Id3-Medicated B Cell Trafficking to the Aorta and VCAM-1 Expression Modulate Inflammation and the Progression of Atherosclerosis

Lipinski, Michael Joseph, Department of Physiology, University of Virginia
McNamara, Coleen, Department of Physiology, University of Virginia
Owens, Gary, Department of Molecular Physiology and Biological Physics, University of Virginia
Leitinger, Norbert, Department of Pharmacology, University of Virginia
Kelly, Kimberly, Department of Biomedical Engineering, University of Virginia
Glover, David, Department of Medicine

Atherosclerosis is a major cause of morbidity and mortality in Western society. While we have made great strides in reducing adverse cardiovascular events, improved understanding of atherosclerosis may lead to further reductions in adverse outcomes. Thus, investigating the role of the immune system in the early progression of atherosclerosis may help in the development of novel therapeutics for atherosclerotic disease. Inhibitor of differentiation-3 (Id3) is a helix-loop-helix transcription factor found to play a role in atherosclerosis. To assess potential mechanisms associated with the loss of Id3 function, the development of atherosclerosis in the Id3 -/- Apoe -/- mice was evaluated. Loss of Id3 resulted in the accelerated initiation and progression of atherosclerosis, an effect attenuated by transplantation of Id3 +/+ bone marrow. FACS analysis of marrow-derived cells identified an Id3- dependent, aortic specific reduction in B lymphocytes. Adoptive transfer of Id3 +/+ but not Id3 -/- B cells into a B cell-deficient mouse model ameliorated the development and progression of atherosclerotic plaque. Molecular imaging techniques demonstrated that Id3 +/+ B cells traffic to the aorta and preferentially to regions of atherosclerosis or regions prone to the development of atherosclerosis while Id3 -/- B cells had reduced trafficking to the aorta. Adoptive transfer of B lymphocytes also led to a reduction in macrophage accumulation in atherosclerotic plaques of B-cell deficient mice. ii Loss of Id3 was also shown to lead to increased atherosclerosis in a Ldlr - /- murine model of atherosclerosis. Loss of Id3 in Ldlr -/- mice resulted in increased intimal macrophage content and a reduction in the aortic B cell content. The loss of Id3 increased intimal expression of VCAM-1. Further molecular studies demonstrated that Id3 regulates VCAM-1 transcription and expression through interaction with E12. These data demonstrate that Id3 plays a key role in regulating B cellmediated atheroprotection and that Id3 regulates inflammation and VCAM-1 expression in mouse models of atherosclerosis. VCAM-1 is known to play an important role in leukocyte recruitment to atherosclerotic plaque. Thus, the increase in VCAM-1 expression seen with loss of Id3 may help explain the increased recruitment of macrophages in atherosclerotic plaque. Interestingly, loss of Id3 also decreases B cell trafficking to the aorta which our data suggests may have a role in macrophage recruitment as well. Thus, Id3 plays a critical role in modulating inflammation and the immune system in atherosclerosis. These data suggest that modulation of Id3 activity may serve as a novel therapeutic target for the treatment and prevention of early atherosclerosis. iii Dedication and Acknowledgements With the completion of my thesis, I conclude one of the most challenging periods of my life. The last four years have represented an enormous period of growth in both my personal life and academic career. An integral part of this growth must be attributed to my mentor Dr Coleen McNamara. She has enabled me to pursue research to further explore a greater understanding behind the mechanisms of atherosclerosis, providing an excellent balance of independent research while guiding and refining my research. However, I truly believe it is her devotion to family and work/life balance that sets her apart as a mentor. For these life lessons, I will be forever grateful. While I have learned a great deal from many individuals, it is the friendships that I have developed that are the most rewarding. Jim Garmey is a loyal and selfless friend who always has time to help and talk. His generosity and incredible morals are inspiring and remind me to continually strive to be a better person. Amanda Doran has been inspiring. Not only is she a great friend but will likely remain a powerhouse in the field. I respect her ability to overcome adversity and to achieve an incredible scientific standard. Hamid Deliri has been a steadfast friend through my time in the lab. Jen Kirby and I have a special relationship forged through both clinical and lab experience. She is like the older sister I never had and has taken to the role perfectly by both watching out for me and mocking me when necessary. The friendships of Stephanie Oldham, Alexis Cutchins, and Marcus Skaflen have also been extremely valuable to me and my family. Finally, I have enjoyed working with Kirsti Campbell. She has an iv amazing work ethic and I feel fortunate to have had the chance to work with her. I think she will go on to incredible things with whatever she decides to do and hope I helped provide a positive educational experience. In addition, I would like to thank the rest of my thesis committee: Dr Gary Owens, Dr Norbert Leitinger, Dr Kim Kelly, and Dr David Glover. It is refreshing to work with individuals so willing to help out and collaborate. I also appreciate their encouragement to continue pursuing research during the challenging times. Finally, I wish to thank my family for all of the support they have given during this challenging time. My parents and family in California have always encouraged me to pursue my academic passions and have made my education a possibility. The Sinesky's have also been amazing in-laws and their devotion to family is inspiring. Finally, I wish to thank my lovely wife Shawn and beautiful daughters Sophia and Josephine. Without their encouragement, I do not believe I would have completed this thesis. The simple logic of my daughter Sophia truly cuts to the heart of my passion for research and medicine, "Daddy is a doctor. He helps sick people". I dedicate this thesis, all that I do, and all of my love to my amazing family.

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PHD (Doctor of Philosophy)
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