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Assessment of Heat Shock Proteins in Human Myometrial Cells Under Pro-Inflammatory Conditions and Their Presence in Extracellular Vesicles

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Assessment of Heat Shock Proteins in Human Myometrial Cells Under Pro-Inflammatory Conditions and Their Presence in Extracellular Vesicles Assessment of Heat Shock Proteins in Human Myometrial Cells under Pro-Inflammatory Conditions and their Presence in Extracellular Vesicles A Thesis Submitted to the College of Graduate and Postdoctoral Studies For the Degree of Master of Science Department of Veterinary Biomedical Sciences University of Saskatchewan By GEORGIA CAMERON BAILEY © Georgia Bailey, 2020. All rights reserved. Unless otherwise noted, copyright of the material in this thesis belongs to the author Permission to Use This thesis is being presented to fulfill the requirements to obtain a Postgraduate Master’s degree from the University of Saskatchewan. The University Library has my permission to make this thesis accessible for review. Furthermore, I give my consent for allowing copying of this document for academic or scholarly purposes granted by the professor who supervised this work, or if absent, the Head of the Department of Veterinary Biomedical Sciences or the Dean of the College of Veterinary Medicine (see below). I understand that using this thesis, or parts of, for financial gain shall not be allowed without written consent from myself and that recognition will be given to myself and to the University of Saskatchewan if used for any scholarly publication. Head of the Department of Veterinary Biomedical Sciences Western College of Veterinary Medicine, 52 Campus Dr University of Saskatchewan Saskatoon, Saskatchewan, S7N 5B4, Canada OR Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan, S7N 5C9, Canada i Abstract During pregnancy the myometrium undergoes a program of differentiation in parallel with immune system transformation. The end result is the production of a powerfully contractile tissue able to deliver a term fetus. Small heat shock proteins (sHSPs) are a family of 10 proteins (HSPB1-10) involved in regulating stress responses in tissues, including inflammation, and may take part in intercellular communication via extracellular vesicles (EVs). Since HSPB1 and HSPB5 are highly expressed in myometrium during late pregnancy and labour, it was hypothesized that HSPB1 and HSPB5 expression would be induced in myometrial cells by a pro- inflammatory stimulus, in correlation with the Nuclear Factor kappa B (NF-kB) signaling pathway. Based on the literature, it was also hypothesized that these proteins would be released extracellularly in EVs. Immortalized human myometrial cells (hTERT-HM) were incubated with 1 ng/ml of IL-1β or vehicle for 0.5, 1, 3, or 6 hours (h). The expression of sHSPs and markers of inflammation in these cells were then assessed by immunoblot analysis. Detection of pSer82- HSPB1 and pSer59-HSPB5 proteins was significantly elevated in cells at 0.5 h of cytokine incubation compared to vehicle control. Activation of NF-kB, marked by phosphorylation on serine-536, was also significantly elevated after 0.5 h. In contrast, inhibitor of kappa B (IkB) expression was significantly decreased after 1 h of cytokine incubation. Thus, pSer82-HSPB1 and pSer59-HSPB5 levels correlate with NF-kB activation. Analysis of cell conditioned culture media in this experimental model also demonstrated myometrial cell secretion of EVs. Scanning electron microscopy and transmission electron microscopy verified EVs as round vesicles of 20 to 200 nm in diameter regardless of whether or not cells were exposed to IL-1β. Immunoblot analysis demonstrated EVs contained the markers cluster of differentiation 63 (CD63), apoptosis linked gene 2-interacting protein X (ALIX), and tumor susceptibility gene 101 (TSG101). ii Furthermore, HSP90, HSP70, HSPB1, and NF-kB were all detected in EVs as cargo for extracellular release. These results suggest a novel mechanism of myometrial cell intercellular communication during pregnancy that could help modulate the pro-inflammatory response in the myometrium at labour. iii Acknowledgements I would like to express deep gratitude to Dr. Daniel MacPhee for providing me with the opportunity to learn under his guidance and participate in the research projects conducted in his lab. I thank him for sharing his in-depth academic knowledge, for being patient while teaching me valuable laboratory skills, and always being available to provide me when help when needed. I would also like to express my thanks to all other members of the MacPhee lab, specifically Dr. Ewa Miskiewicz and Mikayla Waller. Thank you, Dr. Ewa Miskiewicz, for teaching me various laboratory techniques in an incredibly patient and positive environment. Thank you, Mikayla Waller, for brain-storming ideas with me, helping me with experiments, and making the lab a great place to work every day. I would also like to thank Dr. MacPhee's previous students, especially Logan Hahn, for his previous pro-inflammatory work with hTERT-HM cells, providing me with valuable insight into how to conduct and analyze my experiments. Thank you to Dr. Andres Possa- Terranova and John Ching for being incredible teaching assistant supervisors, and to all the students I had the opportunity to teach and mentor. Finally, I would like to thank my parents for endlessly supporting me through this journey. I want to give thanks to the organization supporting this research, NSERC (Natural Sciences and Engineering Research Council), as this work would not have been possible without their grant. iv Table of Contents Permission to Use ............................................................................................................................ i Abstract ......................................................................................................................................... ii Acknowledgements ....................................................................................................................... iv List of Tables ............................................................................................................................... vii List of Figures ............................................................................................................................ viii List of Abbreviations .................................................................................................................... ix 1. Literature Review ...................................................................................................................... 1 1.1 The Significance of Pre-Term Birth .................................................................................................. 1 1.1.1 The Social and Economic Significance of PTB ........................................................................ 2 1.1.2. Challenges in Rural Settings ..................................................................................................... 4 1.2 Pregnancy, Parturition, and PTB ...................................................................................................... 5 1.2.1 Myometrial Differentiation and Adaptations ............................................................................. 7 1.2.2 Immune Activation During Pregnancy .................................................................................... 16 1.3.3 Role of Nuclear Factor Kappa B .............................................................................................. 21 1.4 Small Heat Shock Proteins .............................................................................................................. 26 1.4.1 Expression of HSPB1 and HSPB5 in the Myometrium .......................................................... 32 1.5 Extracellular Vesicles ....................................................................................................................... 34 1.6 Overall Rationale .............................................................................................................................. 40 1.7 Hypotheses ........................................................................................................................................ 41 1.8 Objectives .......................................................................................................................................... 41 2. Materials and Methods ........................................................................................................... 43 2.1 Cell Culture ...................................................................................................................................... 43 2.2 Interleukin-1β Treatment ................................................................................................................. 43 2.2.1 Concentration Titration Experiments ...................................................................................... 44 2.2.2 Time Course Experiments ........................................................................................................ 45 2.3 MTS Viability Assays ....................................................................................................................... 45 2.4 Cytokine Analysis ............................................................................................................................. 45 2.5 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ................................ 46 2.6 Immunoblot Analysis ....................................................................................................................... 47 2.7 Extracellular Vesicles ......................................................................................................................
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