A Dissertation entitled Alpha7 Nicotinic Acetylcholine Receptor: Novel Role in Macrophage Survival and Murine Atherosclerosis by Robert H. Lee Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences _________________________________________ Guillermo Vazquez, PhD, Committee Chair _________________________________________ David Giovannucci, PhD, Committee Member _________________________________________ Joseph Margiotta, PhD, Committee Member _________________________________________ Sandrine Pierre, PhD, Committee Member _________________________________________ R. Mark Wooten, PhD, Committee Member _________________________________________ Patricia R. Komuniecki, PhD, Dean College of Graduate Studies The University of Toledo December 2014 Copyright 2014, Robert Hugh Lee This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Alpha7 Nicotinic Acetylcholine Receptor: Novel Role in Macrophage Survival and Murine Atherosclerosis by Robert H. Lee Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences The University of Toledo December 2014 Atherosclerosis is a chronic inflammatory disease, characterized by infiltration and accumulation of leukocytes within the vascular wall. Macrophages play a particularly crucial role in all stages of atherosclerotic lesion development. These cells react to their local environment and can alter their function accordingly, resulting in polarization of macrophages to divergent phenotypes. Notably, apoptosis of lesional macrophages undergoing chronic endoplasmic reticulum (ER) stress coupled with impaired clearance of dying cells promotes formation of necrotic cores, a preamble for plaque rupture and thromboembolic events and clinical manifestation as myocardial infarction or stroke. The alpha7 nicotinic acetylcholine receptor (α7nAChR) is recognized as an important regulator of macrophage function in inflammation, and activation of this receptor in other cell types can protect against apoptosis. We hypothesized that activation of α7nAChR could protect macrophages against iii apoptosis, and therefore play an anti-atherogenic role in protecting against apoptosis and necrosis. We investigated the potential role of α7nAChR in macrophage survival in bone marrow derived macrophages (BMDMs) polarized to the M1 or M2 phenotype, and found that α7nAChR stimulation preferentially protected M2 BMDMs from ER stress-induced apoptosis. We then investigated the impact of macrophage α7nAChR deficiency on lesion development by utilizing a bone marrow transplantation model using low density lipoprotein receptor knockout (LDLR-/-) mice. Bone marrow deficiency of α7nAChR had no effect on early plaques, but at the advanced stage α7nAChR deficiency resulted in reduced lesion area and macrophage content, and this was accompanied by reduction lesional cell proliferation. These studies represent novel findings in the role of α7nAChR in macrophage survival in vitro and in the development of murine atherosclerotic lesions, and set a foundation for future studies to further investigate the therapeutic potential of macrophage α7nAChR in the progression of atherosclerosis. iv This dissertation is dedicated to my Grandma and Grandpa Lee, and my late Grandma and Grandpa Caverly. You have given me so much love and support and I am lucky to have grown up with grandparents who were so involved in my life. I love you all very much. Acknowledgements I first want to thank my mentor, Dr. Guillermo Vazquez, for giving me the opportunity to earn my Ph.D. under his guidance. I have learned so much more than I ever expected to beyond just the basics of doing scientific research, and I want to thank you for your support, your trust in me, and your tolerance of my excessive use of commas in scientific writing. I hope our scientific and personal relationship continues for a long time to come. To my parents, thank you for your help in the hardest times and in the little moments too. You helped me to become the honest, hardworking and reasonable person I am today, love you both. To Dr. Rande Worth, thank you for your friendship and your sincere interest in my success, I always appreciate your honesty and good advice, and carrying our team when I shoot a bad round. To my current and past labmates and all my other “science friends”, thank you for your friendship and best of luck with everything ahead of you. And to my siblings, cousins, aunts, uncles and all the rest of my family, I am so blessed to have grown up in a fun, loving and close-knit family and I appreciate all the love and support over the years. v Table of Contents Abstract............................................................................................................................. iii Acknowledgements ......................................................................................................... v Table of Contents ............................................................................................................ vi List of Figures .................................................................................................................. ix List of Abbreviations ..................................................................................................... xii List of Symbols .............................................................................................................. xiv 1 Introduction ..........................................................................................................1 1.1 Atherosclerosis: disease overview ...............................................................1 1.2 Early atherosclerosis: role of the monocyte/macrophage……………. ..3 1.3 Advanced atherosclerosis and macrophage apoptosis…………… ........8 1.4 Macrophage differentiation and polarization…………………………..13 1.5 α7 nicotinic acetylcholine receptor……………………………… ............17 1.6 Summary and hypothesis ...........................................................................20 2 Materials and Methods .....................................................................................22 2.1 Experimental Animals .................................................................................22 2.2 Preparation of bone marrow derived macrophages ...............................22 vi 2.3 Macrophage polarization into M1 and M2 phenotypes .........................23 2.4 In vitro TUNEL assay ...................................................................................24 2.5 Cell lysis and immunoblotting ...................................................................24 2.6 Real Time PCR (RT-PCR) ............................................................................25 2.7 Fura-2-based real time fluorescence ..........................................................26 2.8 Bone marrow transplantation (BMT) ........................................................27 2.9 Determination of plasma cholesterol and triglycerides .........................27 2.10 Aortic root sectioning ................................................................................28 2.11 Immunohistochemistry .............................................................................28 2.12 In situ immunostaining for M1 and M2 macrophages ..........................30 2.13 In situ TUNEL ..............................................................................................30 2.14 Necrotic core evaluation and cap thickness ...........................................31 2.15 Statistical analysis ......................................................................................32 3 Results ........................................................................................................33 3.1 α7nAChR is expressed in polarized bone marrow derived macrophages ........................................................................................................33 3.2 α7nAChR mediated Ca2+ influx is undetectable in BMDMs .................38 3.3 α7nAChR stimulation promotes activation of survival signaling pathways ........................................................................................................39 3.4 α7nAChR stimulation reduces ER stress-induced apoptosis in M2 BMDMs ........................................................................................................48 3.5 α7nAChR stimulation is associated with upregulation of Bcl-2 ...........52 vii 3.6 Role of macrophage α7nAChR in development of murine atherosclerosis .....................................................................................................55 3.7 Impact of bone marrow deficiency of α7nAChR on early atherosclerotic lesions ........................................................................................56 3.8 Impact of bone marrow deficiency of α7nAChR on advanced atherosclerotic lesions ........................................................................................61 4 Discussion and Conclusions ............................................................................72 References ........................................................................................................................85 A Publications and select presentations ............................................................105 viii List of Figures 3-1 Expression of polarization