The Role of Inflammation and Immune Responses Robert L Wilensky, MD University of Pennsylvania

I have no real or apparent conflicts of interest to report. Robert L. Wilensky, MD I have no real or apparent conflicts of interest to report.

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

Role of mast cells in plaque instability Kovanen PT Immunol Rev 2007;217:105

Selective inhibition of Lp-PLA2, an important inflammatory mediator, reduces necrotic core area DM/HC Control DM/HC Darapladib Wilensky et al Nat Med 2008;14:1059

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

Hypothesis: Subsequent development of high-risk coronary artery lesions can be predicted using invasive coronary imaging.

Angiography/IVUS/NIRS Angiography/IVUS/NIRS Angiography/IVUS/NIRS Experimental approach Induction DM/HC n=21 Tissue harvest n=13 5 sudden deaths 1m 2m 3m 4m 5m 6m 7m 8m 9m Angiography/IVUS/NIRS Angiography/IVUS/NIRS Angiography/IVUS/NIRS Morphometrics Histology (Movat’s) Fibrous cap thickness (Picro-sirius Red) Activated macrophages (Cathepsin S) Cellular proliferation (Ki-67) Apoptosis (TUNEL) Serum glucose- 304 ± 49 mg/dl Serum cholesterol- 511 ± 64 mg/dl

IVUS distribution of plaques: n(%) IVUS results Months IVUS distribution of plaques: n(%) No advanced disease Pathologic intimal thickening Fibroatheromas 3 388 (96.3%) 15 (3.7%) 6 214 (52.3%) 89 (21.8%) 106 (25.9%) 9 144 (31.5%) 177 (38.7%) 136 (29.8%) P value <0.001 0.019

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 < 0.0001 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

NIRS positivity is associated with increased inflammation

NIRS positive NIRS negative macrophages proliferation apoptosis

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

Correlation of NIRS and histology: A) Plaque area 2 4 6 8 10 12 Plaque area (mm2) NEG POS 3 mth 6 mth 9 mth Correlation of NIRS and histology: A) Plaque area B) Necrotic core area 1 2 3 4 5 6 7 8 NC area (mm2) NEG POS 3 mth 6 mth 9 mth NIRS at 3, 6 and 9 month

Antecedent NIRS positivity is associated with increased incidence of fibroatheromas at 9 months by histology 152 32 38 16 For both 3 and 6 month positive NIRS p= 0.03 for TCFA p= 0.0004 for fibroatheromas For either 3 or 6 month positive NIRS p= 0.007 for TCFA p= 0.0001 for fibroatheromas

Predictors of TCFA and fibroatheroma at 9-m Thin cap fibroatheromas Fibroatheromas Months Univariate Multivariate 9-m NIRS positive P+M area Necrotic core area CSN >40% Positive RI (All p<0.001) Lumen area (p=0.01) NIRS positive (p=0.000, OR= 3.97, 1.88-8.36) CSN>40% (p=0.046, OR= 2.76, 1.01-7.46) Positive RI (p= 0.002, OR=3.81, 1.60-7.05) (P=0.015) OR= 13.37, 5.18-34.45) CSN>40% (p=0.001, OR= 5.14, 1.98-13.35) 6-m NC area (All p=0.001) NIRS positive (p=0.143, OR=1.71, 0.84-3.48) NIRS positive (p=0.005, (OR=2.71,1.34-5.49) 3-m None NIRS positive (p=0.048) NIRS positive (p=0.137, OR=1.63, 0.86-3.09)

A NIRS, IVUS, histology of culprit lesion in animal suffering sudden death, 132 days after DM/HC induction and 42 days after NIRS/IVUS evaluation C B

Summary In DM/HC pigs inflammation is an important driving factor for the development of high-risk lesions. Inhibition of inflammation reduces necrotic core area. In-vivo anatomic and biochemical evaluation predicted the presence of and progression of atherosclerotic lesions with a high-risk phenotype, by histology. NIRS positive fibroatheromas had increased inflammation, cellular proliferation and apoptosis within a thinned fibrous cap; all characteristics of unstable lesions. The persistence of a lipid core plaque within the arterial wall predicted subsequent progressive plaque development, larger necrotic core size and the presence of a TCFA. In this model focal inflammation is dynamic as some lesions, despite persistent high- cholesterol levels did not maintain NIRS positivity or transformed from a NIRS negative to NIRS positive state during the study period.

Summary In DM/HC pigs inflammation is an important driving factor for the development of high-risk lesions. Inhibition of inflammation reduces necrotic core area. In-vivo anatomic and biochemical evaluation predicted the presence of and progression of atherosclerotic lesions with a high-risk phenotype, by histology. NIRS positive fibroatheromas had increased inflammation, cellular proliferation and apoptosis within a thinned fibrous cap; all characteristics of unstable lesions. The persistence of a lipid core plaque within the arterial wall predicted subsequent progressive plaque development, larger necrotic core size and the presence of a TCFA. In this model focal inflammation is dynamic as some lesions, despite persistent high- cholesterol levels did not maintain NIRS positivity or transformed from a NIRS negative to NIRS positive state during the study period.

Summary In DM/HC pigs inflammation is an important driving factor for the development of high-risk lesions. Inhibition of inflammation reduces necrotic core area. In-vivo anatomic and biochemical evaluation predicted the presence of and progression of atherosclerotic lesions with a high-risk phenotype, by histology. NIRS positive fibroatheromas had increased inflammation, cellular proliferation and apoptosis within a thinned fibrous cap; all characteristics of unstable lesions. The persistence of a lipid core plaque within the arterial wall predicted subsequent progressive plaque development, larger necrotic core size and the presence of a TCFA. In this model focal inflammation is dynamic as some lesions, despite persistent high- cholesterol levels did not maintain NIRS positivity or transformed from a NIRS negative to NIRS positive state during the study period.

Summary In DM/HC pigs inflammation is an important driving factor for the development of high-risk lesions. Inhibition of inflammation reduces necrotic core area. In-vivo anatomic and biochemical evaluation predicted the presence of and progression of atherosclerotic lesions with a high-risk phenotype, by histology. NIRS positive fibroatheromas had increased inflammation, cellular proliferation and apoptosis within a thinned fibrous cap; all characteristics of unstable lesions. The persistence of a lipid core plaque within the arterial wall predicted subsequent progressive plaque development, larger necrotic core size and the presence of a TCFA. In this model focal inflammation is dynamic as some lesions, despite persistent high- cholesterol levels did not maintain NIRS positivity or transformed from a NIRS negative to NIRS positive state during the study period.

Summary In DM/HC pigs inflammation is an important driving factor for the development of high-risk lesions. Inhibition of inflammation reduces necrotic core area. In-vivo anatomic and biochemical evaluation predicted the presence of and progression of atherosclerotic lesions with a high-risk phenotype, by histology. NIRS positive fibroatheromas had increased inflammation, cellular proliferation and apoptosis within a thinned fibrous cap; all characteristics of unstable lesions. The persistence of a lipid core plaque within the arterial wall predicted subsequent progressive plaque development, larger necrotic core size and the presence of a TCFA. In this model focal inflammation is dynamic as some lesions, despite persistent high- cholesterol levels did not maintain NIRS positivity or transformed from a NIRS negative to NIRS positive state during the study period.

Clinical results 21 pigs demonstrated consistently elevated glucose and cholesterol levels. 5 animals died unexpectedly due to ischemic sudden death 4 of 5 had multi-vessel disease with thrombus 1 of 5 had single TCFA but no thrombus 2 animals died of procedurally related complications- both with 3 vessel disease. 1 animal of non-cardiac causes 13 animals remained in the study

Excellent correlation of P + M areas by histology and IVUS IVUS: Plaque + media area (mm2) R= 0.87 P<0.001 Histology: Plaque + media area (mm2)

Progression of IVUS/NIRS over time A 1 4 2 3 Progression of IVUS/NIRS over time B 1 4 2 3 A: 3 months B: 6 months C: 9 months 1 2 3 4 C 1 2 3 4 1 2 3 4

IVUS results (2) Months P+M area (mm2) NC area (mm2) EEL area (mm2) Lumen area (mm2) Cross sectional narrowing (%) 3 1.94 ± 0.4 0 ± 0 9.35 ± 2.2 8.1 ± 1.5 21.3 ± 2.1 6 4.29 ± 1.9 1.50 ± 2.88 12.87 ± 6.5 8.8 ± 2.6 32.8 ± 11.8 9 8.19 ± 4.2 2.65 ± 4.88 16.76 ± 6.5 7.9 ± 1.9 40.9 ±15.5 P value <0.001 0.423

NIRS results 303 (82.1) 30 (8.1) 19 (5.2) 17 (4.6) 311 (75.3) Months Block chemogram color threshold Number (%) of sections positive for NIRS Red Dark orange Light orange Yellow 3 303 (82.1) 30 (8.1) 19 (5.2) 17 (4.6) 6 311 (75.3) 44 (10.6) 35 (8.5) 23 (5.6) 9 365 (79.3) 39 (8.5) 35 (7.6) 21 (4.6) P value 0.405 0.841 0.680 0.887