University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Regulation Of The Human Vascular Smooth Muscle Cell Transcriptome By Extracellular Matrix Stiffness Christopher Yu University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, and the Genetics Commons Recommended Citation Yu, Christopher, "Regulation Of The Human Vascular Smooth Muscle Cell Transcriptome By Extracellular Matrix Stiffness" (2017). Publicly Accessible Penn Dissertations. 2710. https://repository.upenn.edu/edissertations/2710 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2710 For more information, please contact [email protected]. Regulation Of The Human Vascular Smooth Muscle Cell Transcriptome By Extracellular Matrix Stiffness Abstract Arterial stiffness is a risk factor for several cardiometabolic diseases and is caused by pathological remodeling of the vascular extracellular matrix (ECM). Vascular smooth muscle cells (VSMCs) respond to ECM stiffness by proliferating, migrating, and further remodeling the vascular ECM, thus contributing to vascular disease like atherosclerosis and hypertension. VSMCs along the vasculature are highly diverse as they arise from different embryologic origins, reside in ECMs of diverse compositions, and are exposed to various mechanical forces. This dissertation aims to understand how ECM stiffness regulates the transcriptional response of VSMCs from different origins, namely aortic (Ao) and coronary (Co) VSMCs. We conducted deep sequencing of RNA from Ao and Co VSMCs grown on engineered polyacrylamide hydrogel surfaces tuned to physiologic and pathologic stiffness. Using several bioinformatic approaches, we compared the transcriptional landscapes in Ao and Co VSMCs by looking at whole-gene level expression, splicing, and conservation properties, with a focus on long non-coding RNAs (lncRNAs), as they compose a significant portion of the unexplored VSMC transcriptome. We found evidence suggesting that the overall transcriptional response to stiffness may be conserved across species and cell types, and that stiffness significantly dictates VSMC transcriptional identity over contributions from embryologic origins. However, we also discovered instances of origin-specific stiffness esponsesr in stiffness-mediated lncRNA expression and stiffness-mediated splicing. We identified a highly correlated network of adjacent stiffness-sensitive lncRNAs-protein coding gene pairs, which led us to experimentally interrogate the lncRNA PACER as a regulator of stiffness-mediate expression. We surprisingly found that PACER’s previously established regulatory pathway is absent in VSMCs. Using enrichment methods, we identified TBX5 and show that it is a stiffness-sensitive transcription factor specific ot Co VSMCs. We also demonstrated a new role for MALAT1 as a lncRNA regulator of stiffness-dependent VSMC proliferation and migration. Thus, this dissertation reveals many novel characteristics of the VSMC stiffness-regulated transcriptome that may have clinical utility in understanding and managing the pathogenesis of arterial stiffening. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Bioengineering First Advisor Daniel J. Rader Second Advisor Richard K. Assoian Keywords Extracellular matrix stiffness, Long non-coding RNAs, Transcriptome, Vascular smooth muscle cells, Vascular stiffness Subject Categories Cell Biology | Genetics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2710 REGULATION OF THE HUMAN VASCULAR SMOOTH MUSCLE CELL TRANSCRIPTOME BY EXTRACELLULAR MATRIX STIFFNESS Christopher Yu A DISSERTATION in Bioengineering Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation Co-Supervisor of Dissertation ________________________ _________________________ Daniel J. Rader, M.D. Richard K. Assoian, Ph.D. Seymour Gray Professor of Molecular Medicine Professor of Pharmacology Graduate Group Chairperson _______________________ Ravi Radhakrishnan, Ph.D. Professor of Bioengineering; Professor of Chemical and Biomolecular Engineering Dissertation Committee: (Chair): Peter F. Davies, Ph.D., Sc.D., Professor of Pathology & Laboratory Medicine; Professor of Bioengineering Rebecca G. Wells, M.D., Professor of Medicine Christopher S. Chen, M.D., Ph.D., Professor of Biomedical Engineering (Boston University) REGULATION OF THE HUMAN VASCULAR SMOOTH MUSCLE CELL TRANSCRIPTOME BY EXTRACELLULAR MATRIX STIFFNESS COPYRIGHT 2017 Christopher Ku Yu This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/3.0/us/ Dedication I dedicate this thesis to my parents (Robin H. Yu, M.D., and Estrelita Ku, M.D.), who prioritized and spared no expense on the education and life opportunities for me and my brothers. This thesis and my pursuit of a combined MD/PhD degree would never have been possible without their life-long support. I also dedicate this thesis to my incredible wife, Sue Lee, who put her own Ph.D studies on hold to take care of her sick mom. She has the biggest heart for family. Without her constant support, I could not have completed my dissertation. En Taro Adun iii ACKNOWLEDGMENTS Early in my Ph.D, I assumed that my prior research experiences in high school and college would have made my Ph.D experience smooth and straightforward. But it became quickly apparent that I had a lot to learn. There’s a lot of people, programs, and organizations to thank along the way for teaching me and providing me scientific, financial, and social support. First, I have to thank all the mentors and teachers I had before starting my Ph.D. who gave me a foundation for critical thinking and the scientific method. Thanks to everyone in the Rader Lab, Assoian Lab, and Chen Lab. I was lucky to be immersed in three different scientific environments utilizing microfabrication/tissue engineering, cell and molecular biology, computation/next-generation sequencing and human genetics. From the Rader Lab: Deepti Abbey, Guha Arunkumar, Rob Bauer, Xin Bi, Linda Carmichael, Stephanie DerOhannessian, Susannah Elwyn, Nicholas Hand, Sumeet Khetarpal, Hyein Kim, Wen Lin, Nicholas Lyssenko, Dawn Marchadier, Minal Mehta, Sylvia Nuremberg, Amirth Rodrigues, Kevin Trindade, Cathy Warford. From the Assoian Lab: Nate Bade, Yong Ho Bae, Paola Castagnino, Beth Hawthorne, Shulin Liu, Keeley Mui, Ziba Razinia, Ryan Von Kleeck, and Tina Xu. From the Chen Lab: Colin Choi, Peter Galie, and Michael Yang. I’d also like to thank others from other labs and cores. The Reily Lab (when they were at Penn), especially, Rachel Ballantyne and Chenyi Xue. The CDB microscopy core and its director Andrea Stout. The ITMAT biomechanics core, especially Jeff Byfield. Thanks my committee members Peter Davies and Rebecca Wells for their never-ending support through both the ups and downs of a PhD Study. Keeping me on track during thesis committee meetings and being very accommodating to me. The NIH funding sources: HL119346 and T32HL007954-17 I would also like to acknowledge all my friends for all their support and for the shared fun times through all the years. All the people I started med school, the group collectively known as zergling rush, especially Mike Sheng, Ray Zhang, James Hui, and Nabil Thalji. My long-time friends from college, Yang Li and Sophie Su. To the friends I met along the Ph.D path, especially Rosa Maria Alvarez, Keeley Mui, Kristy Ou, and Sean Yeldell. All the members of Penn Lions, for keeping me physically active, culturally active, and feel young. Thanks to my family. My mom, my dad, and my brothers, Robinson and Michael. My dad got sick and passed away toward the end of my Ph.D, but our family stuck together to support each other through that difficult time. As this thesis is dedicated to my mom and dad, I literally could not have completed this dissertation without them. Lastly, thank you so so much to my loving wife, Sue Lee. She has given me unlimited support in every aspect of my life, not only during my Ph.D studies, but almost every day in the last 10 years of knowing each other. She will discuss with me ad nauseam any scientific and non-scientific issue that I cannot believe or understand. She has helped iv me with all my grammar issues, even in writing of this dissertation, and practiced presentations with me. She has always been there to keep me sane through all the strings of failed experiments and turbulent times in life. I love her very much and owe her my deepest gratitude. v ABSTRACT REGULATION OF THE HUMAN VASCULAR SMOOTH MUSCLE CELL TRANSCRIPTOME BY EXTRACELLULAR MATRIX STIFFNESS Christopher Yu Daniel J. Rader, M.D. Richard K. Assoian, Ph.D. Arterial stiffness is a risk factor for several cardiometabolic diseases and is caused by pathological remodeling of the vascular extracellular matrix (ECM). Vascular smooth muscle cells (VSMCs) respond to ECM stiffness by proliferating, migrating, and further remodeling the vascular ECM, thus contributing to vascular disease like atherosclerosis and hypertension. VSMCs along the vasculature are highly diverse as they arise from different embryologic origins, reside in ECMs of diverse compositions, and are exposed to various mechanical forces.