The effect of Cold Atmospheric Plasma (CAP) on the molecular mechanism of wound healing I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Greifswald vorgelegt von Debarati Shome geboren am 15.03.1992 in Kolkata, India Greifswald, den 15.06.2020 I Dekan: Prof. Dr. Gerald Kerth 1. Gutachter: Prof. Dr. Katharina Riedel 2. Gutachter: Prof. Dr. Michael Lalk 3. Gutachter: Prof. Dr. Steffen Emmert Tag der Promotion: 02.11.2020 II Contents Contents Contents ............................................................................................................................... III Abbreviations...................................................................................................................... VII 1. Introduction ........................................................................................................................ 1 1.1 Human Skin............................................................................................................... 1 1.2 Wound Healing ......................................................................................................... 2 1.3 Acute Wounds ........................................................................................................... 3 1.4 Chronic Wounds ....................................................................................................... 5 1.5 Signal Transduction .................................................................................................. 7 1.6 HIPPO Signaling Pathway ........................................................................................ 7 1.7 Plasma ..................................................................................................................... 10 1.8 Plasma Medicine ..................................................................................................... 12 1.9 The atmospheric pressure argon plasma jet kINPen MED ..................................... 14 1.10 Wound healing is subject to redox control.............................................................. 16 1.11 Aim of the work ....................................................................................................... 17 2. Materials ........................................................................................................................... 19 Table 2.1 Chemicals used in this study ............................................................................ 19 Table 2. 2 Laboratory equipment ..................................................................................... 20 Table 2. 3 Laboratory instrument ..................................................................................... 21 Table 2.4 Kits/Antibodies ................................................................................................ 22 Table 2.5 Software ........................................................................................................... 23 3. Methods ............................................................................................................................. 25 3.1 Cell Culture ............................................................................................................. 25 3.2 Plasma Source ......................................................................................................... 25 3.3 Indirect Plasma treatment ....................................................................................... 26 3.4 Direct Plasma Treatment: ........................................................................................ 26 3.5 Coculture with keratinocytes & fibroblasts ............................................................ 27 3.6 Harvest of GM Fbs conditioned medium ................................................................ 27 3.7 Metabolic Activity assay ......................................................................................... 28 3.8 Intracellular ROS production with H2DCFDA assay .............................................. 28 III Contents 3.9 Amplex Red Assay for H2O2 measurement ............................................................ 28 3.10 Cell migration assay ................................................................................................ 28 3.11 Gene expression analysis ........................................................................................ 30 3.11.1 RNA isolation .............................................................................................. 30 3.11.2 cDNA transcription ...................................................................................... 31 3.11.3 Quantitative Polymerase Chain Reaction .................................................... 31 2 3.11.4 RT PCR Profiler Array ............................................................................... 33 3.12 Protein expression analysis ..................................................................................... 34 3.12.1 Immunoblotting ............................................................................................ 34 3.12.2 Enzyme linked immunosorbent assay (ELISA) ........................................... 37 3.13 Immunofluorescence ............................................................................................... 37 3.14 Statistics ................................................................................................................... 38 4. Results ............................................................................................................................... 39 4.1 Characterization of H2O2 production in CAP treated medium ............................... 39 4.2 Intracellular ROS production in HaCaT and GM Fbs ............................................ 39 4.3 Influence of CAP on metabolic activity in mono and coculture .............................. 40 4.3.1 Metabolic activity of mono and coculture after 3 and 24 hours of CAP treatment.................................................................................................................. 40 4.3.2 Influence of NAC on metabolic activity in mono and coculture ................... 41 4.4 Influence of CAP on cell migration in mono and coculture ................................... 43 4.4.1 Influence of indirect CAP treatment on cell migration .................................. 43 4.4.2 Influence of NAC on cell migration of HaCaT and coculture ....................... 50 4.4.3 Influence of direct CAP treatment on cell migration ..................................... 53 4.5 CAP activates HIPPO signaling axis ......................................................................... 55 4.5.1 CAP activates YAP-CTGF-Cyr61 signaling pathway ................................... 55 4.5.2 Effect of CAP on other signaling molecules in cross talk with YAP ............ 59 4.5.3 Effect of CAP on nuclear translocation of YAP ............................................ 61 4.5.3 Effect of NAC on YAP-CTGF-Cyr61 signaling cascade .............................. 64 4.5.4 CAP Promotes Migration in coculture through Secreted CTGF and Cyr61 .. 65 4.6 Influence of CAP on regenerative signaling molecules ............................................. 68 4.6.1 CAP activates HIPPO signaling molecules in GM Fbs ................................. 69 4.6.2 CAP activates Extracellular matrix (ECM) and adhesion molecules ............ 74 5. Discussion .......................................................................................................................... 81 5.1 Effect of CAP on cell migration and extracellular matrix molecules ........................ 81 5.2 NAC abrogates CAP mediated cell migration ........................................................... 84 5.3 Effect of CAP on metabolic activity .......................................................................... 85 IV Contents 5.4 Effect of CAP on HIPPO signaling pathway and paracrine signaling ....................... 87 5.4.1 CAP modulated activation of YAP-CTGF-Cyr61 ......................................... 87 5.4.2 Effect of CAP on paracrine signaling between keratinocytes and fibroblasts ................................................................................................................................. 89 6. Outlook .............................................................................................................................. 93 7. Summary ........................................................................................................................... 95 8. Zusammenfassung ............................................................................................................ 97 9. References ......................................................................................................................... 99 List of Tables ...................................................................................................................... 115 List of Figures ..................................................................................................................... 117 Supplementary Data .......................................................................................................... 125 Acknowledgements ............................................................................................................. 141 V Abbreviations ANOVA Analysis of Variances BMP Bone