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Joseph P. McConnell PhD, DABCC The Mayo Clinic and Foundation Health Diagnostic Laboratory, Inc.

Lipoprotein Associated Phospholipase A2 A Novel Cardiovascular Risk Marker and a Potential Therapeutic Target. Joseph P. McConnell PhD, DABCC The Mayo Clinic and Foundation Health Diagnostic Laboratory, Inc. Richmond, Virginia ASCP Teleconference: December 9, 2009. Technology.

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Joseph P. McConnell PhD, DABCC The Mayo Clinic and Foundation Health Diagnostic Laboratory, Inc.

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  1. Lipoprotein Associated Phospholipase A2A Novel Cardiovascular Risk Marker and a Potential Therapeutic Target Joseph P. McConnell PhD, DABCC The Mayo Clinic and Foundation Health Diagnostic Laboratory, Inc. Richmond, Virginia ASCP Teleconference: December 9, 2009

  2. Technology Patient Care Meeting Patient Needs through Laboratory Excellence Research Results Cardiovascular Laboratory Medicine CVLM Cardiovascular Laboratory Medicine Mayo Clinic

  3. Disclosures diaDexus Inc. South San Francisco, CA Past: grant support no current grant support Supplied reagents for Lp-PLA2 analysis to support research and development efforts

  4. Objectives After attending this session, participants will be able to: • Describe differences between Lp-PLA2 and other inflammatory biomarkers like C-reactive protein. • Discuss the possible atherosclerotic mechanism of action of Lp-PLA2. • Summarize primary and secondary cardiovascular (CV) prevention studies that have demonstrated associations between elevated Lp-PLA2, cardiovascular events, and stroke. • Describe how Lp-PLA2 may become a future therapeutic target.

  5. Outline • Novel Cardiovascular Biomarkers “risk markers” • Inflammation and atherosclerosis • Mechanisms by which Lp-PLA2 is Atherogenic • Studies linking elevated Lp-PLA2 to CV events • Is Lp-PLA2 present in atherosclerotic lesions? • Vulnerable lesions? • Can Lp-PLA2 be a therapeutic target? • Lp-PLA2 measurement.

  6. Ischemic Events in the U.S. • 1,500,000 heart attacks: 500,000 deaths • 795,000 strokes: 150,000 deaths • Many individuals who experience an • Ischemic event will die as a result of that event. • Several who have an event have no prior • symptoms. Source: AHA Heart Disease and Stroke Statistics 2009 Update; Circulation, May 2009

  7. Cardiovascular Risk Factors • There is a need for additional risk factors. • Although traditional risk factors produce reasonably accurate estimates of risk in a population, they predict only 50-60% of events in individual patients. • Additional risk factors would improve accuracy of decisions regarding preventative therapies Rader DJ. NEJM. 2000;343:1179-82

  8. Inflamm. hsCRP antibodies chlamidia CMV Lipids/Oxidation Platelet actv. Coagulation Fibrinolysis Endothelium Nutrition Fibrinogen PF 1+2 TAT TFPI TPP TXA2 12-HETE B-TBG P-selectin D-Dimer PAI-I Plasminogen Endothelin Prostacyclin Nitric oxide soluble ICAM VCAM LDL subclasses Lp (a) F2 isoprostanes Conj. dienes HCY B12,B6 Folate Vit. C,E, Carotene Homocysteine Thrombin monocyte chemotaxis ICAM VCAM E-selectin TXA2 Tissue factor VWF Native LDL Growth Factors slow O2 radicals smooth muscle cell mitogenesis Vasoconstriction rapid Oxidized LDL

  9. Can have utility in selected individuals to guide intensity of risk reduction therapy. No guidelines for measurement NCEP: Emerging Risk Factors Lipoprotein (a) Homocysteine Prothrombotic factors Proinflammatory factors Impaired fasting glucose Evidence of subclinical atherosclerotic disease

  10. Cardiovascular Risk Technology Team Cardiovascular Health Clinic Laboratory Cardiology Working Group

  11. Proposed Markers of Inflammation Adhesion Molecules ICAM, VCAM, E-selectin, P-selectin Cytokines IL-6, IL-1beta, TNF-alpha Acute Phase reactants C-reactive protein, Fibrinogen, Serum amyloid A Matrix Metalloproteinases and Others MMP-9, sCD-40 Ligand, myeloperoxidase Lp-PLA2

  12. Different Inflammatory Markers: Different Roles Adapted from: Rader D. N Engl J Med. 2000

  13. Lp-PLA2 and CRP: Two Independent and Distinct Inflammatory Markers

  14. Women: N = 56 Men: N = 56 Frequency of Lp-PLA2 in Men and Women N = 56

  15. Biological Variability of Lp-PLA2 Blood from 43 healthy adults each drawn 7 times over 4 weeks *Wolfert RL, et al. Circulation. 2004.

  16. 4.2 (95% CI 1.7-10.3, p=0.001) Risk Ratios 1.4 1.2 1.0 Additive Risk for Incident CHD for LDL <130 by Lp-PLA2 and hsCRP Tertiles Adjusted for demographics, current smoking status, blood pressure, diabetes, and HDL Ballantyne et al., Circulation 2004

  17. 11.4 (95% CI 3.1–41.4, p<0.001) Risk Ratios 5.5 5.8 1.0 Additive Risk for Incident Ischemic Stroke by Lp-PLA2 and hsCRP Tertiles Adjusted for demographics, current smoking status, blood pressure, diabetes, LDL and HDL Ballantyne et al. (Scientific Sessions of the AHA, November, 2004)

  18. Lipoprotein Associated Phospholipase A2 (Lp-PLA2) AKA: Platelet-activating factor acetylhydrolase 50kDa, Ca-insensitive lipase Produced predominantly by macrophages > 80% bound to LDL Not responsive to IL-1, IL-6, TNF-alpha. Hydrolyzes oxidized phospholipids

  19. Phosphatidylcholine (PC) Oxidation Oxidatively-Modified PC Lp-PLA2 Putative Inflammatory Mediators + Oxidized Fatty Acid Lyso-PC Lp-PLA2 and LDL Oxidation

  20. Lyso-PC Expression of adhesion molecules Upregulation of cytokines and CD40 ligand Stimulation of macrophage proliferation Induction of MCP-1 Migration of vascular smooth muscle cells Inhibition of endothelial derived nitric oxide Chemoattractant for macrophage and T-cells 1. Dada N, et al. Expert Rev Mol Diagn. 2002;2(1):89-94. 2. Quinn MT, et al. Proc Natl Acad Sci USA. 1988;85:2805-2809.. 3. MacPhee CH, et al. Biochem J. 1999;338:479-487. 4. Carpenter KL, et al. FEBS Lett. 2001;505:357-363 Pro-Atherogenic Activities of Lysophosphatidylcholine (Lyso-PC)

  21. The Role of Lp-PLA2 in CVD LUMEN Monocytes Atheroma Cytokines Adhesion Molecules Oxidized LDL Lp-PLA2 Lyso-PC OxFA Foam Cell INTIMA • Macrophage MEDIA

  22. When arterial blood touches the necrotic lipid pool – it clots… Fibrous cap Lumen Lipid core Lumen Unstable Plaque Stable Plaque • May have significant stenosis • Thick fibrous cap /high collagen content • Small lipid pool • Few inflammatory cells • Low Lp-PLA2 content • May have minimal stenosis • Thin fibrous cap / low collagen content • Large lipid pool • Active inflammatory cells • High Lp-PLA2 content

  23. Lp-PLA2 in the atherosclerotic lesion Kolodgie et.al, ATVB 2006;26:2523-29

  24. West of Scotland Coronary Prevention Study (WOSCOPS): NEJM, Oct. 2000 Hypercholesterolemic subjects 6,595 men, aged 45 to 65 mean follow-up of 5 year 580 coronary events 1160 event free controls Matched for age and smoking status

  25. C-ReactiveProtein (mg/L) Lipoprotein-AssociatedPhospholipase A2 (mg/L) White-Cell Count(x10-3/mm3) 3.0 2.0 Risk ratio 1.0 0.5 <1.80 2.08-2.36 >2.72 <1.80 6.01-6.90 >8.10 <0.76 1.45-2.52 >4.59 1.45-2.52 2.53-4.59 1.81-2.07 2.37-2.72 5.21-6.00 6.91-8.10 Univariate Other Inflamm. markers NEJM 343(16):1148, 2000 CP1009715-53 Age, BP, Lipids Inflammatory Markers in Ischemia (WOSCOPS)

  26. Elevated Lp-PLA2 is Consistently Associated with Increased Risk for Cardiovascular Disease 0.5 1 1.5 2 2.5 3 3.5 4 4.5

  27. 504 patients with coronary angiography Vessel Disease 3V None 2V Mild 1V 46% patients have none or mild coronary occlusion 54% patients have significant occlusion (>50% stenosis)) Brilakis, McConnell et.al, European Heart Journal, Jan. 2005 Lp-PLA2: Relation to Angiographic CAD and Cardiovascular Events

  28. Lp-PLA2 and Angiographic Coronary Disease Univariate Analysis

  29. Multivariate Logistic Regression Analysis After adjusting for age, gender, smoking history, hypertension, cholesterol, HDL cholesterol, and triglyceride, Lp-PLA2 was no longer was no longer independently predictive of angiographic CAD. CRP was not predictive of CAD in either the univariate or multivariate model.

  30. Lp-PLA2 and Clinical Outcomes Median Follow-up was 4 years 58 cardiovascular events occurred in 49 of 466 contacted patients (11%) cardiac death in 6 myocardial infarction in 14 coronary revascularization in 28 stroke in 10 Clinical, lipid, and inflammatory (CRP and fibrinogen) variables were assessed.

  31. Lp-PLA2 CRP Fibrinogen 1.33(1.08-1.64) P = 0.006 1.36 (1.06-1.75) P = 0.015 1.41(1.14-1.74) P = 0.002 1.32(1.06-1.63) P = 0.012 1.09(0.78-1.51) P = 0.624 1.32(1.00-1.75) P = 0.05 Single analyte Lp-PLA2 + CRP + Fibrinogen Adjusted for Clinical (age, gender, smoking, HTN) and lipid (total and HDL cholesterol, Lp(a), and triglycerides) variables All Cause Death/AMI/Revasc/Stroke Brilakis et al., EHJ 2005 Hazard Ratio Associated with Markers of Inflammation (per 1 SD)

  32. Variable HR 95% CI Age 1.3 0.96 - 1.88 Male Gender 1.2 0.62 - 2.49 Hypertension 1.3 0.74 - 2.30 Smoking 1.2 0.68 - 2.23 LDL cholesterol 1.0 0.69 - 1.45 HDL cholesterol 0.83 0.61 - 1.13 Log triglyceride 0.97 0.70 - 1.34 Log CRP 1.1 0.73 - 1.55 Log HCY 1.2 0.92 - 1.59 LDL size 1.9 0.72 - 1.37 Fibrinogen 1.6 1.16 - 2.29 Lp-PLA2 1.3 1.05 - 1.57 HR For events in 425 non-AMI: Multivariate

  33. HR For Angiographic CAD: Multivariate Variable HR 95% CI P-Value Age 1.05 1.03 - 1.07 <0.0001 Male Gender 4.03 2.46 – 6.60 <0.0001 Hypertension 1.60 1.06 - 2.42 0.026 Smoking 1.45 0.95 - 2.22 0.087 Total cholesterol 1.01 1.004 - 1.016 0.001 HDL cholesterol 0.96 0.94 - 0.98 0.0002 Log triglyceride 0.84 0.52 - 1.37 0.481 Log CRP 1.06 0.87 - 1.30 0.532 Fibrinogen 1.16 0.95 - 1.42 0.154 Lp-PLA2 0.91 0.71 - 1.17 0.466 Lp(a) cholesterol (per 5 mg/dL) 1.56 1.15 - 2.11 0.004

  34. Atherosclerosis Burden vs.Vulnerable Plaque Risk markers for atherosclerotic buildup and plaque formation may be different than those which predict events. This needs to be studied in more detail, but may provide insight into most appropriate measures in given clinical situations.

  35. Other Published Mayo Studies • Lp-PLA2 in Peripheral Arterial Disease • Lp-PLA2 and Endothelial Dysfunction • Lp-PLA2 and Cardiac Allograft Vasculopathy in Heart Transplant Patients • Production of Lp-PLA2 and LPC in coronary arteries and presence in carotid plaques • Lp-PLA2 prognosis after MI • Lp-PLA2 recommended clinical cut-points • Effect of Pioglitizone and Glipizide on Lp-PLA2 • Importance of Lp-PLA2 distribution on Lipoproteins

  36. Coronary Ostium Coronary gradient Myocardial Production CS levels - aorta levels X CBF = Coronary sinus Production of Lp-PLA2 and LysoPC across the coronary circulation

  37. Coronary atherosclerosis No atherosclerosis Lp-PLA2 netproduction(ng/min) CRP netproduction(g/min) P<0.01 P=NS Lp-PLA2 CRP Lp-PLA2 Enters the Coronary Circulation When Coronary Atherosclerosis (by IVUS) is Present Lavi S, McConnell JP, Rihal CS, Prasad A, Mathew V, Lerman LO, Lerman A. Local Production of Lp-PLA2 and LysoPC in the Coronary Circulation: Association With Early Coronary Atherosclerosis and Endothelial Dysfunction in Humans. Circulation 2007

  38. Myocardial Production of Lp-PLA2 and LysoPC 15 patients with and 15 patients without early atherosclerosis by angiography and or IVUS Lavi, McConnell, Lerman, et.al. Circulation 2007

  39. Measurement of Lp-PLA2 • FDA approved microtiter plate ELISA • Requires batch sampling • Reported to be automatable • Not easily adapted in a routine clinical laboratory • Automated immunoturbidimetric method has been developed and now has been approved by the FDA

  40. Automated immunoturbidimetric method for Lp-PLA2 (Auto-PLAC) • Performed on multiple automated chemistry analyzers (Roche, Hitachi 912, etc.) • Improved performance characteristics • Precision, Linearity, Recovery • Correlation: Gen 3 ELISA to Auto=PLAC • (y = 1.1609x +12.11, R2 = 0.5363, (n = 452) • Sample and Reagent Stability Issues exist that we are working on in collaboration with diaDexus

  41. Lp-PLA2 activity assays • Some commercially available methods and home brews • Currently no FDA approved methods • As methods are developed issues to be addressed will be • Standardization • What is the most appropriate substrate? Platelet activating factor Oxidized phosphatidylcholine • Influence/interference form other phospholipases

  42. Therapeutic Reduction of Lp-PLA2 • Can Lp-PLA2 be a therapeutic target? • If so, will reduction reliably predict outcome?

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