HDL and Its Galaxy of Proteins: What Do they Do? Jay Heinecke, University of Washington

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1 HDL and Its Galaxy of Proteins: What Do they Do? Jay Heinecke, University of Washington

2 Disclosures NIH AHA Insilicos Consultant Merck Research Support GSK Research Support Pfizer Research Support Corcept Consultant

3 Lipoproteins Play Critical Roles in Macrophage Cholesterol Homeostasis LDL HDL + apoa-i Delivers Cholesterol Scavenger Receptors ABCA1 ABCG1 Removes Cholesterol Macrophage

4 Many Lines of Evidence Support this Model Epidemiology and clinical trials Low HDL Strongly and consistently associated with CAD risk Genetically engineered mice Compelling links to atherosclerosis ApoA-I, SRB-1, Macrophage ABC transporters Current Issues in HDL Biology HDL Elevating Therapies CETP inhibition Increased risk of CAD events and death? Aim High No benefit?

5 The Dysfunctional HDL Hypothesis HDL Dysfunctional-HDL Cholesterol Efflux Anti-Inflammatory Cholesterol Efflux? Pro-Inflammatory?

6 HDL-C Does Not Necessarily Predict HDL Function or CAD Risk New HDL Metrics are Critical for Evaluating CAD Risk and HDL Therapies

7 Is the Dysfunctional HDL Hypothesis Clinically Relevant?

8 Cholesterol Efflux from Macrophages to Serum HDL of Humans Rothblatt and Rader de la Llera-Moya et al. ATVB 2010;30:

9 Is Dysfunctional HDL Clinically Relevant? Cholesterol efflux capacity, HDL function, and atherosclereosis Khera et al. N Engl J Med. 2011;364:127. Macrophage Cholesterol Efflux to Serum HDL Associates with CAD status Independent of HDL-C Independent of ApoA-I Better predictor of CAD status than HDL-C or apoa-i

10 Hypothesis: Remodeling of the HDL Proteome by Inflammation Impairs Sterol Efflux from Macrophages HDL ABCA1 ABCG1 Inflammation Dysfunctional HDL Oxidation? Altered Protein Cargo? Sterol Effux Macrophage

11 Do Changes in the Protein Cargo of HDL Impair Its Cardioprotective Effects?

12 ApoF ApoE ApoD ApoL-1 PLTP ApoM ApoA-II Gene Ontology Analysis of HDL Proteins CETP Clusterin ApoA-IV LCAT ApoC-I Lipid Metabolism Vaisar et al. JCI 2006 ApoC-II ApoC-III SAA2 SAA1 ApoA-I ApoH PON1 ApoC-IV PON3 SAA4 AHSG AGT HRP SERF2 Proteinase Inhibitor SERA1 SERF1 AMP KNG 1 Complement Regulation C3 C4A C4B C9 VTN ORM2 TTR Acute Phase Response ITIH4 RBP4 HPX FGA TF

13 Proteolysis Complement Activation Peter Libby

14 What is HDL? X

15 Davidson Model PNAS 2009 What is HDL?

16 Why Do We Have Lipoproteins? Sex Nutrition Disease Infections

17 Trypansome Lytic Factor ApoA-I + HRP + ApoL Human Trypanosoma evansi Infection Linked to a Lack of Apolipoprotein L-I Vanhollebeke et al. New Eng J Med 2006

18 New Paradigm for HDL Old Paradigm for HDL: Cholesterol Transport BA FC HDL FC CE Immunity Tissue Repair Proteins Lipids Macrophage FC CE Liver CE TG VLDL/LDL Nutrient Delivery Adipocyte

19 What is the Impact of Acute Inflammation on the Proteome and Function of Human HDL?

20 Study Design: Inflammation and Human HDL Anthony Suffredini Apparently healthy subjects LPS 1 ng/kg 8 subjects 2 ng/kg 4 subjects Isolate HDL by ultracentrifugation Quantify HDL proteome Cholesterol efflux capacity

21 LPS Markedly Increases Plasma Levels of SAA and CRP in Humans SAA (µg/dl) CRP (ng/dl) 3 45 No Change In Plasma or Lipoprotein Lipids Control 1 LPS 2 LPS Control 1 LPS 2 LPS

22 Inflammatory Human HDL Exhibits Impaired Cholesterol Efflux to Macrophages J774 Cells P<0.01 Cholesterol Efflux (%) P<0.03 Control LPS 1 LPS 2

23 Protein Identification and Quantification by MS/MS Tryptic Digest of Proteins MS/MS and Protein LC-ESI-MS/MS Nomenclature Gene Name Spectral Counts MS/MS spectrum (unique peptides) Analysis Quantification Dual Statistical Criteria G-test and t-test

24 The HDL Proteome in Humans Challenged with LPS APOA1 APOA2 APOC3 SERPINA1 SAA4 APOC2 ALB APOM APOC1 APOE APOD PON1 TTR SAA1 APOA4 Control LPS 1ng LPS 2ng 86 Proteins Identified SAA 1 and SAA 2 Increase 20-fold

25 SAA1 and SAA2 Acute-phase proteins Synthesized by liver in response to inflammatory stimuli SAA4 Constitutively synthesized Increased plasma levels predict CAD events Apolipoproteins Associate primarily with HDL

26 Cholesterol Efflux Correlates Inversely with SAA Content of HDL 24.0 Cholesterol Efflux R 2 = 0.42 P = µg SAA / mg HDL protein

27 Hypothesis: Remodeling of the HDL Proteome by SAA Impairs Sterol Efflux to Macrophages HDL Inflammation SAA-enriched HDL ABCA1 ABCG1 Macrophage

28 SAA Deficiency and Inflammation-induced HDL Dysfunction Fred and Maria debeer Wild-type and SAA-/- Mice SAA-/- mice lack both SAA1 and SAA2 Inflammation model (Kisilevsky) PBS Silver nitrate Isolate HDL by ultracentrifugation Quantify HDL proteome Cholesterol efflux capacity

29 Is HDL that Lacks SAA1 and SAA2 Protected from Inflammation-induced Dysfunction?

30 SAA Deficiency Blocks Inflammation-induced Impaired Cholesterol Efflux to Macrophages Fred and Maria de Beer

31 SAA Remodels the HDL Proteome and Impairs Sterol Efflux from Macrophages HDL ABCA1 ABCG1 SAA Dysfunctional HDL Altered Protein Cargo Sterol Effux Macrophage

32 Does Oxidation Impair the Anti-Atherogenic Effects of HDL?

33 HDL-mediated Removal of Cellular Cholesterol HDL Apo A-IA PL FC ABCA1 PL FC FC CE FC Macrophage Foam Cell

34 Myeloperoxidase (MPO): A Macrophage Pathway for the Generation of HOCl NADP + NADPH MPO O 2 O 2 - H 2 O 2 MPO HOCl NO 2 Tyr Proteins Nucleic Acids Lipids BACTERIAL KILLING HOST INJURY Macrophage

35 Chlorotyrosine is a Specific Product of Myeloperoxidase (MPO)

36 ApoA-I Oxidation by HOCl A Specific MPO Product Impairs ABCA1 Activity Oxidation 3 Met Residues 6 Cholesterol Efflux 4 2 Chlorination Tyr HOCl:ApoA-I (m ol:mol)

37 Oxidation of specific amino acid residues in apoa-i impairs reverse cholesterol transport Tyr192 Major site of chlorination Tyr192Phe mutation protective Met residues Methionine sulfoxide reductase Met(S=O) Met(S) Reverses inhibition

38 Proposed Model: Oxidation of Hinge Domains in ApoA-I Impairs HDL Remodeling Impaired Refolding

39 Is HDL Targeted for Oxidation In Vivo by Myeloperoxidase?

40 3-Chlorotyrosine Levels are Markedly Increased in HDL Isolated from Blood of Patients with CAD 100 Chlorotyrosine/Tyr µmol/mol 50 0 Healthy CAD

41 Site Specific Damage of ApoA-I: A Pathway for Generating Dysfunctional HDL in Humans

42 Cardioprotective HDL VLDL/LDL CE X CETP HDL CE X Dysfunctional HDL Oxidation Altered Protein Composition CETP Inhibition? FC X Inflammation X ApoA-I X ABCA1 X Cholesterol Efflux

43 Acknowledgements Baohai Shao, PhD Tomas Vaisar, PhD Jack Oram, PhD Michael Oda Anthony Suffredini Chongren Tang Maria de Beer Fred de Beer

High density lipoprotein metabolism

High density lipoprotein metabolism Lipoprotein classes and atherosclerosis Chylomicrons, VLDL, and their catabolic remnants Pro-atherogenic LDL HDL Anti-atherogenic Plasma lipid transport Liver VLDL FC