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Shear Stress Modulates Gene Expression in Normal Human Dermal Fibroblasts

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Shear Stress Modulates Gene Expression in Normal Human Dermal Fibroblasts University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2017 Shear Stress Modulates Gene Expression in Normal Human Dermal Fibroblasts Zabinyakov, Nikita Zabinyakov, N. (2017). Shear Stress Modulates Gene Expression in Normal Human Dermal Fibroblasts (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/27775 http://hdl.handle.net/11023/3639 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Shear Stress Modulates Gene Expression in Normal Human Dermal Fibroblasts by Nikita Zabinyakov A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOMEDICAL ENGINEERING CALGARY, ALBERTA JANUARY 2017 © Nikita Zabinyakov 2017 Abstract Applied mechanical forces, such as those resulting from fluid flow, trigger cells to change their functional behavior or phenotype. However, there is little known about how fluid flow affects fibroblasts. The hypothesis of this thesis is that dermal fibroblasts undergo significant changes of expression of differentiation genes after exposure to fluid flow (or shear stress). To test the hypothesis, human dermal fibroblasts were exposed to laminar steady fluid flow for 20 and 40 hours and RNA was collected for microarray analysis. Gene expression data was processed using gene network analysis, pathway analysis, and gene functional analysis with comparison to data from publicly available data sets. Additional treatment with PI3K/mTOR pathway inhibitor, PI- 103, was performed to evaluate pathway involvement in flow modulation of gene expression. Results from overall transcription analysis demonstrated that fluid flow modulated many genes in fibroblasts including those related to differentiation, development and TGF-β pathway regulation. ii Acknowledgments I would like to express my respect and gratitude for my supervisor Dr. Kristina D. Rinker for her advice, suggestions, guidance and encouragement throughout my research. I would also like to thank the members of my thesis committee, Dr. Roman Krawetz, and Dr. Derrick Rancourt, for their great input and feedback during our meetings. Special thanks to Dr. Deborah Studer for being always a remarkably patient and understanding mentor and for her time spent helping me with the research design and methodology. I am thankful to Dr. Robert Shepherd for the training and guiding me through time- and labor-consuming lab techniques. Moreover, I want to recognize program director, Dr. Michael Kallos, and program coordinator, Lisa Mayer for their help in receiving administrative and financial support. Finally, to my friends and colleagues from Cellular and Molecular Bioengineering Research Lab, Maria Juliana Gomez, Christopher Sarsons, Linda Tamez, Hagar Labouta, and Kenneth Fuh for backing me up along the way. iii Table of contents Abstract ........................................................................................................................................ iii Acknowledgements ...................................................................................................................... iv List of Tables .............................................................................................................................. vii List of Figures ............................................................................................................................ viii List of Symbols, Abbreviation, Nomenclature ............................................................................. x Chapter 1. Introduction Hypothesis ..................................................................................................................................... 2 Thesis Objective............................................................................................................................. 3 Thesis Overview ............................................................................................................................ 3 Chapter 2. Background and literature review 2.1. Production and expansion of cell lines through de-, re-, and trans-differentiation ........... 5 2.2. Fibroblasts possess a significant plasticity to de-differentiate ......................................... 10 2.3. Dermal fibroblast niche and origin ................................................................................... 13 2.4. Mechanotransduction in cells. .......................................................................................... 16 2.5. Fibroblast response to low rate interstitial flow and wound healing ................................ 21 2.6. Exposure to fluid flow influences pluripotency and differentiation ................................. 22 2.7. Chapter highlights ............................................................................................................ 23 Chapter 3. Methods and materials 3.1. Cell culture ....................................................................................................................... 24 3.2. Flow experiment ............................................................................................................... 26 3.3. Sample preparation for RNA extraction. .......................................................................... 29 3.4. RNA quantification and reverse transcription .................................................................. 30 3.5. Real-time PCR and 2-ΔΔСт analysis ................................................................................... 31 3.6. Microarray and data analysis ............................................................................................ 32 3.7. Live/dead and nuclear/cytoskeleton staining ................................................................... 33 3.8. Comparison with existing microarray data from GEO repository ................................... 34 3.9. Statistical analysis ............................................................................................................ 36 Chapter 4. Results 4.1. NHDF survival and morphology under shear stress ........................................................ 37 4.2. Gene expression changes under the flow iv 4.2.1. Microarray data analysis ........................................................................................... 40 4.2.2. GEO microarray data sets comparison 4.2.2.1. Flow effect (NHDF) vs effect of TGFβ1 treatment (gingival FBs) .................. 47 4.2.2.2. Flow effect (NHDF) vs effect of TGFβ1 treatment (ovarian FBs) ................... 49 4.2.2.3. Flow effect (NHDF) vs lung FB-derived iPSCs gene expression profile ......... 50 4.2.2.4. Flow effect (NHDF) vs natural killer cell gene expression profile .................. 51 4.3. PI-103 inhibitor treatment effect on gene expression 4.3.1. Genes affected by PI-103 only .................................................................................. 52 4.3.2. Genes affected by PI-103 under static conditions ..................................................... 53 4.3.3. Inhibitor overrides flow effect on gene expression................................................... 54 4.3.4. Inhibitor impairs flow effect on gene expression ..................................................... 55 4.3.5. Inhibitor enhances flow effect on gene expression ................................................... 56 Chapter 5. Discussion and summary 5.1. Adaptation response of NHDF to shear stress .................................................................. 58 5.2. Shear stress does not modulate the expression of epithelial and mesenchymal markers but downregulates fibroblastic ones ....................................................................................... 59 5.3. Shear stress positively modulates SOX9 and RUNX2 expression in NHDF ................... 61 5.4. Fluid flow alleviates the expression of genes related to anti-fibrotic, anti-inflammatory, anti-apoptotic, anti-proliferative and anti-migratory effect in NHDF ............................. 62 5.5. Fluid flow modulates expression of genes, which are conserved during differentiation . 64 5.6. Upregulation of OCT4 (POU5F1), as a reciprocal regulation of AKT, may not indicate pluripotency after co-exposure to flow and PI-103 ......................................................... 66 5.7. Limitations ........................................................................................................................ 67 5.8. Future work ...................................................................................................................... 68 References ................................................................................................................................... 70 Appendix ..................................................................................................................................... 79 v List of Tables Table 1. Fluid flow simulation models for fibroblast study. Table 2. Sequences of designed primers for genes of interest. Table 3. Secondary microarray data used from
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