Inflammation Infection and C PowerPoint Presentation

Slide 1 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine

Slide 2 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic

Slide 3 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126.

Slide 4 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors?

Slide 5 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable

Slide 6 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter

Slide 7 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter

Slide 8 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims.

Slide 9 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials.

Slide 10 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality.

Slide 11 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen.

Slide 12 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients.

Slide 13 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8).

Slide 14 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality

Slide 15 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209

Slide 16 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile

Slide 17 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin

Slide 18 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B

Slide 19 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181.

Slide 20 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker

Slide 21 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events.

Slide 22 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability

Slide 23 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4

Slide 24 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4

Slide 25 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9

Slide 26 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk

Slide 27 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43

Slide 28 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies?

Slide 29 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese

Slide 30 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970.

Slide 31 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo

Slide 32 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235.

Slide 33 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline

Slide 34 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * *

Slide 35 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2

Slide 36 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165

Slide 37 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol

Slide 38 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l

Slide 39 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0

Slide 40 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome?

Slide 41 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397.

Slide 42 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome

Slide 43 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome 0 2 4 6 8 Years of Follow-Up 0.95 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability CRP <1 mg/L CRP 1-3 mg/L CRP >3 mg/L Event Free Survival According to hs-CRP Levels: Analysis Limited to Participants with Metabolic Syndrome at Baseline Ridker et al, Circulation 2003;107:391-7

Slide 44 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome 0 2 4 6 8 Years of Follow-Up 0.95 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability CRP <1 mg/L CRP 1-3 mg/L CRP >3 mg/L Event Free Survival According to hs-CRP Levels: Analysis Limited to Participants with Metabolic Syndrome at Baseline Ridker et al, Circulation 2003;107:391-7

Slide 45 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome 0 2 4 6 8 Years of Follow-Up 0.95 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability CRP <1 mg/L CRP 1-3 mg/L CRP >3 mg/L Event Free Survival According to hs-CRP Levels: Analysis Limited to Participants with Metabolic Syndrome at Baseline Ridker et al, Circulation 2003;107:391-7 AHA / CDC Scientific Statement Markers of Inflammation and Cardiovascular Disease: Applications to Clinical and Public Health Practice Circulation January 28, 2003 “Measurement of hs-CRP is an independent marker of risk and may be used at the discretion of the physician as part of global coronary risk assessment in adults without known cardiovascular disease. Weight of evidence favors use particularly among those judged at intermediate risk by global risk assessment”.

Slide 46 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome 0 2 4 6 8 Years of Follow-Up 0.95 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability CRP <1 mg/L CRP 1-3 mg/L CRP >3 mg/L Event Free Survival According to hs-CRP Levels: Analysis Limited to Participants with Metabolic Syndrome at Baseline Ridker et al, Circulation 2003;107:391-7 AHA / CDC Scientific Statement Markers of Inflammation and Cardiovascular Disease: Applications to Clinical and Public Health Practice Circulation January 28, 2003 “Measurement of hs-CRP is an independent marker of risk and may be used at the discretion of the physician as part of global coronary risk assessment in adults without known cardiovascular disease. Weight of evidence favors use particularly among those judged at intermediate risk by global risk assessment”. 1 mg/L 3 mg/L 10 mg/L Low Risk Moderate Risk High Risk Acute Phase Response Ignore Value, Repeat Test in 3 weeks >100 mg/L Ridker PM. Circulation 2003;107:363-9 Clinical Application of hs-CRP for Cardiovascular Risk Prediction

Slide 47 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome 0 2 4 6 8 Years of Follow-Up 0.95 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability CRP <1 mg/L CRP 1-3 mg/L CRP >3 mg/L Event Free Survival According to hs-CRP Levels: Analysis Limited to Participants with Metabolic Syndrome at Baseline Ridker et al, Circulation 2003;107:391-7 AHA / CDC Scientific Statement Markers of Inflammation and Cardiovascular Disease: Applications to Clinical and Public Health Practice Circulation January 28, 2003 “Measurement of hs-CRP is an independent marker of risk and may be used at the discretion of the physician as part of global coronary risk assessment in adults without known cardiovascular disease. Weight of evidence favors use particularly among those judged at intermediate risk by global risk assessment”. 1 mg/L 3 mg/L 10 mg/L Low Risk Moderate Risk High Risk Acute Phase Response Ignore Value, Repeat Test in 3 weeks >100 mg/L Ridker PM. Circulation 2003;107:363-9 Clinical Application of hs-CRP for Cardiovascular Risk Prediction Inflammatory and Infections Agents in CHD Belgian epidemiologic study included 446 of 16307 male workers aged 35-39 who had evidence of CHD vs. 892 controls. CRP, but none of the infectious agents (H. pylori, C. pneumoniae, CMV, and EBV) were associated with CHD, even after adjustment for other risk factors. De Backer et al. Atherosclerosis 2002; 160: 457-63.

Slide 48 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome 0 2 4 6 8 Years of Follow-Up 0.95 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability CRP <1 mg/L CRP 1-3 mg/L CRP >3 mg/L Event Free Survival According to hs-CRP Levels: Analysis Limited to Participants with Metabolic Syndrome at Baseline Ridker et al, Circulation 2003;107:391-7 AHA / CDC Scientific Statement Markers of Inflammation and Cardiovascular Disease: Applications to Clinical and Public Health Practice Circulation January 28, 2003 “Measurement of hs-CRP is an independent marker of risk and may be used at the discretion of the physician as part of global coronary risk assessment in adults without known cardiovascular disease. Weight of evidence favors use particularly among those judged at intermediate risk by global risk assessment”. 1 mg/L 3 mg/L 10 mg/L Low Risk Moderate Risk High Risk Acute Phase Response Ignore Value, Repeat Test in 3 weeks >100 mg/L Ridker PM. Circulation 2003;107:363-9 Clinical Application of hs-CRP for Cardiovascular Risk Prediction Inflammatory and Infections Agents in CHD Belgian epidemiologic study included 446 of 16307 male workers aged 35-39 who had evidence of CHD vs. 892 controls. CRP, but none of the infectious agents (H. pylori, C. pneumoniae, CMV, and EBV) were associated with CHD, even after adjustment for other risk factors. De Backer et al. Atherosclerosis 2002; 160: 457-63. Infection and CHD - is there a connection? Local or systemic infections resulting from gram negative bacteria such as Chlamydia pneumoniae and Helicobacter pylori, including cytomegalovirus (CMV) have been implicated in atheroscelosis While several case control studies have shown increased titers of C.pneumoniae and H. Pylori in those with vs. without CHD, convincing evidence from prospective studies is lacking.

Slide 49 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome 0 2 4 6 8 Years of Follow-Up 0.95 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability CRP <1 mg/L CRP 1-3 mg/L CRP >3 mg/L Event Free Survival According to hs-CRP Levels: Analysis Limited to Participants with Metabolic Syndrome at Baseline Ridker et al, Circulation 2003;107:391-7 AHA / CDC Scientific Statement Markers of Inflammation and Cardiovascular Disease: Applications to Clinical and Public Health Practice Circulation January 28, 2003 “Measurement of hs-CRP is an independent marker of risk and may be used at the discretion of the physician as part of global coronary risk assessment in adults without known cardiovascular disease. Weight of evidence favors use particularly among those judged at intermediate risk by global risk assessment”. 1 mg/L 3 mg/L 10 mg/L Low Risk Moderate Risk High Risk Acute Phase Response Ignore Value, Repeat Test in 3 weeks >100 mg/L Ridker PM. Circulation 2003;107:363-9 Clinical Application of hs-CRP for Cardiovascular Risk Prediction Inflammatory and Infections Agents in CHD Belgian epidemiologic study included 446 of 16307 male workers aged 35-39 who had evidence of CHD vs. 892 controls. CRP, but none of the infectious agents (H. pylori, C. pneumoniae, CMV, and EBV) were associated with CHD, even after adjustment for other risk factors. De Backer et al. Atherosclerosis 2002; 160: 457-63. Infection and CHD - is there a connection? Local or systemic infections resulting from gram negative bacteria such as Chlamydia pneumoniae and Helicobacter pylori, including cytomegalovirus (CMV) have been implicated in atheroscelosis While several case control studies have shown increased titers of C.pneumoniae and H. Pylori in those with vs. without CHD, convincing evidence from prospective studies is lacking. Prospective Studies of CHD and Infectious Pathogens Physician’s Health Study (nested case-control) shows RR 1.1 (0.8-1.5) for C. Pneumoniae, 0.94 (0.7-1.2) for cytomegalovirus, and 0.72 (0.6-0.9) for Herpes simplex virus. H. pylori also shows mixed results. Whincup showed a nonsignificant 1.3 OR when adjusted for other risk factors, the large ARIC study showed no relation, and the Caerphilly Prospective study showed RR=1.05 in 1796 men followed 14 years.

Slide 50 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome 0 2 4 6 8 Years of Follow-Up 0.95 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability CRP <1 mg/L CRP 1-3 mg/L CRP >3 mg/L Event Free Survival According to hs-CRP Levels: Analysis Limited to Participants with Metabolic Syndrome at Baseline Ridker et al, Circulation 2003;107:391-7 AHA / CDC Scientific Statement Markers of Inflammation and Cardiovascular Disease: Applications to Clinical and Public Health Practice Circulation January 28, 2003 “Measurement of hs-CRP is an independent marker of risk and may be used at the discretion of the physician as part of global coronary risk assessment in adults without known cardiovascular disease. Weight of evidence favors use particularly among those judged at intermediate risk by global risk assessment”. 1 mg/L 3 mg/L 10 mg/L Low Risk Moderate Risk High Risk Acute Phase Response Ignore Value, Repeat Test in 3 weeks >100 mg/L Ridker PM. Circulation 2003;107:363-9 Clinical Application of hs-CRP for Cardiovascular Risk Prediction Inflammatory and Infections Agents in CHD Belgian epidemiologic study included 446 of 16307 male workers aged 35-39 who had evidence of CHD vs. 892 controls. CRP, but none of the infectious agents (H. pylori, C. pneumoniae, CMV, and EBV) were associated with CHD, even after adjustment for other risk factors. De Backer et al. Atherosclerosis 2002; 160: 457-63. Infection and CHD - is there a connection? Local or systemic infections resulting from gram negative bacteria such as Chlamydia pneumoniae and Helicobacter pylori, including cytomegalovirus (CMV) have been implicated in atheroscelosis While several case control studies have shown increased titers of C.pneumoniae and H. Pylori in those with vs. without CHD, convincing evidence from prospective studies is lacking. Prospective Studies of CHD and Infectious Pathogens Physician’s Health Study (nested case-control) shows RR 1.1 (0.8-1.5) for C. Pneumoniae, 0.94 (0.7-1.2) for cytomegalovirus, and 0.72 (0.6-0.9) for Herpes simplex virus. H. pylori also shows mixed results. Whincup showed a nonsignificant 1.3 OR when adjusted for other risk factors, the large ARIC study showed no relation, and the Caerphilly Prospective study showed RR=1.05 in 1796 men followed 14 years. Other Studies of Infectious Agents In South Asian persons with CHD vs. controls, C. pneumoniae specific IgG antibody was seropositive in similar proportions; risk factors appeared to mediate any relations (Mendis et al. Int J Cardiol 2001; 79: 191-6). Cross-sectional survey of 704 individuals of C. pneumoniae and CMV with risk factors did nto show significant associations (Danesh et al., J Cardiovasc Risk 1999; 6: 387-90). Meta-analysis of 24 articles involving H. pylori infection and CHD showed a pooled odds ratio of 1.55 (95% CI: 1.38-1.74) (p<0.001), suggested a weak relation, but high hetrogeneity between studies precludes clear demonostration (Pellicano et al., Eur J Epidemiol 1999; 15: 611-9). ARIC Study failed to show clear relation between IgG antibodies for C. pneumoniae and incident CHD occurring over average 3.3 years. (Nieto et al. Am J Epidemiol 1999; 150: 149-56).

Slide 51 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome 0 2 4 6 8 Years of Follow-Up 0.95 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability CRP <1 mg/L CRP 1-3 mg/L CRP >3 mg/L Event Free Survival According to hs-CRP Levels: Analysis Limited to Participants with Metabolic Syndrome at Baseline Ridker et al, Circulation 2003;107:391-7 AHA / CDC Scientific Statement Markers of Inflammation and Cardiovascular Disease: Applications to Clinical and Public Health Practice Circulation January 28, 2003 “Measurement of hs-CRP is an independent marker of risk and may be used at the discretion of the physician as part of global coronary risk assessment in adults without known cardiovascular disease. Weight of evidence favors use particularly among those judged at intermediate risk by global risk assessment”. 1 mg/L 3 mg/L 10 mg/L Low Risk Moderate Risk High Risk Acute Phase Response Ignore Value, Repeat Test in 3 weeks >100 mg/L Ridker PM. Circulation 2003;107:363-9 Clinical Application of hs-CRP for Cardiovascular Risk Prediction Inflammatory and Infections Agents in CHD Belgian epidemiologic study included 446 of 16307 male workers aged 35-39 who had evidence of CHD vs. 892 controls. CRP, but none of the infectious agents (H. pylori, C. pneumoniae, CMV, and EBV) were associated with CHD, even after adjustment for other risk factors. De Backer et al. Atherosclerosis 2002; 160: 457-63. Infection and CHD - is there a connection? Local or systemic infections resulting from gram negative bacteria such as Chlamydia pneumoniae and Helicobacter pylori, including cytomegalovirus (CMV) have been implicated in atheroscelosis While several case control studies have shown increased titers of C.pneumoniae and H. Pylori in those with vs. without CHD, convincing evidence from prospective studies is lacking. Prospective Studies of CHD and Infectious Pathogens Physician’s Health Study (nested case-control) shows RR 1.1 (0.8-1.5) for C. Pneumoniae, 0.94 (0.7-1.2) for cytomegalovirus, and 0.72 (0.6-0.9) for Herpes simplex virus. H. pylori also shows mixed results. Whincup showed a nonsignificant 1.3 OR when adjusted for other risk factors, the large ARIC study showed no relation, and the Caerphilly Prospective study showed RR=1.05 in 1796 men followed 14 years. Other Studies of Infectious Agents In South Asian persons with CHD vs. controls, C. pneumoniae specific IgG antibody was seropositive in similar proportions; risk factors appeared to mediate any relations (Mendis et al. Int J Cardiol 2001; 79: 191-6). Cross-sectional survey of 704 individuals of C. pneumoniae and CMV with risk factors did nto show significant associations (Danesh et al., J Cardiovasc Risk 1999; 6: 387-90). Meta-analysis of 24 articles involving H. pylori infection and CHD showed a pooled odds ratio of 1.55 (95% CI: 1.38-1.74) (p<0.001), suggested a weak relation, but high hetrogeneity between studies precludes clear demonostration (Pellicano et al., Eur J Epidemiol 1999; 15: 611-9). ARIC Study failed to show clear relation between IgG antibodies for C. pneumoniae and incident CHD occurring over average 3.3 years. (Nieto et al. Am J Epidemiol 1999; 150: 149-56). Clinical Trial Evidence for Antibiotic Treatment and Prevention of CVD ACADEMIC Study of 302 patients with CHD seropositive to C. Pneumoniae randomized to azithromycin 500 mg/wk or placebo for 3 months showed no significant treatment difference (HR=0.89, p=0.74) for recurrent events (Muhlestein et al., Circulation 2000; 102: 1755-60). AZACS Multicenter study of 1439 pts with unstable angina randomized to 250 mg azithromycin/day for up to 6 months showed no significant benefit for death, recurrent MI, or recurrent ischemia (Cercek et al., Lancet 2003; 361: 809-13). WIZARD trial of 7,747 pts post-MI randomized to 12 week of therapy with azithromycin or placebo showed no significant reduction in reinfarction, revascularization, hospitalization for angina, or death (O’Connor et al., JAMA 2003; 290: 1459-66).

Slide 52 -

Inflammation, Thrombosis, Infection, and CARDIOVASCULAR DISEASE Nathan D Wong, PhD, FACC Professor and Director Heart Disease Prevention Program University of California, Irvine Beyond Cholesterol: Predicting Cardiovascular Risk In the 21st Century Cardiovascular Risk Lipids HTN Diabetes Behavioral Hemostatic Thrombotic Inflammatory Genetic Inflammation and Atherosclerosis Inflammation may determine plaque stability - Unstable plaques have increased leukocytic infiltrates - T cells, macrophages predominate rupture sites - Cytokines and metalloproteinases influence both stability and degradation of the fibrous cap Lipid lowering may reduce plaque inflammation - Decreased macrophage number - Decreased expression of collagenolytic enzymes (MMP-1) - Increased interstitial collagen - Decreased expression of E-selectin - Reduced calcium deposition Libby P. Circulation 1995;91:2844-2850. Ross R. N Engl J Med 1999;340:115-126. Is there clinical evidence that inflammatory markers predict future coronary events and provide additional predictive information beyond traditional risk factors? Evaluating Novel Risk Factors for CAD Consistency of prospective data Strength of association Independence of association Improve predictive value Standardized measure Low variability High reproducibility Biologic plausibility Low cost Modifiable Biomarkers for Venous and Arterial Thrombosis +++ – hs-CRP / SAA / IL-6 / TNF + – Lp(a) ++ – Platelet function ++ – PAI-1: ag +++ – tPA: ag ++ – vWF: ag + – Factor VII +++ – Fibrinogen Arterial Venous Parameter Biomarkers for Venous and Arterial Thrombosis (cont’d) ++ ++ D-dimer ++ ++ Homocysteine – + Protein S – + Protein C – ++ Anti-thrombin III – ++ Factor VIII – + Prothrombin – ++ Prothrombin mutation – +++ Factor V Leiden Arterial Venous Parameter Thrombosis and Cardiovascular Risk Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction, as well as occlusion during or following angioplasty. Often preceded by platelet aggregation and activation of the coagulation system. A thrombus may develop at sites of only mild to moderate coronary stenosis. The majority of coronary events occur where there is less than 70% stenosis. Occlusive coronary thrombosis plays a role in over 80% of myocardial infarctions and about 95% of sudden death victims. Fibrinogen and Atherosclerosis Promotes atherosclerosis Essential component of platelet aggregation Relates to fibrin deposited and the size of the clot Increases plasma viscosity May also have a proinflammatory role Measurement of fibrinogen, incl. Test variability, remains difficult. No known therapies to selectively lower fibrinogen levels in order to test efficacy in CHD risk reduction via clinical trials. Fibrinogen and CHD Risk: Epidemiologic Studies Recent meta-analysis of 18 studies involving 4018 CHD cases showed a relative risk of CHD of 1.8 (95% CI 1.6-2.0) comparing the highest vs lowest tertile of fibrinogen levels (mean .35 vs. .25 g/dL) ARIC study in 14,477 adults aged 45-64 showed relative risks of 1.8 in men and 1.5 in women, attenuated to 1.5 and 1.2 after risk factor adjustment. Scottish Heart Health Study of 5095 men and 4860 women showed fibrinogen to be an independent risk factor for new events--RRs 2.2-3.4 for coronary death and all-cause mortality. Fibrinogen and CHD Risk Factors Fibrinogen levels increase with age and body mass index, and higher cholesterol levels Smoking can reversibly elevated fibrinogen levels, and cessation of smoking can lower fibrinogen. Those who exercise, eat vegetarian diets, and consume alcohol have lower levels. Exercise may also lower fibrinogen and plasma viscosity. Studies also show statin-fibrate combinations (simvastatin-ciprofibrate) and estrogen therapy to lower fibrinogen. Other Thrombotic Factors and CHD Mixed reports of coagulation factor VIIc in cardiovascular disease. PROCAM study showed no association with CHD events, CHS also showed no relation to subclinical CVD. Endogenous tissue-type plasminogen activator (tPA) shown in some studies to relate to increased cardiovascular risk--Physician’s Health Study showed RR for MI 2.8, stroke 3.5 in those in 5th vs. 1st quintile of tPA. Plasminogen activitor inhibitor type 1 (PAI-1) shown associated with increased cardiovascular risk, esp in diabetic patients. Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Primary Prevention US Physician’s Health Study- 22,071 male physicians - 44% reduction in MI risk, 13% nonsignificant increase in risk of stroke British Doctor’s Study of 5139 male physicians showed nonsignificant 3% reduction in MI risk,13% nonsignificant increase in stroke Hypertension Optimal Treatment (HOT) study among 18,790 pts w/htn showed 15% reduction in CVD events, 36% reduction in MI Women’s Health Study (n=39,876 women aged 45+) randomized to 100 mg asprin/day vs placebo, 10 years follow-up – results recently released and asprin preventive only for stroke (17% reduction overall, p=0.04; 24% ischemic stroke, p<.001); nonfatal MI RR=1.02, CVD death 0.95, ns) (NEJM 2005; 352: 1366-8). Aspirin and Cardiovascular Risk: Clinical Trial Evidence for Secondary Prevention Antiplatelet Trialists Collaboration of 54,000 patients with cardiovascular disease (10 trials post-MI) showed 31% reduction in MI, 42% reduction in stroke, 13% reduction in total vascular mortality International Study of Infarct Survival of 17,187 pts w/evolving MI showed 49% reduction in reinfarction, 26% reduction in nonfatal stroke, and 23% reduction in total vascular mortality Antiplatelet Therapy: Targets Collagen Thrombin TXA2 ADP (Fibrinogen Receptor) ADP = adenosine diphosphate, TXA2 = thromboxane A2, COX = cyclooxygenase clopidogrel bisulfate TXA2 phosphodiesterase ADP Gp IIb/IIIa Activation COX ticlopidine hydrochloride aspirin Gp 2b/3a Inhibitors dipyridamole Schafer AI. Am J Med 1996;101:199–209 Antiplatelet Therapy: Common Oral Agents 1Topol EJ et al. Circulation. 2003;108:399-406 2Diener H-C et al. Lancet 2004;364;331-7 3Plavix® package insert. www.sanofi-synthelabo.us 4Peters RJ et al. Circulation 2003;108:1682-7 5Hass WK. NEJM 1989;321:501-7 6Urban P. Circulation. 1998;98:2126-32 7Ticlid® package insert. www.rocheusa.com *Clopidogrel is generally given preference over Ticlopidine because of a superior safety profile Aspirin: Mechanism of Action Membrane Phospholipids ARACHIDONIC ACID Prostaglandin H2 COX-1 Thromboxane A2  Platelet Aggregation Vasoconstriction Prostacyclin  Platelet Aggregation Vasodilitation Aspirin Aspirin Recommendations Aspirin (75-162 mg daily) for intermediate risk men with a 10 year risk of CHD >10%. Aspirin (75-162 mg daily) for intermediate risk women with a 10 year risk of CHD >10%. Aspirin for low risk women with a 10 year risk of CHD<10%. Aspirin (75-325 mg daily) for those with known CHD. Primary Prevention Secondary Prevention I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III I I I IIa IIa IIa IIb IIb IIb III III III IIa IIa IIa IIb IIb IIb III III III B Rader, NEJM 2000; 343: 1181. P. Ridker CRP vs hs-CRP CRP is an acute-phase protein produced by the liver in response to cytokine production (IL-6, IL-1, tumor necrosis factor) during tissue injury, inflammation, or infection. Standard CRP tests determine levels which are increased up to 1,000-fold in response to infection or tissue destruction, but cannot adequately assess the normal range High-sensitivity CRP (hs-CRP) assays (i.e. Dade Behring) detect levels of CRP within the normal range, levels proven to predict future cardiovascular events. Potential Mechanisms Linking CRP to Atherothrombosis Confounding by cigarette consumption Innocent bystander - Acute phase response Cytokine surrogate - IL-6, TNF-, IL-1 Direct effects of CRP - Innate immunity - Complement activation - CAM induction Prior infection - Chlamydia, H pylori, CMV Marker for subclinical atherosclerosis - EBCT / IMT / ABI Marker for insulin resistance/ obesity Marker for endothelial dysfunction Marker for dysmetabolic syndrome Marker for plaque vulnerability hs-CRP and Risk of Future MI in Apparently Healthy Men P<0.001 P<0.001 P=0.03 Quartile of hs-CRP P Trend <0.001 Relative Risk of MI Ridker et al, N Engl J Med. 1997;336:973–979. 0 1 2 3 1 2 3 4 hs-CRP and Risk of Future Stroke in Apparently Healthy Men P<0.02 P=0.02 Relative Risk of Ischemic Stroke P Trend <0.03 Ridker et al, N Engl J Med. 1997;336:973–979. Quartile of hs-CRP 0 1 2 1 2 3 4 hs-CRP as a Risk Factor For Future CVD : Primary Prevention Cohorts 0 1.0 2.0 3.0 4.0 5.0 6.0 Kuller MRFIT 1996 CHD Death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRHS 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events* Ridker AFCAPS 2001 CV Events* Rost FHS 2001 Stroke Pradhan WHI 2002 MI,CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI Relative Risk (upper vs lower quartile) Ridker PM. Circulation 2003;107:363-9 hs-CRP Adds to Predictive Value of TC:HDL Ratio in Determining Risk of First MI Total Cholesterol:HDL Ratio Ridker et al, Circulation. 1998;97:2007–2011. hs-CRP Relative Risk Risk Factors for Future Cardiovascular Events: WHS 0 1.0 2.0 4.0 6.0 Lipoprotein(a) Homocysteine IL-6 TC LDLC sICAM-1 SAA Apo B TC: HDLC hs-CRP hs-CRP + TC: HDLC Relative Risk of Future Cardiovascular Events Ridker et al, N Engl J Med. 2000;342:836-43 Is there clinical evidence that inflammation can be modified by preventive therapies? Elevated CRP Levels in Obesity: NHANES 1988-1994 Visser M et al. JAMA 1999;282:2131-2135. Normal Percent with CRP 0.22 mg/dL Overweight Obese Effects of Weight Loss on CRP Concentrations in Obese Healthy Women 83 women (mean BMI 33.8, range 28.2-43.8 kg/m2) placed on very low fat, energy-restricted diet (6.0 MJ, 15% fat) for 12 weeks Baseline CRP positively associated with BMI (r=0.281, p=0.01) CRP reduced by 26% (p<0.001) Average weight loss 7.9 kg, associated with change in CRP Change in CRP correlated with change in TC (r=0.240, p=0.03) but not changes in LDL-C, HDL-C, or glucose At 12 weeks, CRP concentration highly correlated with TG (r=0.287, p=0.009), but not with other lipids or glucose Heilbronn LK et al. Arterioscler Thromb Vasc Biol 2001;21:968-970. Effect of HRT on hs-CRP: the PEPI Study 3.0 2.0 1.0 hs-CRP (mg/dL) Months 0 12 36 Cushman M et al. Circulation 1999;100:717-722. 1999 Lippincott Williams & Wilkins. CEE + MPA cyclic CEE + MPA continuous CEE + MP CEE Placebo Long-Term Effect of Statin Therapy on hs-CRP: Placebo and Pravastatin Groups Pravastatin Placebo Median hs-CRP Concentration (mg/dL) -21.6% (P=0.004) 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 Baseline 5 Years Ridker et al, Circulation. 1999;100:230-235. hs-CRP (mg/L) Effect of Statin Therapy on hs-CRP Levels at 6 Weeks Jialal I et al. Circulation 2001;103:1933-1935. 2001 Lippincott Williams & Wilkins. 6 5 4 3 2 1 0 Baseline Prava (40 mg/d) Simva (20 mg/d) Atorva (10 mg/d) *p<0.025 vs. Baseline Effect of Bezafibrate with and without Fluvastatin on Plasma Fibrinogen, PAI-1, and CRP in Patients with CAD and Mixed Hyperlipidemia Beza 400 mg/d Beza 400 mg/d + fluva 20 mg/d Beza 400 mg/d + fluva 40 mg/d Cortellaro M et al. Thromb Haemost 2000;83:549-553. Change at 24 weeks, % n: 81 Fibrinogen PAI-1 CRP 80 74 70 72 63 83 80 75 P<0.05 vs. baseline * * * No History of CAD Men > 55, Women > 65 LDL-C <130 mg/dL hs-CRP >2 mg/L Rosuvastatin (N =7500) Placebo (N =7500) MI Stroke Unstable Angina CVD Death CABG/PTCA 4 week Run-in JUPITER Randomized Trial of Rosuvastatin in the Primary Prevention of Cardiovascular Events Among Individuals with Low Levels of LDL-C and Elevated Levels of hs-CRP hs-CRP Lipids hs-CRP LFTs UA hs-CRP LFTs UA Lipids HbA1C Screening Visit 1 Randomization Visit Safety Visit Bi-Annual Follow-Up Visits End of Study Visit hs-CRP LFTs HbA1C Screening Visit 2 N Engl J Med. 2002;347:1157-1165 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 Quintiles of LDL 0 2 4 6 8 Years of Follow-Up 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability Quintiles of CRP Ridker et al, N Engl J Med. 2002;347:1157-1165. 5 4 3 2 1 5 4 3 2 1 Event-Free Survival According to Baseline Quintiles of C-Reactive Protein and LDL Cholesterol 1.00 0.99 0.98 0.97 0.96 0.00 0 2 4 6 8 Years of Follow-up Low CRP-low LDL Low CRP-high LDL High CRP-low LDL High CRP-high LDL CV Event-Free Survival Using Combined hs-CRP and LDL-C Measurements Ridker et al, N Engl J Med. 2002;347:1157-1165. Probability of Event-free Survival Median LDL 124 mg/dl Median CRP 1.5mg/l hs-CRP Adds Prognostic Information at all Levels of LDL-C and at all Levels of the Framingham Risk Score 0-1 25 20 15 10 5 0 Relative risk Multivariable relative risk 2-4 5-9 10-20 130-160 <130 >160 Framingham estimate of 10-year risk (%) LDL cholesterol (mg/dL) C-Reactive Protein (mg/L) C-Reactive Protein (mg/L) 1 0 2 3 <1.0 1.0-3.0 >3.0 Ridker et al, N Engl J Med. 2002;347:1557. <1.0 1.0-3.0 >3.0 What is the role of hs-CRP with regard to diabetes and the metabolic syndrome? Circulation. 2003;107:391-397. 0 1 2 3 4 5 0 2 4 6 8 C-reactive protein (mg/L) Number of Components of the Metabolic Syndrome Ridker et al, Circulation 2003;107:391-7 Plasma hs-CRP Levels According to Severity of the Metabolic Syndrome 0 2 4 6 8 Years of Follow-Up 0.95 0.96 0.97 0.98 0.99 1.00 CVD Event-Free Survival Probability CRP <1 mg/L CRP 1-3 mg/L CRP >3 mg/L Event Free Survival According to hs-CRP Levels: Analysis Limited to Participants with Metabolic Syndrome at Baseline Ridker et al, Circulation 2003;107:391-7 AHA / CDC Scientific Statement Markers of Inflammation and Cardiovascular Disease: Applications to Clinical and Public Health Practice Circulation January 28, 2003 “Measurement of hs-CRP is an independent marker of risk and may be used at the discretion of the physician as part of global coronary risk assessment in adults without known cardiovascular disease. Weight of evidence favors use particularly among those judged at intermediate risk by global risk assessment”. 1 mg/L 3 mg/L 10 mg/L Low Risk Moderate Risk High Risk Acute Phase Response Ignore Value, Repeat Test in 3 weeks >100 mg/L Ridker PM. Circulation 2003;107:363-9 Clinical Application of hs-CRP for Cardiovascular Risk Prediction Inflammatory and Infections Agents in CHD Belgian epidemiologic study included 446 of 16307 male workers aged 35-39 who had evidence of CHD vs. 892 controls. CRP, but none of the infectious agents (H. pylori, C. pneumoniae, CMV, and EBV) were associated with CHD, even after adjustment for other risk factors. De Backer et al. Atherosclerosis 2002; 160: 457-63. Infection and CHD - is there a connection? Local or systemic infections resulting from gram negative bacteria such as Chlamydia pneumoniae and Helicobacter pylori, including cytomegalovirus (CMV) have been implicated in atheroscelosis While several case control studies have shown increased titers of C.pneumoniae and H. Pylori in those with vs. without CHD, convincing evidence from prospective studies is lacking. Prospective Studies of CHD and Infectious Pathogens Physician’s Health Study (nested case-control) shows RR 1.1 (0.8-1.5) for C. Pneumoniae, 0.94 (0.7-1.2) for cytomegalovirus, and 0.72 (0.6-0.9) for Herpes simplex virus. H. pylori also shows mixed results. Whincup showed a nonsignificant 1.3 OR when adjusted for other risk factors, the large ARIC study showed no relation, and the Caerphilly Prospective study showed RR=1.05 in 1796 men followed 14 years. Other Studies of Infectious Agents In South Asian persons with CHD vs. controls, C. pneumoniae specific IgG antibody was seropositive in similar proportions; risk factors appeared to mediate any relations (Mendis et al. Int J Cardiol 2001; 79: 191-6). Cross-sectional survey of 704 individuals of C. pneumoniae and CMV with risk factors did nto show significant associations (Danesh et al., J Cardiovasc Risk 1999; 6: 387-90). Meta-analysis of 24 articles involving H. pylori infection and CHD showed a pooled odds ratio of 1.55 (95% CI: 1.38-1.74) (p<0.001), suggested a weak relation, but high hetrogeneity between studies precludes clear demonostration (Pellicano et al., Eur J Epidemiol 1999; 15: 611-9). ARIC Study failed to show clear relation between IgG antibodies for C. pneumoniae and incident CHD occurring over average 3.3 years. (Nieto et al. Am J Epidemiol 1999; 150: 149-56). Clinical Trial Evidence for Antibiotic Treatment and Prevention of CVD ACADEMIC Study of 302 patients with CHD seropositive to C. Pneumoniae randomized to azithromycin 500 mg/wk or placebo for 3 months showed no significant treatment difference (HR=0.89, p=0.74) for recurrent events (Muhlestein et al., Circulation 2000; 102: 1755-60). AZACS Multicenter study of 1439 pts with unstable angina randomized to 250 mg azithromycin/day for up to 6 months showed no significant benefit for death, recurrent MI, or recurrent ischemia (Cercek et al., Lancet 2003; 361: 809-13). WIZARD trial of 7,747 pts post-MI randomized to 12 week of therapy with azithromycin or placebo showed no significant reduction in reinfarction, revascularization, hospitalization for angina, or death (O’Connor et al., JAMA 2003; 290: 1459-66). Infectious Agents and the Future Individuals with greater infectious burdens may be at greater risk, because they are older, have poorer health habits, less access to care. Observed associations often may be due to selection biases or confounding from age and other factors Prospective clinical trials under way examining role of certain antibiotics such as azithromycin on reduction of recurrent events in CHD patients. Until these data are available, no role for measurement or treatment of infectious burden.