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Proteomic Analysis of Three Dimensional Organotypic Liver Models

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Proteomic Analysis of Three Dimensional Organotypic Liver Models Proteomic Analysis of Three Dimensional Organotypic Liver Models Lucas Trung Vu Dissertation submitted to the faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of: Doctor of Philosophy in Chemical Engineering Chair: Padmavathy Rajagopalan Co-chair: Richard F. Helm T.M. Murali Chang Lu Richey M. Davis September 15th 2015 Blacksburg, VA Keywords: Shotgun Proteomics, Liver, In vitro Proteomic Analysis of Three Dimensional Organotypic Liver Models Lucas Trung Vu Abstract In vitro liver models that closely mimic the in vivo microenvironment are central for understanding hepatic functions and intercellular communication processes. Bottom- up shotgun proteomic analysis of the hepatic cells can lend insight into such processes. This technique employs liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of protein abundances by measuring intensities of their corresponding peptides. Organotypic 3D liver models have been developed in our laboratory that consist of hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM), which serves as a mimic for the Space of Disse. Each component within these models is easily separable allowing for systematic evaluation of the cells and PEMs. In this study, proteomes of hepatocytes from PEM containing models, cultured with and without LSECs, were compared to those from monolayers. Changes in core metabolism were evaluated among all culture conditions. Overall, all cultures were ketogenic and performed gluconeogenesis. The presence of the PEM led to increases in proteins associated with mitochondrial-based β-oxidation and peroxisomal proteins. The PEMs also limited production of structural proteins, which are linked to dedifferentiation of hepatocytes, suggesting that cell-ECM interactions are essential for maintenance of their liver-like state. The presence of LSECs increased levels of carboxylesterases and other phase I and phase II detoxification enzymes suggesting that intercellular signaling mediates enzyme abundance. Taken together, these results suggest that the cell-cell (from the LSECs) and cell-ECM (from the PEMs) interactions exert different, yet crucial effects, and both are required for the preservation of metabolic liver functions and differentiated phenotypes. Changes in the PEMs as a result of cell culture were also evaluated but exhibited minimal differences at this time point. Proteomes of LSECs monolayers were also characterized. Enzymes related to the metabolism of amino acids, lipids, oxidative phosphorylation and phase I and phase II detoxification processes were all identified in LSECs monolayers highlighting their role in these processes. Characterization of 3DHL LSECs was not possible due to ion suppression resulting from the presence of excess contaminant proteins. Nonetheless, this study provides a foundation in which LSECs from 3D liver models can be compared against in future studies. iii Acknowledgments First and foremost, I would like to thank my family. Mom and Dad, thanks for always being there to listen to me and giving me the best advice that you could in any situation. This degree is not only my achievement but also yours as well. To my brothers, Ivan and Jeffrey, thank you for always being there, I love you both very much and without you guys there to calm me down while I was doing this, I might not have been able to do it. I also offer my deepest thanks to my primary advisor, Professor Padma Rajagopalan, for all of your guidance, support and advice throughout my graduate studies here at Virginia Tech. I sincerely thank you for everything that you have taught me but most importantly, I am very grateful to you for pushing me to always be better and giving me the opportunity to grow as a scientist in your group. To my co-advisor, Professor Rich Helm, thank you for giving me the opportunity to learn in your lab and for taking the time to teach and advise me over the past couple of years. I have gained several skills that I know will serve me well in the future. I would also like to thank you for supporting me throughout my graduate studies here at Virginia Tech. Collectively, I thank both of my advisors for their patience throughout these past few years, I realize now that it was not an easy task but I am forever grateful that you both took a chance on me. To all of my lab mates in the Rajagopalan research group, past and present, including Dr. Adam Larkin, Dr. Era Jain, Dr. Gaurav Jain, Cigdem Arca, Brandon Veres, Sophia Orbach, Margaret Cassin, Andrew Ford, Rebekah Less and Kristen Sheerer, thank you for being there and listening to me with both personal and research related issues. All of iv those late nights, afternoon coffees, and scientific debates are memories that I will cherish for the rest of my life. I wish you all nothing but the best. To my lab mates in the Helm research group, Dr. Keith Ray, Dr. Sherry Hildreth, Evan Foley and Jody Jervis. Keith and Sherry, thank you for all of the help with proteomics and helping me learn mass spectrometry. Jody, thanks for your help with reagents and sample prep. Finally, thank you to all of you for all around good conversations. v Table of contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv List of figures ................................................................................................................... viii List of tables ........................................................................................................................ x Chapter 1: Introduction ....................................................................................................... 1 1.1 Background and significance .................................................................................... 1 1.2 In vitro hepatic cultures ............................................................................................ 5 1.3 Mass spectrometry .................................................................................................. 11 1.4 Data dependent and data independent analysis ....................................................... 13 1.5 Protease digestion ................................................................................................... 15 1.6 Quantification methods ........................................................................................... 17 1.7 Liver proteomics ..................................................................................................... 18 Chapter 2: Studying the changes in the hepatocyte proteome and PEMs in the presence and absence of LSECs in 3DHL organotypic liver models .............................................. 22 2.1 Introduction ............................................................................................................. 22 2.2 Materials and Methods ............................................................................................ 25 2.2.1 Chemicals and Reagents .................................................................................. 25 2.2.2 Isolation and culture of hepatocytes and non-parenchymal cells .................... 25 2.2.3 Type I collagen isolation .................................................................................. 26 2.2.4 Polyelectrolyte multilayer (PEM) assembly .................................................... 26 2.2.5 Liver model assembly ...................................................................................... 27 2.2.6 Hepatocyte isolation and processing of lysates ............................................... 27 2.2.7 PEM isolation and processing .......................................................................... 29 2.2.8 LC-MS analysis ............................................................................................... 29 2.2.9 Data analysis .................................................................................................... 30 2.2.10 Ketone body measurements ........................................................................... 31 2.2.11 Glucose measurements ................................................................................... 32 2.2.12 Statistical analyses ......................................................................................... 33 2.3 Results ..................................................................................................................... 33 2.3.1 Overview and liver model functions ................................................................ 33 2.3.2 PEM-containing liver constructs and hepatocyte monolayers have different proteomic signatures favoring β-oxidation and ketogenesis ..................................... 35 2.3.3 HMs contain higher levels of several glycolytic and gluconeogenic enzymes 40 2.3.4 Structural and trafficking proteins in HMs are indicative of dedifferentiation 41 vi 2.3.5 3DHL liver models exhibit higher levels of proteins associated with drug metabolism ................................................................................................................ 42 2.3.6 Proteomic analysis of the PEMs show limited matrix remodeling .................. 44 2.4 Discussion ..............................................................................................................
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