The University of Maine DigitalCommons@UMaine Honors College 5-2012 The Haptotactic Motility of Angiogenic Endothelial Cells Breana E. Bennett University of Maine, [email protected] Follow this and additional works at: https://digitalcommons.library.umaine.edu/honors Part of the Biochemistry Commons Recommended Citation Bennett, Breana E., "The Haptotactic Motility of Angiogenic Endothelial Cells" (2012). Honors College. 39. https://digitalcommons.library.umaine.edu/honors/39 This Honors Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Honors College by an authorized administrator of DigitalCommons@UMaine. For more information, please contact [email protected]. THE HAPTOTACTIC MOTILITY OF ANGIOGENIC ENDOTHELIAL CELLS by Breana E. Bennett A Thesis Submitted in Partial Fulfillment of the Requirements for a Degree with Honors (Biochemistry) The Honors College University of Maine May 2012 Advisory Committee: Sharon L. Ashworth, Assistant Research Professor of Cell Biology, Co-advisor Alireza Sarvestani, Assistant Professor, Mechanical Engineering, Co-advisor Mark E. Haggerty, Rezendes Preceptor for Civic Engagement, Honors College Dorothy E. Croall, PhD., Professor of Biochemistry Robert Gundersen, Chair and Associate Professor of Molecular and Biomedical Sciences ABSTRACT Angiogenesis, an essential but often pathological biological process, requires complex cellular coordination. This coordination is achieved through numerous extracellular signals. We investigated the role of the extracellular matrix (ECM) protein fibronectin in the support of angiogenesis and endothelial cell motility. The directed migration in response to increasing concentration of ECM proteins is termed haptotaxis. We created a preliminary, two-dimensional model of the haptotactic motility of endothelial cells using a step change in fibronectin density as a substrate. Bovine aortic endothelial cells (BAECs) exhibited directional motility towards the higher fibronectin concentration side. In addition, the physical and mechanical properties of BAECs varied in response to fibronectin concentration. Higher fibronectin concentrations led to decreased cell motility and increased adherence. Our studies also examined the properties of the actin cytoskeleton and the focal adhesion scaffold protein paxillin during this haptotactic migration. These studies and the two-dimensional model will serve as a baseline for future analysis of the haptotactic motility of angiogenic endothelial cells. ACKNOWLEDGEMENTS This work could not have been completed without funding from the NCRR Maine IDeA Network of Biomedical Research Excellence (INBRE) (grant number NIH P20- RR016463), which funded my summer research fellowship through the Mount Desert Island Biological Laboratory (MDIBL). My research throughout the fall and spring semesters was funded in part by the INBRE senior thesis fellowship award sponsored by the Honors College, supported by grants from the National Center for Research Resources (5P20RR016463-12) and the National Institute of General Medical Sciences (8 P20 GM103423-12) from the National Institutes of Health. The Institute of Molecular Biophysics provided additional funding. I would like to acknowledge the support and scientific guidance provided to me by my co-advisors, Sharon Ashworth and Ali Sarvestani. Your unwavering faith in my abilities has certainly helped to make this all possible. I also extend my gratitude to my fellow lab members in both the Sarvestani and Ashworth Labs, especially Alexa Mitchell for ‘babysitting’ some of my longer time-lapse videos and Morgan Gilman for her general guidance and being the cell culture guru. I would also like to thank the remaining members of my Honors Thesis Committee, Dorothy Croall, Bob Gundersen and Mark Haggerty for their genuine interest in and support of this project. My family, specifically my parents, likewise deserves thanks. Not only have you encouraged me throughout my entire life, but you were reasonably understanding when I was in the lab rather than visiting home. iii TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................ vi CHAPTER 1: INTRODUCTION ................................................................................... 1 1.1 Blood Vessels – The Heart of Angiogenesis ........................................................... 3 1.2 Angiogenesis and the Formation of New Blood Vessels ......................................... 4 1.3 Angiogenesis and its Role in Cancer ...................................................................... 5 1.4 Cell Migration........................................................................................................ 6 1.5 Actin Treadmilling is Required for Cell Migration ................................................. 6 1.6 Small GTPases Regulate Cell Motility ................................................................... 7 1.7 Directional Endothelial Cell Migration ................................................................... 8 1.7.1 Chemotactic Motility ................................................................................... 8 1.7.2 Haptotactic Motility .................................................................................... 9 1.8 Focal Adhesions and the Scaffold Protein Paxillin ............................................... 10 1.9 Proposed Model of the Role of Haptotaxis in Angiogenesis ................................. 11 CHAPTER 2: MATERIALS AND METHODS ............................................................ 12 2.1 Cell Culture ......................................................................................................... 12 2.2 Transfection of BAECs ........................................................................................ 13 2.3 Fibronectin Step-Gradient Preparation ................................................................. 13 2.4 Time-lapse Video Microscopy of BAECs ............................................................ 15 2.5 Cell Staining and Visualization ............................................................................ 16 2.6 Analysis of Confocal Images................................................................................ 17 CHAPTER 3: RESULTS .............................................................................................. 17 3.1 BAECs Were Successfully Transfected With Actin-pEGFP and Paxillin-pEGFP . 17 iv 3.2 Fibronectin Steps Were Successfully Generated on Glass-Bottomed Dishes ......... 18 3.3 BAECs Exhibited Directional Motility in Response to Step Change in Fibronectin Concentration ...................................................................................................... 19 3.4 BAECs Demonstrated Varying Motilities on High and Low Fibronectin Concentrations..................................................................................................... 20 3.5 Actin and Paxillin Localization and Morphology Vary Based on Fibronectin Concentration ...................................................................................................... 21 CHAPTER 4: DISCUSSION ........................................................................................ 22 4.1 Generated Fibronectin Steps Contain a Distinct Change in Fibronectin Concentration ...................................................................................................... 22 4.2 Differential Localization and Morphology of Actin and Paxillin in BAECs Based on Fibronectin Concentration............................................................................... 23 4.3 The Haptotactic Motility of BAECs ..................................................................... 24 4.4 Physical Properties of BAECs Vary Based on Fibronectin Concentration ............. 26 4.5 Concluding Remarks ............................................................................................ 28 REFERENCES .............................................................................................................. 31 FIGURES ...................................................................................................................... 34 BIOGRAPHY OF THE AUTHOR ................................................................................ 47 v LIST OF FIGURES Figure 1. Schematic of blood vessel structure Figure 2. Overview of small GTPase signaling involved in cell migration Figure 3. 1% agarose gel of plasmid DNA used for BAEC transfections Figure 4. BAECs transfected with actin-pEGFP and paxillin-pEGFP Figure 5. Control paxillin-EGFP expressing cell fixed and stained on glass Figure 6. Fibronectin step generated on glass-bottomed MatTek dishes Figure 7. Time course frames taken from cells seeded on a fibronectin step Figure 8. BAEC motility in response to fibronectin step Figure 9. Average net distance gain of BAECs located near and far from the border between high and low fibronectin concentrations Figure 10. Average distance traveled over 15 minute time period of BAECs located near and far from the border between high and low fibronectin concentrations Figure 11. Average area of migration for BAECs located on the high and low fibronectin concentration sides of the step Figure 12. Average cell size of BAECs located on the high and low fibronectin concentration sides of the step Figure 13. Actin localization varied based on fibronectin concentration