.

5. Hypercholesterolemic DM/HC
Non DM/HC
Diabetic
Hypercholesterolemic
DM/HC
Hamamdzic et al Am J Physiol
Heart Circ Physiol 2010;299:699

6. Mohler et al. Arterioscler Thromb Vasc Biol 2008;28:850
120
MMP9 6
3.0
100%
CCR1 5
leptin
ILIRN
75%
leptin
IL8 4
2.0
APOE
ILIRN
Fold increase in gene expression
% Advanced lesions
Mean intimal area (mm2)
50% 3
ABCA1
PTGS2
ICAM-1
ICAM-1
PLA2G7
NOS2A,
ABCA1
ABCA1
CCL2,
ICAM-1
adiponectin
NOS2A
ITGB2
VCAM1
1.0 2
CD163,
PPARg
CD68,
UCP2
PLAUR
HMOX1,
HLA,
preproadipsin
HMOX1
25%
TM7SF1 1 1m 3m 6m 9m
Mohler et al. Arterioscler Thromb Vasc Biol 2008;28:850

7. Akt signaling

8. DM/HC arteries demonstrate hypo-activation of Akt pathway- a central signaling node
Hamamdzic et al Am J Physiol Heart Circ Physiol 2010;299:699

9. Synergistic actions of DM/HC increase cellular proliferation,
A. Proliferation
Synergistic actions of DM/HC increase cellular proliferation,
apoptosis and inflammation.
C. NF-κB
B. Apoptosis
Hamamdzic et al Am J Physiol
Heart Circ Physiol 2010;299:699

10. Lipoprotein-phospholipase A2 (Lp-PLA2)
Source: enzyme secreted by leukocytes, T lymphocytes and mast cells
Circulation: ~70% bound to atherogenic lipoproteins (LDL)
Vessel wall: high specificity toward polar phospholipids in oxidized LDL
Result: formation of inflammatory mediators
sn-2 oxidation
Lp-PLA2

11. Darapladib reduced plasma Lp-PLA2 activity* * * * * *
Initiation of treatment
Nat Med 2008;14:1059

12. -2 most severe proximal LAD lesions
Coronary artery lesion complexity was reduced in the treatment group
-2 most severe proximal LAD lesions
Control
Treated
Nat Med 2008;14:1059

13. Selective inhibition of Lp-PLA2 reduces necrotic core area
Nat Med 2008;14:1059

14. Darapladib reduced macrophage content
-Cathepsin S staining
Control
Treated
Nat Med 2008;14:1059

15. Normalized Expression
Darapladib reduced inflammatory gene expression in coronary arteries: Macrophage markers
CD68
Lp-PLA2
6e5
3e5
4e5
Normalized Expression
Intensity
2e5
p<0.05
p<0.05 * *
2e5
1e5
No DM/HC
DM/HC Controls
DM/HC Darapladib
Nat Med 2008;14:1059

16. Normalized Expression
Darapladib reduced inflammatory gene expression in coronary arteries: Leukocyte subsets
CXCR3
CCR2
1e4
4e3
Normalized Expression
Intensity
p<0.05
p<0.05 * *
5e3
2e3
No DM/HC
DM/HC Controls
DM/HC Darapladib
CXCR3 (IP-10 receptor): A Th1 lymphocyte marker which regulates Th1-induced inflammation and lymphocyte chemotaxis.
CCR2 (MCP-1 receptor):
A marker of a subset of monocytes which
preferentially accumulate within atherosclerotic lesions

17. Upregulated with DM/HC (fold) Influence of Darapladib
Gene Name
Human
Entrez GeneID
Upregulated with DM/HC (fold)
Influence of Darapladib
% Change
P value
b-Arrestin 2
409
3.0
-47
0.004
HLA-DMA
3108
3.6
-38
0.007
PTAFR
5724
5.8
-49
0.009
BIN2
51411
7.1
-53
Lp-PLA2
7941
14.6
-55
0.012
CXCR3
2833
4.0
-68
0.015
GM2A
2760
5.4
-50
0.016
LAIR1
3903
4.8
-42
IL1RN
3557
21.1
-56
0.019
Cat. S
1520
16.0
0.020
gp91phox
1536
19.4
0.024
p47phox
653361
10.4
-54
0.025
CCR2
1231
1.9
-86
0.026
PLAUR
5329
6.7
-39
CD18
3689
14.3
0.029
UCP2
7351
6.1
0.030
HMOX1
3162
6.4
-44
0.033
DENND2D
79961
6.9
-45
0.034
CD68
968
16.7
-48
0.040
EVI2A
2123
3.7
0.041
EVI2B
2124
7.2
-41
0.043
CCL5
6352
0.046
SLC27A4
10999
3.2
0.047
NPL
80896
10.7
0.048
All coronary genes altered by Lp-PLA2 inhibition
Nat Med 2008;14:1059

18. Upregulated with DM/HC (fold) Influence of Darapladib
Gene Name
Human
Entrez GeneID
Upregulated with DM/HC (fold)
Influence of Darapladib
% Change
P value
b-Arrestin 2
409
3.0
-47
0.004
HLA-DMA
3108
3.6
-38
0.007
PTAFR
5724
5.8
-49
0.009
BIN2
51411
7.1
-53
Lp-PLA2
7941
14.6
-55
0.012
CXCR3
2833
4.0
-68
0.015
GM2A
2760
5.4
-50
0.016
LAIR1
3903
4.8
-42
IL1RN
3557
21.1
-56
0.019
Cat. S
1520
16.0
0.020
gp91phox
1536
19.4
0.024
p47phox
653361
10.4
-54
0.025
CCR2
1231
1.9
-86
0.026
PLAUR
5329
6.7
-39
CD18
3689
14.3
0.029
UCP2
7351
6.1
0.030
HMOX1
3162
6.4
-44
0.033
DENND2D
79961
6.9
-45
0.034
CD68
968
16.7
-48
0.040
EVI2A
2123
3.7
0.041
EVI2B
2124
7.2
-41
0.043
CCL5
6352
0.046
SLC27A4
10999
3.2
0.047
NPL
80896
10.7
0.048
All coronary genes altered by Lp-PLA2 inhibition
Monocyte/T cell recruitment
Oxidative stress
Markers of unstable human atheroma

19. IVUS, NIRS in DM/HC pig LAD

20. NIRS of LCx from pig that died suddenly weeks thereafter

21. Progression of disease by IVUS
3 Month
6 Month
9 Month
102 53
109
Fibroatheroma 49 34 7 86
149
Pathological intimal thickening 12 12 5 51 1 95 22 81 49
Normal / Intimal Hyperplasia
370
370
194
194
124
123 23 23

22. NIRS positivity predicts lesion type by histology
NIRS pos (n= 66)
NIRS neg (n= 238)
P value
TCFA n (%)
29 (43.9)
34 (14.3)
0.0001
Fibroatheroma n(%)
38 (16.0)
PIT n (%)
2 (3.0)
64 (26.9)
Intimal hyperplasia n(%)
6 (9.2)
61 (25.6)
0.004
Non-atherosclerotic n(%)
0 (0)
41 (17.2)
Plaque area (mm2)
6.3 ± 4.9
2.7 ± 3.0
<
Plaque + Media area (mm2)
8.8 ± 5.3
4.6 ± 3.4
Necrotic core area (mm2)
4.5 ± 8.8
0.93 ± 2.1
0.002
% necrotic core area
40 ± 64
11 ± 19
0.0004
Fibrous cap thickness (μ)
145.9 ± 131.5
206.7 ± 199.9
0.04

23. NIRS positivity at 6 months predicts progression of plaque and necrotic core area by IVUS

24. Increased coronary artery lipid deposition (by NIRS) predicts increased necrotic core area at 9 months (by IVUS) 9
P< 0.01 8 7
3m Pos/6m Pos 6
P=0.38 5
3m Neg/6m Pos
P=0.002
P<0.0001
NC area (mm2) 4
P= 0.09
P=0.008
P<0.0001 3
3m Pos/6m Neg 2
3m Neg/6m Neg 1
3 month
6 month
9 month
NIRS POS at 3 months
NIRS NEG at 3 months

26. NIRS positivity at 6 months associated with increased incidence of fibroatheromas at 9 months by histology.
For both 3 and 6 month positive NIRS
p= 0.03 for TCFA
p= for fibroatheromas)
For either 3 or 6 month positive NIRS
p= for TCFA
p= for fibroatheromas)

27. NIRS positivity correlates with increased macrophage infiltration, cellular proliferation and apoptosis
Stain positivity
NIRS positive
(n= 34)
NIRS negative
(n= 83)
P value
Cathepsin (per mm2 of plaque)
0.158 ± 0.08
0.123 ± 0.08
0.036
Cathepsin (per mm2 of cap)
0.056 ± 0.04
0.034 ± 0.03
0.006
Ki67 (per mm2 of plaque)
± 0.002
± 0.002
0.0001
Ki67 (per mm2 of cap)
± 0.004
± 0.003
0.0162
TUNEL (per mm2 of plaque)
0.042 ± 0.05
0.026 ± 0.03
0.108
TUNEL (per mm2 of cap)
0.058 ± 0.07
0.031 ± 0.038
0.042

28. NIRS positive
NIRS negative
macrophages
proliferation
apoptosis

29. Summary
There is strong evidence that immune responses play a role in development of atherosclerosis and/or vascular instability. Hypoactivation of Akt by increasing NF-κB may be playing a major role.

30. Summary
There is strong evidence that immune responses play a role in development of atherosclerosis and/or vascular instability. Hypoactivation of Akt by increasing NF-κB may be playing a major role.
Inhibiting the immunomodulatory/ inflammatory response (effecting efferocytosis?) by inhibiting Lp-PLA2 reduces disease progression but more importantly development of complex coronary artery disease in DM/HC pigs.

31. Summary
There is strong evidence that immune responses play a role in development of atherosclerosis and/or vascular instability. Hypoactivation of Akt by increasing NF-κB may be playing a major role.
Inhibiting the immunomodulatory/ inflammatory response (effecting efferocytosis?) by inhibiting Lp-PLA2 reduces disease progression but more importantly development of complex coronary artery disease in DM/HC pigs.
Hence, modulating the inflammatory/immune response may play a particularly important role in inhibiting the development of unstable lesions.

32. Summary
There is strong evidence that immune responses play a role in development of atherosclerosis and/or vascular instability. Hypoactivation of Akt by increasing NF-κB may be playing a major role.
Inhibiting the immunomodulatory/ inflammatory response (effecting efferocytosis?) by inhibiting Lp-PLA2 reduces disease progression but more importantly development of complex coronary artery disease in DM/HC pigs.
Hence, modulating the inflammatory/immune response may play a particularly important role in inhibiting the development of unstable lesions.
The combination of IVUS & NIRS predicts lesion progression and increased inflammation in DM/HC pig coronary arteries.