The role of high density lipoprotein compositional and functional heterogeneity in metabolic disease By Scott M. Gordon B.S. State University of New York College at Brockport October, 2012 A Dissertation Presented to the Faculty of The University of Cincinnati College of Medicine in partial fulfillment of the requirements for the Degree of Doctor of Philosophy from the Pathobiology and Molecular Medicine graduate program W. Sean Davidson Ph.D. (Chair) David Askew Ph.D. Professor and Thesis Chair Professor Department of Pathology Department of Pathology University of Cincinnati University of Cincinnati Francis McCormack M.D. Gangani Silva Ph.D. Professor Assistant Professor Department of Pathology Department of Pathology University of Cincinnati University of Cincinnati Jason Lu Ph.D. Assistant Professor Division of Bioinformatics Cincinnati Children’s Hospital i Abstract High density lipoproteins (HDL) are complexes of phospholipid, cholesterol and protein that circulate in the blood. Epidemiological studies have demonstrated a strong inverse correlation between plasma levels of HDL associated cholesterol (HDL-C) and the incidence of cardiovascular disease (CVD). Clinically, HDL-C is often measured and used in combination with low density lipoprotein cholesterol (LDL-C) to assess overall cardiovascular health. HDL have been shown to possess a wide variety of functional attributes which likely contribute to this protection including anti-inflammatory and anti- oxidative properties and the ability to remove excess cholesterol from peripheral tissues and deliver it to the liver for excretion, a process known as reverse cholesterol transport. This functional diversity might be explained by the complexity of HDL composition. Recent studies have taken advantage of advances in mass spectrometry technologies to characterize the proteome of total HDL finding that over 50 different proteins can associate with these particles. This adds to a growing body of evidence that supports the global hypothesis of this thesis which is that the total pool of HDL in an individual is composed of numerous subspecies with distinct protein and lipid compositions and therefore will have distinct functional properties. Additionally, we believe that the composition of HDL is dynamic and can change in response to changes in the environment of the blood, as can occur in disease. To test these hypotheses we devised an approach based on three aims. Aim 1: Identify and characterize HDL subspecies based on protein composition. Aim 2: Analyze functional heterogeneity across separated plasma HDL fractions. Aim 3: Examine the effect of type 2 diabetes on HDL subspecies distribution in young adults. To accomplish these goals we have developed novel methods for the separation and fractionation of HDL subspecies from human plasma and their subsequent proteomic and functional analysis. Our findings support the existence of a diverse array of HDL species with distinct functionalities which correlate strongly with specific lipid or protein components of the particle. Additionally, we found that the composition of specific subfractions of HDL are altered in type 2 diabetes and that these fractions can predict vascular health better than LDL-C or HDL-C, suggesting that these may prove to be better biomarkers for cardiovascular risk than the current clinical standard. A detailed understanding of HDL subspeciation will be invaluable in the further development of HDL as a biomarker and therapeutic target not only for cardiovascular disease but a variety of disease states. ii Copyright Notice This dissertation is based, in part, on the following published manuscripts. Permission to include the information contained within these manuscripts was obtained from the publishers (Appendix 1). 1. Gordon, S., Durairaj, A., Lu, J., and Davidson, W.S. HDL Proteomics: Identifying New Drug Targets and Biomarkers by Understanding Functionality. (2010) Cur. Cardiovasc. Risk Reports., 4: 1-8. 2. Gordon, S.M., Jingyuan Deng, L. Jason Lu, and W. Sean Davidson. Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography. (2010) J. Proteome Res., Oct. 1; 9(10):5239-49. 3. Gordon, S.M., Hofmann, S., Askew, D., Davidson, W.S. High Density Lipoprotein: It’s Not Just About Lipid Transport Anymore. (2011) Trends in Endocrinology and Metabolism. 22, 9-15. iii Acknowledgements This work was supported by an American Heart Association Pre-doctoral Fellowship, a University Research Council Graduate Student Research Fellowship from the University of Cincinnati and by a pilot grant from the Cincinnati Diabetes and Obesity Center. I want to thank my thesis advisor Sean Davidson for his outstanding guidance and encouragement and for helping me to obtain the best possible opportunities to develop as a successful independent scientist. Adam Rich: My undergraduate research advisor who introduced me to the world of laboratory research and the scientific community. I cannot express enough appreciation for the way you took me in and allowed me to think and grow in your lab. Kathy and Phil Gordon: My amazing parents who have always encouraged me to reach for the stars and told me that there is nothing I cannot accomplish if I put my mind to it and work hard. iv Table of Contents Abstract……………………………………………………………………..………… ii Copyright notice………………………………………………...……………………. iii List of Figures………………………………………………………………………… vii List of Tables…………………………………………………………………………. ix Chapter 1. Introduction and background A. High density lipoproteins 1. Clinical importance of HDL………………………………….. 1 2. Functions of HDL…….…………………………………...….. 4 3. Current measures of HDL subfractions……………….…... 17 4. Applications of modern proteomics to HDL…….……..…... 20 5. Evidence for HDL subspeciation based on protein content ……………………………………………………..…. 24 6. HDL subspeciation and its impact on the treatment of cardiovascular disease……………………………………... 27 B. Goals of thesis research 1. Aim 1: To identify and characterize HDL subspecies based on protein composition…………………………….. 30 2. Aim 2: Analyze functional heterogeneity across separated plasma HDL fractions…………………………. 31 3. Aim 3: Examine the effect of type 2 diabetes on HDL subspecies distribution in young adults…………………… 31 Chapter 2. Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography A. Introduction……………………………………………….…..………. 57 B. Experimental………………………………………………….………. 60 C. Results……………………………………………………..…….……. 63 D. Discussion…………………………………………………………….. 69 E. Conclusions…………………………………………………………… 74 Chapter 3. A novel correlation analysis to determine HDL subspecies protein composition A. Introduction……………………………………………………..…….. 91 B. Experimental………………………………………………………….. 92 C. Results………………………………………………………………… 95 D. Discussion……………………………………………………………. 99 v Chapter 4. Using apolipoprotein deficient systems to study HDL composition A. Introduction…………………………………………………………… 114 B. Experimental………………………………………………………….. 116 C. Results………………………………………………………………… 117 D. Discussion…………………………………………………………….. 121 Chapter 5. Functional analysis of HDL subfractions A. Introduction…………………………………………………………... 145 B. Methods…………………..…………………………………………... 146 C. Results………………………………………………………………… 148 D. Discussion……………………………………………………………. 152 Chapter 6. Effects of type 2 diabetes on lipoprotein composition and arterial stiffness in adolescents and young adults A. Introduction…………………………………………………………... 162 B. Methods…………………..…………………………………………… 164 C. Results………………………………………………………………… 168 D. Discussion……………………………………………………………. 174 Chapter 7. Discussion A. Thesis Summary..………………………………………………….... 200 B. Contributions of this work.………………………………………….. 203 C. The future of HDL subspecies research and clinical implications. 207 Appendix I. Reproduction Rights…………………………………………………. x Appendix II. Supplemental Data………………………………………………..…. xiii vi List of Figures Chapter 1. 1-1. The increasing functional heterogeneity of high density lipoprotein. Chapter 2. 2-1. Elution profiles from Superose 6 (2x) and Superdex 200 (3x) size exclusion chromatography configurations. 2-2. SDS PAGE comparison of total HDL preparations derived from ultracentrifugation (UC) and gel filtration (GF) chromatography. 2-3. Ability of calcium silicate hydrate (CSH) to bind ultracentrifugally isolated human plasma lipoproteins. 2-4. Ability of CSH to bind phospholipid-containing particles from fractions collected by gel filtration chromatography. 2-5. Examples of elution profile shifts for proteins upon ether delipidation of fresh human plasma. 2-6. Lipid-associated proteins identified in the plasmas of 3 normolipidemic donors. 2-7. Distribution patterns of common HDL associated proteins across gel filtration fractions. 2-8. Triple Superdex distribution profiles for identified lipid- associated proteins. 2-9. Gene Ontology functional associations of newly identified lipoprotein associated proteins. Chapter 3. 3-1. Correlation strategy for the identification of phospholipid particle subspecies. 3-2. Lipid and protein distribution profiles produced by gel filtration chromatography. 3-3. Lipid and protein distribution profiles produced by anion exchange chromatography. 3-4. Lipid and protein distribution profiles produced by isoelectric focusing. 3-5 Heat map displaying protein distribution patterns by anion exchange chromatography. 3-6. Heat map displaying protein distribution patterns by isoelectric focusing. 3-7. Tracking co-migratory patterns of protein pairs across different