Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Epigenetic modifications in vascular disease and regenerative medicine Mihaela Culmes aus Racari, Rumänien 2012 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn PD Dr. Jaroslav Pelisek betreut und von Herrn Prof. Dr. Ernst Wagner von der Fakultät für Chemie und Pharmazie vertreten. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, 22.09.2012 . ......................... Mihaela Culmes Dissertation eingereicht am 22.09.2012 1. Gutachter: Prof. Dr. Ernst Wagner 2. Gutachter: PD Dr. Jaroslav Pelisek Mündliche Prüfung am 05.12.2012 From far away to us you came to improve your ken and knowledge it was your dream and inner aim to graduate and end the college foreign country, different manner the start wasn’t easy, not at all but still faithful under your banner you always followed your own call persistent and beyond the doubt you went on with lifted head in hope of fortune looking out high spirits around did you spread scientist with heart and soul all the time you have been challenging the others goal to see more beyond the scene always open, ready to aid to explain, improve the others never mind the price you paid as you’d help your own brothers So I esteemed to work with you and helped you to find your fate ‘cause you’re the one to have a clue. And now here you stand to pass the gate dedicated by J.P. I TABLE OF CONTENTS 1 INTRODUCTION 1 1.1 Epigenetics ................................................................................................................................ 1 1.1.1 DNA methylation and DNMTs ................................................................................................. 1 1.1.2 Hydroxymethylation, TET1 ...................................................................................................... 3 1.1.3 Histone modifications – methylation and acetylation .............................................................. 4 1.1.4 Histone methyltransferases and their role ............................................................................... 5 1.2 Epigenetic changes in vascular disease ................................................................................ 9 1.2.1 Atherosclerosis is the leading cause of stroke and heart attack ............................................. 9 1.2.2 Stages of atherosclerosis – histological classification ........................................................... 10 1.2.3 Role of epigenetics in vascular disease ................................................................................ 14 1.2.4 Aim of the study – epigenetic changes in vascular disease .................................................. 16 1.3 Epigenetic changes in regenerative medicine ..................................................................... 18 1.3.1 Reprogramming of mesenchymal stem cells (MSCs) through epigenetics .......................... 19 1.3.2 Epigenetic modifying drugs and cell reprogramming ............................................................ 20 1.3.2.1 5-azacytidine, inhibitor of global DNA methylation ........................................................ 21 1.3.2.2 BIX-01294, a small molecule able to inhibit the G9a methyltransferase ....................... 22 1.3.2.3 Valproic acid as a histone deacethylase inhibitor ......................................................... 24 1.3.3 Adipose tissue more than a fat storage ................................................................................. 24 1.3.4 Cellular components of adipose tissue and relevant CD markers expression pattern ......... 25 1.3.5 Source of mesenchymal stem cells and their differentiation potential .................................. 27 1.3.6 Regenerative medicine and vascular disease ....................................................................... 27 1.3.7 Biological grafts ..................................................................................................................... 28 1.3.8 Aim of the study - epigenetics in regenerative medicine ....................................................... 29 2 MATERIALS AND METHODS 31 2.1 Cell culture experiments ......................................................................................................... 31 2.1.1 Isolation of primary adipose derived mesenchymal stem cells (adMSCs) ............................ 31 2.1.2 Cell culture ............................................................................................................................. 33 2.1.2.1 Culture of adipose-derived mesenchymal stem cells .................................................... 33 2.1.2.2 Osteogenic differentiation .............................................................................................. 33 2.1.2.3 Adipogenic differentiation .............................................................................................. 33 2.1.2.4 Endothelial differentiation .............................................................................................. 34 2.1.2.5 Cell line used as positive control ................................................................................... 34 2.1.2.6 Cell propagation ............................................................................................................ 34 2.1.2.7 Cell counting .................................................................................................................. 35 TABLE OF CONTENTS II 2.1.3 Cell culture analysis ............................................................................................................... 36 2.1.3.1 Cytochemistry ................................................................................................................ 36 2.1.3.1.1 Von Kossa staining ................................................................................................... 36 2.1.3.1.2 Alkaline phosphatase staining .................................................................................. 37 2.1.3.1.3 Oil red O staining ...................................................................................................... 38 2.1.3.2 Assays ........................................................................................................................... 38 2.1.3.2.1 Viability assay ........................................................................................................... 38 2.1.3.2.2 Alkaline phosphatase activity assay ......................................................................... 39 2.1.3.2.3 Oil Red O quantification assay ................................................................................. 40 2.1.3.2.4 Sulphorodamine B – protein measurement .............................................................. 40 2.1.3.2.5 Ac-LDL uptake .......................................................................................................... 41 2.2 Immunohistochemistry ........................................................................................................... 41 2.2.1 LSAB method ........................................................................................................................ 41 2.2.2 APAAP method ...................................................................................................................... 42 2.3 Flow cytometry (FACS analysis) ............................................................................................ 43 2.4 Tissue sampling, processing, and analysis ......................................................................... 44 2.4.1 Study group, atherosclerotic plaque processing, and characterization ................................. 44 2.4.2 Decellularisation and recellularization ................................................................................... 45 2.4.3 Histochemistry ....................................................................................................................... 46 2.4.3.1 Haematoxylin – Eosin staining ...................................................................................... 46 2.4.3.2 Elastica van Giesson staining ....................................................................................... 46 2.4.4 Immunohistochemistry........................................................................................................... 47 2.4.5 Microscopy and digitalization ................................................................................................ 47 2.5 Gene expression analysis at mRNA level using PCR.......................................................... 48 2.5.1 RNA extraction from cells ...................................................................................................... 48 2.5.2 RNA extraction from FFPE tissue samples ........................................................................... 48 2.5.3 cDNA synthesis ..................................................................................................................... 48 2.5.4 SYBR Green-based real-time PCR ....................................................................................... 49 2.6 Epigenetic analysis of methylated DNA using PCR ............................................................ 52 2.6.1 DNA isolation from cells .......................................................................................................