SMOOTH MUSCLE CELL CHARACTERIZATION AND TRANSCRIPTOMIC ANALYSIS IN HUMAN CAROTID ATHEROSCLEROTIC PLAQUES HAIZE GOICURIA 2018 UNIVERSIDAD DEL PAÍS VASCO (UPV / EHU) Departamento de Neurociencias SMOOTH MUSCLE CELL CHARACTERIZATION AND TRANSCRIPTOMIC ANALYSIS IN HUMAN CAROTID ATHEROSCLEROTIC PLAQUES TESIS DOCTORAL Haize Goicuria Directores: Dra. Iraide Alloza y Dr. Koen Vandenbroeck Leioa, 2018 (c)2018 HAIZE GOICURIA IRIONDO AKNOWLEDGEMENTS I would like to thank all those people who have participated so much and who have made this thesis possible. First, thanks to the directors of this thesis Koen Vandenbreock and Iraide Alloza, for their direction, support and enthusiasm during these years of thesis in the laboratory of Neurogenomiks. Also thanks to the University of the Basque Country (UPV / EHU) as well as ACHUCARRO center which have facilitated the development of this thesis. I would also like to thank all the members of the laboratory for their support during all these years. In one way, to the colleagues of the past as Aitzkoa, Pepe and Ianire and, in another way, to those of the present as Nerea, Jorge, Cristina and Andoni, and especially to Paloma, my laboratory partner, for her unconditional help and because the fun moments with her helped me to get here. I am immensely grateful to my parents and relatives for their support and especially to my mother for her motivation, empathy and patience during these years, her understanding has been very important to me. I would like also give thanks to my friends Bea and Ana for their understanding during my PhD and to Alberto, for be my breath and bring me a piece of happiness to my life every day. And finally I wish to thank patients of this study for their sample donation without whom this study would be impossible. TABLE OF CONTENTS LIST OF PUBLICATIONS ABBREVIATIONS 1. INTRODUCTION ............................................................................................................... 1 1.1. ATHEROSCLEROSIS DISEASE ................................................................................ 1 1.1.1. Epidemiology ........................................................................................................ 1 1.1.2. Carotid atherosclerosis .......................................................................................... 2 1.1.3. Biology of atherosclerotic plaque .......................................................................... 3 1.1.4. Unstable vs Stable plaque – Symptomatic vs. Asymptomatic patients ................. 9 1.1.5. Risk factors .......................................................................................................... 11 1.1.6. Biomarkers of carotid atherosclerotic plaque rupture ......................................... 15 1.2. VASCULAR SMOOTH MUSCLE CELLS ............................................................... 23 1.2.1. SMC phenotypic switching ................................................................................. 24 1.2.2. SMCs in carotid atherosclerosis .......................................................................... 26 1.2.3. SMC-specific markers ......................................................................................... 28 2. OBJECTIVES ................................................................................................................... 31 3. SUBJECTS & METHODS ............................................................................................... 32 3.1. SMC CHARACTERIZATION ................................................................................... 32 3.1.1. Patient selection ................................................................................................... 32 3.1.2. Isolation of human vascular smooth muscle cells ............................................... 33 3.1.3. Flow cytometry ................................................................................................... 34 3.1.4. Gene expression analysis .................................................................................... 34 3.1.5. Western blot ........................................................................................................ 36 3.1.6. Confocal microscopy ........................................................................................... 37 3.2. RNA-seq TECHNOLOGY ......................................................................................... 37 3.2.1. Patient selection for SMCs isolation ................................................................... 37 3.2.2. RNA library assembly ......................................................................................... 38 3.2.3. RNA transcriptomics analysis ............................................................................. 38 3.2.4. Gene Set functional enrichment analysis and network analysis .......................... 39 3.2.5. Digital PCR on Fluidigm Biomark platform ....................................................... 39 4. RESULTS ........................................................................................................................... 42 4.1. SMC ANALYSIS ........................................................................................................ 42 4.1.1. Selection of SMC culture method ....................................................................... 42 4.1.2. Purity control ....................................................................................................... 43 4.1.3. SMC characterization .......................................................................................... 47 4.2. RNA-SEQ STUDY DESIGN ...................................................................................... 54 4.2.1. Primary analysis .................................................................................................. 59 4.2.2. Secondary analysis .............................................................................................. 70 4.2.3. Tertiary analysis .................................................................................................. 72 4.3. TRANSCRIPTOME SEQUENCING ANALYSIS .................................................... 75 4.3.1. DEG and DEI analysis ........................................................................................ 75 4.3.2. Functional enrichment and network analysis ...................................................... 82 4.3.3. Effect of cell culture and passaging on gene expression ..................................... 96 4.3.4. Confirmation of RNAseq identified biomarkers by digital PCR ........................ 97 4.3.5. Inflammatory pathways in carotid atheroma SMCs ............................................ 99 4.3.6. Cytokines and chemokines expression based on patient symptomatology ....... 102 5. DISCUSSION .................................................................................................................. 104 5.1. ATHEROMA PLAQUE SMC CHARACTERIZATION ......................................... 104 5.2. SMC TRANSCRIPTOMIC ANALYSIS .................................................................. 106 5.3. CYTOKINE PATHWAYS UNEARTHED BY RNASEQ IN CAROTID ATHEROMA SMCs ............................................................................................................. 109 6. CONCLUSION ................................................................................................................ 114 7. REFERENCES ................................................................................................................ 114 LIST OF PUBLICATIONS Goikuria H., Freijo Guerrero M. del M., Vega R., Sastre M., Elizagary E., Lorenzo A., Vandenbroeck K., Alloza I., Characterization of Carotid Smooth Muscle Cells during Phenotypic Transition, Cells, 7 (2018) 23. doi: 10.3390/cells7030023. Goikuria H., Vandenbroeck K., Alloza I., Inflammation in human carotid atheroma plaques, Cytokine Growth Factor Rev. 39 (2018) 62-70. doi: 10.1016/j.cytogfr.2018.01.006 Alloza Moral I., Goikuria H., Idro J.L., Triviño J.C., Férnandez Velasco J.M., Elizagaray E., García Barcina M., Montoya G., Sarasola E., Vega R., Freijo Guerrero M. del M., Vandenbroeck K., RNAseq based transcriptomics study of SMCs from carotid atherosclerotic plaque: BMP2 and IDs proteins are crucial regulators of plaque stability, Sci. Rep. 7 (2017) 3470. doi: 10.1038/s41598-017-03687-9. Alloza Moral I., Goikuria H., Freijo Guerrero M. del M., Vandenbroeck K., A role for autophagy in carotid atherosclerosis, European stroke journal 1 (2016) 255 - 263. doi: 10.1177/2396987316674085. Swaminathan B., Goikuria H., Rodríguez-Antigüedad A., López Medina A., Freijo Guerrero M. del M., Vandenbroeck K., Alloza I, Autophagic marker MAP1LC3B expression levels are associated with carotid atherosclerosis symptomatology, Plos One (2014) 12 - 9. doi: 10.1371/journal.pone.0115176. ABBREVIATIONS SMC smooth muscle cells EC endothelial cells CV cardiovascular disease ROS reactive oxygen species ECM extracellular matrix ICAM-1 intracellular adhesion molecule 1 VCAM-1 vascular cell adhesion molecule 1 MCP-1 monocyte chemoattractant protein 1 CCL2 C-C motif chemokine ligand 2 LDL low density lipoprotein oxLDL oxidized low density lipoprotein IFN-γ interferon-gamma TNF-α tumor necrosis factor-alpha HDL high density lipoprotein PDGF platelet-derived growth factor EGF endothelial growth factor NFκβ nuclear factor kappa beta IL interleukin G-CSF granulocyte colony-stimulating factor M-CSF macrophage colony-stimulating factor GM-CSF granulocyte-macrophage