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THE USE OF A TISSUE ENGINEERED MEDIA EQUIVALENT IN THE STUDY OF A NOVEL SMOOTH MUSCLE CELL PHENOTYPE A Dissertation Presented to The Academic Faculty by JoSette Leigh Briggs Broiles In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Bioengineering Georgia Institute of Technology April 2008 COPYRIGHT 2008 BY JOSETTE LEIGH BRIGGS BROILES THE USE OF A TISSUE ENGINEERED MEDIA EQUIVALENT IN THE STUDY OF A NOVEL SMOOTH MUSCLE CELL PHENOTYPE Approved by: Dr. Robert M. Nerem, Advisor Dr. Thomas N. Wight School of Mechanical Engineering Hope Heart Program Georgia Institute of Technology Benaroya Research Institute at Virginia Mason Department of Pathology University of Washington Dr. Raymond P. Vito Dr. Elliot Chaikof School of Mechanical Engineering Department of Biomedical Engineering Georgia Institute of Technology Georgia Institute of Technology and Emory University Dr. W. Robert Taylor Department of Biomedical Engineering Georgia Institute of Technology and Emory University Date Approved: December 18, 2007 to Yvette Louise Briggs, the perfect example of a wife-mother-student ACKNOWLEDGEMENTS Before I proceed with my long list of thank-you’s, I must praise God for blessing me with the opportunity to pursue a Ph.D. and surrounding me with wonderful people that have encouraged me throughout this process. My tenure at Georgia Tech has been quite a challenging voyage. This was the first time I experienced repeated failures, was not at the top of the class, and not in complete control of my fortune. I’ve often said that in high school I learned how to navigate social pressures and in undergrad I discovered the meaning of true friendship. But, it has been my graduate school experience that has taught me the most about myself. In the last 6.5 years, I’ve married my best friend and confidante, given birth to two beautiful boys, mourned the loss of nine loved ones, learned how to fail without being a failure, and became inspired to reach and teach others. These moments of joy and tests of faith have humbled me beyond explanation, mellowed my Type A personality, and put my life in proper perspective. OK. Now that I’ve finished my testimony, I will begin my expressions of gratitude. I would like to thank Dr. Robert Nerem for welcoming me into his research lab and to his bottomless well of wisdom, advice, and optimism. I would also like to thank Dr. Thomas Wight for readily providing his expertise and encouragement. Thank you to the members of my committee, Drs. Elliot Chaikof, W. Robert Taylor, and Raymond Vito, for your research direction. I would also like to acknowledge my sources of funding: the National Science Foundation, The National Consortium for Graduate Degrees for Minorities in Engineering and Science, Inc. (GEM), Facilitating Academic - iv - - iv - Careers in Engineering and Science (FACES) Fellowship, George Family Foundation, and the Georgia Tech/Emory Center for the Engineering of Living Tissues. To the Nerem lab members, past and present, thank you for the hundreds of hours of stimulating as well as mindless conversation. Special thanks to Steve Woodard, Dr. Tabassum Ahsan, and Stacey Schutte for the brainstorming sessions and discussions about life philosophies. I would be remiss if I did not acknowledge the hardworking IBB/GTEC staff. I must thank Tracey Couse, Aqua Asberry, Jonafel Crowe, Lisa Cox, Kathy Huggins, Chris Ruffin, and Gloria at the Petit Café’ for your assistance with histology, training on core equipment, the last minute acquisition of signatures, student advisement, administrative assistance, and the hot cups of chicken noodle soup! If it weren’t for my network of Georgia Tech friends, I probably would have made good on one of my many threats to quit school. To Dr. Lori Lowder, Dr. Onyi Irrechukwu, Ima Ebong, Dr. Manu Platt, Drs. Brian and Annica Wayman, and Lola Brown, I say a resounding THANK YOU for your friendship and patient ears. Thanks for providing motivation when I felt incompetent after a botched experiment. Thanks for the distractions when I really should have been working. HA! I’d also like to thank Dr. Gilda Barabino, Dr. Andrew Williams, and Dr. Felicia Benton-Johnson for your mentorship. Your visibility as African American PhDs has inspired me and many others. To my church family and Delta sorors, thanks for providing a balance in my life. Special thanks go to Cindy (Snow) Walker, childcare extraordinaire. You are truly gifted when it comes to working with children. I want to thank your for giving me the peace of mind knowing that Winston was in capable, loving hands while I kept long hours at school. I must thank my sisters Michelle Omari, Crystal Johnson, and v Brandy Nolan. Even though I don’t keep in touch like I should, I know that you “have my back” and will be available whenever I need you. Thanks for being such great and understanding friends. To my extended family, thank you for your prayers and phone calls. Thank you for the road trips to Atlanta and hot plates of mashed potatoes and macaroni-and-cheese awaiting my return home. Thanks for bragging on me even if you don’t know what I do exactly. Finally, I conclude by thanking the most important people in my life. To my dad and brother, Joseph and Jarret Briggs, thanks for your prayers, support and the semiannual marathon phone calls to convince me that all of this is worth it. To my sons, Winston and Solomon, you bring me immense joy and you are my motivation to do better and be better. Damon, you have always been my biggest cheerleader. From the day I received my acceptance letter to those long days and nights I slaved at my laptop writing this dissertation, you have been my rock! I love you more than words could convey. To my mother, Yvette Louise Briggs, even though you are absent in body, I continue to carry your spirit with me. Everyday I strive to emulate you by excelling in my roles as a Christian wife, mother, and servant. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iv LIST OF FIGURES ix LIST OF ABBREVIATIONS xii SUMMARY xv CHAPTER 1 INTRODUCTION 1 Motivation 1 Hypothesis and Specific Aims 3 Significance 4 2 BACKGROUND 5 Arterial Physiology 5 Smooth Muscle Cells 8 Medial Extracellular Matrix 9 Tissue Engineered Blood Vessels 19 3 EXTRACELLULAR MATRIX ARCHITECTURE AND COMPOSITION INFLUENCE TROPOELASTIN EXPRESSION IN SMOOTH MUSCLE CELLS THAT OVEREXPRESS VERSICAN V3 26 Introduction 26 Materials and Methods 28 Results 39 Discussion 64 vii 4 TROPOELASTIN SYNTHESIS BY SMOOTH MUSCLE CELLS THAT OVEREXPRESS V3 VERSICAN IS SENSITIVE TO STIMULATION BY MEDIUM ADDITIVES 68 Introduction 68 Materials and Methods 69 Results 76 Discussion 100 5 MECHANICAL STIMULATION INCREASES TROPOELASTIN SYNTHESIS BY VERSICAN V3 OVEREXPRESSING SMOOTH MUSCLE CELLS IN TISSUE ENGINEERED MEDIA EQUIVALENTS 104 Introduction 104 Materials and Methods 105 Results 115 Discussion 127 6 CONCLUSION AND RECOMMENDATIONS 132 APPENDIX A: SELECTED PROTOCOLS 139 Preparation of Type I Collagen Media Equivalents 140 Preparation of Fibrin Media Equivalents 141 Mechanical Stimulation of Tissue Engineered Media Equivalents 142 Intracellular Staining for Flow Cytometry 144 RNA Isolation from Media Equivalents 146 cDNA Synthesis for Quantitative RT-PCR 148 REFERENCES 150 VITA viii LIST OF FIGURES Page Figure 2.1: Anatomy of the artery 6 Figure 2.2: Forces exerted within the blood vessel 7 Figure 2.3: Elastic fiber conformation 11 Figure 2.4: Elastin binding protein chaperones tropoelastin to the extracellular space 13 Figure 2.5: Tropoelastin incorporation into elastic fibers 14 Figure 2.6: Structure of versican isoforms 17 Figure 3.1: Schematic of TEME fabrication 31 Figure 3.2: Light microscopy images of LXSN and LV3SN cells 40 Figure 3.3: Confocal imaging of intracellular tropoelastin. 41 Figure 3.4: Disk shaped collagen and fibrin TEMEs have distinct appearances 43 Figure 3.5: Compaction of collagen TEMEs 44 Figure 3.6: Compaction of fibrin TEMEs 44 Figure 3.7: Modified Movat’s Pentachrome staining of collagen TEMEs 45 Figure 3.8: Orcein staining of collagen TEMEs 46 Figure 3.9: Immunohistochemical staining for tropoelastin in collagen TEMEs 47 Figure 3.10: Versican and tropoelastin gene expression varies with matrix structure and composition after one day in culture 50 Figure 3.11: Versican and tropoelastin gene expression varies with matrix structure and composition after seven days in culture 51 Figure 3.12: LXSN and LV3SN cells contain equal amounts of DNA per cell 52 Figure 3.13: DNA content in collagen TEMEs 54 Figure 3.14: DNA content in fibrin TEMEs 54 Figure 3.15: Changes in LXSN DNA content relative to matrix type 55 ix Figure 3.16: Changes in LV3SN DNA content relative to matrix type. 55 Figure 3.17: Western analysis of tropoelastin protein expression in whole lysates of cell monolayers, collagen TEMEs and fibrin TEMEs 58 Figure 3.18: Western analysis of tropoelastin secreted into the spent media of monolayer, collagen TEMEs, and fibrin TEMEs cultures 58 Figure 3.19: Flow cytometry assessment of versican and tropoelastin in monolayers 60 Figure 3.20: Flow cytometry assessment of versican and tropoelastin in collagen TEMEs 61 Figure 3.21: Flow cytometry assessment of versican and tropoelastin in fibrin TEMEs 62 Figure 4.1: Experimental timeline for stimulation with medium additives 71 Figure 4.2: Light microscopy of monolayers after one day of chondroitin sulfate and chondroitinase stimulation 77 Figure 4.3: Light microscopy of monolayers after one day of TGF-β1 stimulation. 78 Figure 4.4: DNA content in TEMEs after one day of exposure to medium additives 80 Figure 4.5: DNA content in TEMEs after seven days of exposure to medium additives 80 Figure 4.6: Versican gene expression in monolayers after one day of exposure to medium additives 82 Figure 4.7: Tropoelastin gene expression in monolayers after one day of exposure to medium additives 83 Figure 4.8: Confocal microscopy of intracellular tropoelastin in LV3SN monolayers treated with medium additives.