RECONSTRUCTING THE VASCULAR REGENERATIVE NICHE WITH OXYGEN-CONTROLLABLE HYDROGELS By Michael R. Blatchley A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland July 2019 © 2019 Michael R. Blatchley All Rights Reserved 1 Abstract A thorough understanding of the molecular and microenvironmental regulators of blood vessel formation is key to designing new therapeutic strategies to treat two of the leading causes of death worldwide, cardiovascular disease and cancer. Two key parameters of the vascular regenerative niche that play broad and potent roles in endothelial cell (EC) morphogenesis are matrix stiffness and hypoxia (defined as <5% O2). To recapitulate these cues, we designed hydrogels comprised of gelatin and dextran backbones to study the effects of O2 and matrix stiffness on ECs. For both of these hydrogel backbones, we conjugated phenolic moieties, which can be enzymatically crosslinked in an O2-consuming reaction to form a hypoxic hydrogel. By altering the concentration of each polymer component and adding or omitting a secondary crosslinker, we generated a set of hydrogels that spanned both a range of physiological stiffnesses and hypoxic conditions. We studied the mechanism of cluster-based vasculogenesis that had previously been reported in vivo, but never studied in vitro because of the limitations with existing cell culture platforms. Using our O2-controllable hydrogels, we observed and quantified the kinetics of cluster formation, and determined key regulators of the process. EC exposure to hypoxia led to upregulation of reactive oxygen species and proteases that facilitated rapid matrix degradation and cluster formation. ECs sprouted from clusters via cell-matrix interactions to form expansive vascular networks, which could be further enhanced through secondary crosslinking to increase cell-matrix interactions. In vivo studies in a mouse model confirmed the proposed mechanism. Finally, in depth analysis of specific signaling involved in cluster formation included genes associated with cell cycle, cell survival, and metabolism. This work has illuminated key regulators of a complex mechanism for vasculogenesis, and represents a powerful in vitro tool to study cells in an environment that closely matches the niche in which they reside in vivo. Thesis advisor: Dr. Sharon Gerecht Thesis committee members: Dr. Warren Grayson and Dr. Akrit Sodhi ii Acknowledgements I would first like to thank my thesis advisor, Dr. Sharon Gerecht, for providing me with exemplary mentorship. From the first meeting I had with her to discuss a rotation in the lab, she listened to my interests and helped guide me to learn the skills necessary to become a successful PhD student, as well as set myself up well for the next stages of my career. She believed in me as a scientist and writer from the early stages of my time in the lab, and has been invaluable in helping me refine and expand my skillset both in the lab and in disseminating my work. There were high points and low points during my time as a PhD student, and it was great to know I would always have Dr. Gerecht’s support to work through the difficult times and celebrate the successes. I would also like to thank my thesis committee members, Dr. Warren Grayson and Dr. Akrit Sodhi, for helping guide and refine my lines of inquiry. They have challenged me to think critically about my work, have asked thoughtful questions, and have suggested a number of important experiments. Our discussions have also helped me refine the best ways to present my work, and have helped me improve how I design an experimental plan. Letters of recommendation are a crucial component in attaining fellowships, awards, and future jobs. I have applied for many fellowships and awards over the last six years and am incredibly thankful for all those professors that have written letters for me since applying for graduate school: Dr. Kinam Park, Dr. Clint Chapple, Dr. Irina Petrache, Dr. Jennifer Elisseeff, Dr. Sharon Gerecht, Dr. Hai-Quan Mao, Dr. Honggang Cui, Dr. Jason Burdick, and Dr. Michael Miller. I also want to thank the past and current members of the Gerecht lab. We are a wonderfully diverse and ceaselessly entertaining and wonderful collection of young scientists. Our conversations in the office range from incredibly productive discussions of science and experimental design, to venting about reviewers, to talking about new restaurants and movies. It is great to have a group of people so willing talk science as well as life outside of science. iii When I first joined the lab, I learned from many lab members, but Dr. Kyung Min Park served as my primary mentor. Kyung was a great mentor to me and served as a role model through his work ethic and his commitment to always better himself as a scientist, communicator, and husband and father. I have had many productive scientific discussions with members of the lab and am specifically thankful for fruitful discussions with Dr. Xin Yi Chan, Dr. Tom Shen, Dr. Quinton Smith, Dr. Hawley Pruitt, Dr. Zhao Wei, Dr. Dan Lewis, Matt Davenport, Bria Macklin, Justin Lowenthal, Morgan Elliott, John Jamieson, Eugenia Volkova, and Franklyn Hall. The members of the lab have become good friends of mine and I have really enjoyed getting to know everyone better through game nights and happy hours. It has also been wonderful to have a competitive outlet throughout my time in graduate school, so I am thankful for the many opportunities to play soccer, basketball, football, and softball with friends from the lab, the BME and ChemBE programs, and outside of Hopkins in various city leagues. I have had the opportunity to mentor a number of students: Brooke Smith, Songnan Wang, Franklyn Hall, Arianne Papa, and Vidur Kailash, over the last six years and am thankful for the opportunity to improve myself as a mentor and very thankful for their patience as I improved my mentorship skills. The INBT and BME administration have been instrumental to my success in graduate school. Hong Lan has been a wonderful resource in the BME department, and I am grateful to have had the opportunity to work closely with her as president of the PhD council. Conversations with Hong never fail to be entertaining and she is an incredibly positive presence in the department and Hopkins community. I am also thankful to the INBT staff, including Kierra Suggs, Ellie Boettinger-Heasley, Gregg Nass, Carla Dodd, Christine Duke, Jon French, Camille Mathis, Lateai Jones, Ada Simari, Sky Tharp, Luke Thorstenson, and Gina Wadas. I will always be grateful to have received such wonderful mentorship from my time as an undergraduate until now. Dr. Kinam Park, John Garner, Dr. Clint Chapple, Dr. Kelly Schweitzer, Dr. Irina Petrache, and Dr. Jennifer Elisseeff all provided amazing research opportunities and helped me develop new research skills each step of the way. iv I would be nowhere without the continued support of my family. I am extremely fortunate to have a family who has an empathetic view towards the rigors of a PhD program. My dad was my first research mentor, and I really want to thank him for giving me the opportunity to work in his lab in high school, and will be forever thankful for our discussions of science and how to best set myself up for career success on our long bike rides. I have learned the importance of a long bike ride, run, hike, or walk from my dad, and these times have been valuable in organizing my thoughts, planning a paper or figures, or simply clearing my head after a long and frustrating day. Finally, I want to give thanks to my wonderful wife, Kaylee. She has supported me through my time in graduate school and has encouraged me to take a break or two from working and email for a number of long weekend trips from Shenandoah to the Adirondacks. I am grateful for her patience and understanding through the busy grant and paper writing times, and for listening to my complaints. There have been times that I have been overwhelmed with work, and she is always there to support me and encourage me. She always challenges me to become a better person, and I couldn’t ask for a better partner in life. Thank you! v Table of Contents ABSTRACT .................................................................................................................................................. ii ACKNOWLEDGEMENTS ......................................................................................................................iii-v LIST OF TABLES ...................................................................................................................................... xii LIST OF FIGURES ............................................................................................................................ xiii-xiv 1 Reconstructing the Vascular Developmental Milieu In Vitro .............................................................. 1 1.1 Teaser ...................................................................................................................................................... 1 1.2 Introduction ............................................................................................................................................. 1 1.3 Evolution of the vascular system ............................................................................................................ 2 1.4 Early vascular development – vasculogenesis .......................................................................................