UNDERSTANDING THE EFFECTS OF CYCLIC STRAIN ON ANGIOGENESIS by Jacob Ceccarelli A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biomedical Engineering) in the University of Michigan 2013 Doctoral Committee: Associate Professor Andrew James Putnam, Chair Professor Joseph L. Bull Assistant Professor Darnell Kaigler Associate Professor Jan Phillip Stegemann © Jacob Ceccarelli 2013 Acknowledgements First and foremost I would like to thank my advisor, Professor Andrew Putnam, for accepting me into the lab amid stiff competition and guiding me through six years of toil to reach this point. His enthusiasm for science and patience during my setbacks were instrumental in keeping me motivated and productive. I would also like to thank him for taking me to Michigan, which I viewed as an adventure and opportunity. I would also like to thank the other members of my dissertation committee, Professors Jan Stegemann, Darnell Kaigler Jr., and Joseph Bull. Their collaborative efforts and insights have helped to improve as a scientist since the establishment of the committee. Several funding sources were critical to the success of my project. NIH R01-HL085339 and R01HL-085339-S1, the University of Michigan first year BME fellowship, Rackham Graduate School travel and research awards, and two 1-year graduate student fellowships from UC Irvine allowed me to focus solely on research throughout graduate school. Next I would like to thank those who contributed to my scientific ii success: my colleagues in the lab and collaborators in others. In Irvine, Peter Kim welcomed me into the lab and taught me the basics, which I have used throughout my dissertation work. Suraj Kachgal was an excellent source of information and discussion with respect to molecular biology techniques, and most notably his insights contributed my proficiency with Western blotting. In addition, Suraj, Bita Carrion, and Isaac Janson moved with me from Irvine and made the transition to new state and school very smooth. I would also like to thank Albert Cheng, an undergraduate student who I worked closely with, for his hard work on my project. Of special note are Ram Rao, Stephanie Grainger, Marina Vigen, Yan Peng Kong, David Lai, and Rahul Singh, who allowed me to collaborate with them on their respective projects, solved key problems by providing useful data or techniques, or both. I am extremely grateful to them for including me in their work and helping me with mine. The friends I made in Ann Arbor were extremely important for my mental health over the years, and I am eternally grateful for their company. I am glad to have been able to distract myself with them when I needed a break, train and compete with excellent teammates, and celebrate milestones in our lives and careers. No one has been more supportive during my time here than my iii parents, Gary and Laurie Ceccarelli. Without their discipline and guidance I would not have been able to accomplish anything. They have generously given me countless gifts, plane tickets home, visits, and updates about family and friends that have made me feel like I have a place to come back to after I finished here. Finally, I would like to thank my patient and understanding girlfriend and best friend, Ita Nagy, who has endured having a country between us for four years. iv Table of Contents Acknowledgements ..................................................................................... ii List of Figures ........................................................................................... viii List of Appendices ...................................................................................... x List of Abbreviations................................................................................... xi Abstract .................................................................................................... xii Chapter 1. Introduction ......................................................................................... 1 1.1 Tissue Engineering.......................................................................... 1 1.2 Angiogenesis and Vascularization of Engineered Tissues ............... 3 1.3 Control of Vascular Patterning via Mechanotransduction ................ 7 1.4 Hypothesis .................................................................................... 11 1.5 Specific Aims ................................................................................. 11 1.6 Translational Potential ................................................................... 12 1.7 Overview ....................................................................................... 13 1.8 References .................................................................................... 14 2. Literature Review .............................................................................. 21 v 2.1 Introduction ................................................................................... 21 2.2 Structure and Degradation of Native Fibrin .................................... 22 2.3 Using fibrin to study angiogenesis ................................................. 28 2.4 Protease-mediated migration and angiogenesis in fibrin ............... 32 2.5 Studying mechanotransduction with fibrin ..................................... 35 2.6 Clinical Applications of Fibrin ......................................................... 40 2.7 Re-engineering fibrin for specific applications ............................... 43 2.8 Conclusion .................................................................................... 47 2.9 References .................................................................................... 49 3. Using Cyclic Strain to Pattern Angiogenesis ..................................... 60 3.1 Introduction ................................................................................... 60 3.2 Methods ........................................................................................ 62 3.3 Results .......................................................................................... 71 3.4 Discussion ..................................................................................... 78 3.5 References .................................................................................... 83 4. Endothelial Response to Cyclic Strain is Unhindered by Modulation of Cell Traction Force ........................................................................... 87 4.1 Introduction ................................................................................... 87 4.2 Methods ........................................................................................ 89 4.3 Results .......................................................................................... 95 vi 4.4 Discussion ................................................................................... 100 4.5 References .................................................................................. 106 5. Conclusions and Future Directions ................................................. 111 5.1 Contributions of This Dissertation ................................................ 111 5.2 Expanded Summary .................................................................... 112 5.2 Future Directions ......................................................................... 116 5.4 References .................................................................................. 123 Appendices ..................................................................................... 124 vii List of Figures Figure 1-1 The number of people waiting for transplants exceeds the number of available organs. ........................................................................ 2 Figure 2-1. Schematic of fibrinogen and its assembly into fibrin. ............... 24 Figure 2-1. Fibrin hydrogels are useful as 3D model systems of angiogenic sprouting. .................................................................................................. 29 Figure 2-1. Engineering fibrin for controlled growth factor presentation. ... 44 Figure 3-1. Devices used to house and apply strain to hydrogels. ............ 63 Figure 3-2. Relationship between applied strain and strain within the fibrin gel. ............................................................................................................ 68 Figure 3-1. Effects of cyclic strain on angiogenesis. .................................. 72 Figure 3-2. Effects of a change in strain regimen on angiogenesis. .......... 74 Figure 3-3. Confocal reflectance images of the fibrin matrix at days 2-5 of the angiogenesis assay. ........................................................................... 75 Figure 3-4. The effects of strain on ECM architecture. .............................. 76 Figure 4-1. RhoA construct expression is modulated by doxycycline concentration. ........................................................................................... 96 viii Figure 4-2. Presence of RhoA mutants affects network length but not directional sprouting at day 4. ................................................................... 97 Figure 4-3. Presence of RhoA mutants affects network length but not directional sprouting at day 8. ................................................................... 99 Figure 4-4. Modulation of cell traction force affects both network length and sprout angle. ..........................................................................................