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Transcript
HDL and Its Galaxy of Proteins: What Do they Do? Jay Heinecke, University of Washington Disclosures NIH AHA Insilicos Consultant Merck Research Support GSK Research Support Pfizer Research Support Corcept Consultant Lipoproteins Play Critical Roles in Macrophage Cholesterol Homeostasis LDL Delivers Cholesterol HDL + apoA-I Scavenger Receptors Macrophage ABCA1 ABCG1 Removes Cholesterol 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? The Dysfunctional HDL Hypothesis HDL Dysfunctional-HDL Cholesterol Efflux Cholesterol Efflux ? Anti-Inflammatory Pro-Inflammatory ? HDL-C Does Not Necessarily Predict HDL Function or CAD Risk New HDL Metrics are Critical for Evaluating CAD Risk and HDL Therapies Is the “Dysfunctional HDL Hypothesis” Clinically Relevant? Cholesterol Efflux from Macrophages to “Serum HDL” of Humans Rothblatt and Rader de la Llera-Moya et al. ATVB 2010;30:796-801 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 Hypothesis: Remodeling of the HDL Proteome by Inflammation Impairs Sterol Efflux from Macrophages Inflammation HDL ABCA1 ABCG1 Sterol Effux Macrophage Dysfunctional HDL Oxidation? Altered Protein Cargo? Do Changes in the Protein Cargo of HDL Impair Its Cardioprotective Effects? Gene Ontology Analysis of HDL Proteins Vaisar et al. JCI 2006 PLTP ApoM CETP LCAT ApoC-I ApoC-II ApoC-III ApoF AGT SERF2 SERF1 ApoC-IV ApoE ApoD Proteinase Inhibitor PON3 Lipid Metabolism SAA4 ApoL-1 SAA2 ApoA-II SAA1 ApoA-I Clusterin ApoA-IV ApoH PON1 Complement Regulation C3 AHSG HRP SERA1 AMP KNG 1 Acute Phase Response C4A C4B C9 VTN ORM2 TTR ITIH4 RBP4 TF HPX FGA Peter Libby Proteolysis Complement Activation What is HDL? X What is HDL? Davidson Model PNAS 2009 Why Do We Have Lipoproteins? Sex Nutrition Disease Infections 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 HDL Old Paradigm forNew HDL:Paradigm Cholesterolfor Transport Immunity Tissue Repair HDL BA FC Liver Macrophage Proteins Lipids FC CE CE FC CE TG VLDL/LDL Nutrient Delivery Adipocyte What is the Impact of Acute Inflammation on the Proteome and Function of Human HDL? 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 LPS Markedly Increases Plasma Levels of SAA and CRP in Humans SAA (µg/dL) 10 → 300 CRP (ng/dL) 3 → 45 60.0 400 300 No Change In Plasma or Lipoprotein Lipids 40.0 200 20.0 100 0 0.0 Control 1 LPS 2 LPS Control 1 LPS 2 LPS Inflammatory Human HDL Exhibits Impaired Cholesterol Efflux to Macrophages J774 Cells Cholesterol Efflux (%) P<0.01 P<0.03 20 10 0 Control LPS 1 LPS 2 Protein Identification and Quantification by MS/MS Tryptic Digest of Proteins LC-ESI-MS/MS MS/MS and Protein Nomenclature Gene Name Spectral Counts MS/MS spectrum (unique peptides) Analysis Quantification Dual Statistical Criteria G-test and t-test The HDL Proteome in Humans Challenged with LPS APOA1 APOA2 APOC3 SERPINA1 86 Proteins Identified SAA4 APOC2 ALB APOM Control LPS 1ng LPS 2ng APOC1 APOE APOD PON1 TTR SAA1 APOA4 SAA 1 and SAA 2 Increase 20-fold 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 Cholesterol Efflux Correlates Inversely with SAA Content of HDL 24.0 24 Cholesterol Efflux R2 = 0.42 22.0 22 2 P = 0.006 20.0 20 18.0 18 0.0 0.2 0.4 0.6 0.8 µg SAA / mg HDL protein Hypothesis: Remodeling of the HDL Proteome by SAA Impairs Sterol Efflux to Macrophages Inflammation HDL SAA-enriched HDL ABCA1 ABCG1 Macrophage 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 Is HDL that Lacks SAA1 and SAA2 Protected from Inflammation-induced Dysfunction? SAA Deficiency Blocks Inflammation-induced Impaired Cholesterol Efflux to Macrophages Fred and Maria de Beer SAA Remodels the HDL Proteome and Impairs Sterol Efflux from Macrophages SAA HDL ABCA1 ABCG1 Sterol Effux Macrophage Dysfunctional HDL Altered Protein Cargo Does Oxidation Impair the Anti-Atherogenic Effects of HDL? HDL-mediated Removal of Cellular Cholesterol HDL Apo A-I PL ABCA1 PL FC CE FC FC FC Macrophage Foam Cell Myeloperoxidase (MPO): A Macrophage Pathway for the Generation of HOCl + NADP NADPH MPO Macrophage O2 2 O H2O2 MPO HOCl Proteins BACTERIAL KILLING Nucleic Acids NO2 Tyr Lipids HOST INJURY Chlorotyrosine is a Specific Product of Myeloperoxidase (MPO) ApoA-I Oxidation by HOCl –A Specific MPO Product– Impairs ABCA1 Activity Oxidation 3 Met Residues 6 Cholesterol Efflux 4 2 0 Chlorination Tyr-192 0 25 50 H O C l: A p o A - I ( m o l: m o l) 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 Proposed Model: Oxidation of Hinge Domains in ApoA-I Impairs HDL Remodeling Impaired Refolding Is HDL Targeted for Oxidation In Vivo by Myeloperoxidase? 3-Chlorotyrosine Levels are Markedly Increased in HDL Isolated from Blood of Patients with CAD Chlorotyrosine/Tyr µmol/mol 100 50 0 Healthy CAD Site Specific Damage of ApoA-I: A Pathway for Generating Dysfunctional HDL in Humans Cardioprotective HDL VLDL/LDL HDL X Dysfunctional HDL Oxidation Altered Protein Composition CETP Inhibition? CETP CE CE FC X X X ABCA1 ApoA-I X Inflammation X Cholesterol Efflux Acknowledgements Baohai Shao, PhD Tomas Vaisar, PhD Jack Oram, PhD Michael Oda Anthony Suffredini Chongren Tang Maria de Beer Fred de Beer
