7-AAD 7-Aminoactinomycin D ANOVA Analysis of variance
Ambra1 Autophagy and beclin 1 regulator 1 AMPK 5′-AMP-activated protein Kinase ATG/APG Autophagy related protein ATPase Adenosine triphosphatase BrdU Bromodeoxyuridine
Cdk-2 Cyclin-dependent protein kinase cDNA Complementary deoxyribonucleic acid CKII Cdk inhibitor
CPT Camptothecin
COL1A1 Alpha-1 type I collagen COL3A1 Alpha-1 type 3 collagen CRM Caloric restriction mimetics CTGF Connective tissue growth factor CTS Cardiotonic steroids
CVD Cardiovascular diseases DAP1 Death associated protein 1 DEPC Diethyl pyrocarbonate
DGR8 DiGeorge syndrome chromosomal region 8 DMEM Dulbecco´s Modified Eagle Medium
DMSO Dimethyl sulfoxide DTT Dithiothreitol
EDTA Ethylene diamine tetraacetic acid ELISA Enzyme linked immunosorbent assay
Abbreviations 84
EP300 Acetyl transferase E1A-binding protein p300 FBS Fetal bovine serum
FBM Fibroblast basal medium
FIP200 Focal adhesion kinase family-interacting protein of 200kDa FOXO3a Forkhead-box-protein O3
GAPDH Glyceraldehyde-3-phosphate dehydrogenase HCFs Human cardiac fibroblasts
HEK293 Human embryonic kidney HF Heart failure
HFmrEF Heart failure with mid-range ejection fraction HFpEF Heart failure with preserved ejection fraction HFrEF Heart failure with reduced ejection fraction
HL-1 Mus musculus cardiac cell line derived from AT-1 mouse atrial cardiomyocyte tumor lineage
HRP Horseradish peroxidase hsp90 Heat shock protein 90 IL-6 Interleukin 6
JNK/SAPK c-Jun N-terminal kinase/stress-activated protein kinase LC3 Microtubule-associated protein 1A/1B-light chain 3 LNA Locked nucleic acid
LV Left ventricle
LVEF Left ventricular ejection fraction MAPK Mitogen-activated protein kinase MCP-1 Monocyte chemotactic protein-1 MMP Matrix metalloproteinase
miRNA Micro-RNA
miRnome miR transcriptome
mRNA Messenger RNA
mTORC Mechanistic target of rapamycin complex NBR-1 Neighbour of BRCA1 gene 1 protein NF-κB Nuclear factor-kappa B
noHF No heart failure
NOXA Phorbol-12-myristate-13-acetate-induced protein PCR Polymerase Chain Reaction
PBS Phosphate buffered saline RISC RNA-induced silencing complex
PI3K Phosphatidylinositol-4,5-bisphosphate 3-kinase PKA Protein kinase A
pre-miR Precursor-microRNA pri-miRNA Primary-microRNA
PVDF Polyvinylidene difluoride membrane RNA Ribonucleic acid
RNU48 Small-nucleolar RNA48 ROS Reactive oxygen species
RT Room temperature
αSMA Alpha smooth muscle actin SCGF-β Stem cell growth factor beta
Scr Scrambled
siRNA Small interfering RNA SIRT1 Sirtuine 1
SDS Sodium dodecyl sulfate
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SQSTM1 Sequestosome 1
Abbreviations 86
TBST Tris-buffered saline with Tween20 TGF-ß Transforming growth factor beta-ß ULK1 Unc-51-like kinase 1
UVRAG UV irradiation resistance-associated tumour suppressor gene VEGF Vascular endothelial growth factor
WIPI2 WD repeat domain phosphoinositide-interacting protein 2; ATG21 18S rRNA 18S ribosomal RNA
9 Register of illustrations
Tables
Table 3.1: Devices used for this work 17
Table 3.2: Material 18
Table 3.3: Chemicals 18
Table 3.4: Natural compounds for treatment of cells 19
Table 3.5: Provided kit systems 19
Table 3.6: Cell type used in this work 20
Table 3.7: Cell culture medium and components 20
Table 3.8: Fibroblast Growth Medium-3 (FGM-3) for HCFs 21
Table 3.9: Claycomb medium for HL-1 21
Table 3.10: DMEM medium for HEK-293 21
Table 3.11: Gel recipe for collection gel (SDS-PAGE) 21
Table 3.12: 10x SDS PAGE electrophoresis buffer 22
Table 3.13: 10x Western Blot transfer buffer 22
Table 3.14: 1x TBST Western blot washing buffer 22
Table 3.15: Luminol reagent for Western blot detection 22
Table 3.16: siRNA applied in this work 22
Table 3.17: mirVana miRNA mimic applied in this work 23
Table 3.18: TaqMan miRNA/gene detection assays applied in this work 23 Table 3.19: QuantiTect Primer assays applied in this work 23
Table 3.20: PCR Primers 24
Table 3.21: Primary antibodies applied in this work 24
Table 3.22: Secondary antibodies applied in this work 24
Table 3.23: Concentration of primary and secondary antibodies used for western blot 29
Figures
Figure 2.1: The versatile roles of cardiac fibroblasts.. ... 3
Figure 2.2: Correlation between proliferation/growth arrest and autophagy modulated by mTORC pathway.. ... 7
Figure 2.3: Six natural compounds inhibit cell proliferation of HCFs. ...10
Figure 2.4: Two known autophagy-influencing compounds inhibit cell proliferation of HCFs. 11 Figure 4.1: Torin 1 increases autophagic flux. ...35
Figure 4.2: Torin 1 presents cardiac fibroblast specific anti-proliferative impact. ...36
Figure 4.3: Torin 1 potently represses collagen 1 protein level. ...36
Figure 4.4: Quercetin increases autophagic flux. ...37
Figure 4.5: Quercetin does not induce apoptosis. ...38
Register of illustrations 88
Figure 4.6: Quercetin has no influence on expression of fibrotic genes. ...38
Figure 4.7: Quercetin potently decreases collagen 1 protein level. ...39
Figure 4.8: Spermidine reduces expression of fibrotic genes. ...40
Figure 4.9: Spermidine has slight anti-fibrotic effects on protein level. ...40
Figure 4.10: Spermidine does not induce apoptosis. ...41
Figure 4.11: Spermidine does not significantly induce autophagic flux. ...42
Figure 4.12: Blocking endogenous autophagy machinery causes indications of pro-fibrotic response. ...43
Figure 4.13: Investigated natural compounds do not induce apoptosis. ...45
Figure 4.14: 10-Hydroxycamptothecin presents no distinct anti-fibrotic character. ...46
Figure 4.15: Piplartine presents no distinct anti-fibrotic character. ...47
Figure 4.16: Anisomycin, Geldanamycin, Bufalin and Gitoxigenin significantly decrease collagen 1 protein level. ...48
Figure 4.17: Anisomycin reduces fibrotic gene expression on mRNA level. ...49
Figure 4.18: Geldanamycin presents opposing fibrotic effects on mRNA and protein level. ..50
Figure 4.19: Bufalin potently represses TGFβ and collagen 3 mRNA expression level. ...50
Figure 4.20: Gitoxigenin significantly reduces α-SMA gene expression but has no influence on CTGF protein expression. ...51
Figure 4.21: Anisomycin, Geldanamycin and Bufalin induce cellular senescence.. ...52
Figure 4.22: Gitoxigenin does not influence cell cycle conditions. ...53
Figure 4.23: Geldanamycin, Bufalin and Gitoxigenin present cardiac fibroblast specific proliferative influence...54
Figure 4.24: Anisomycin exhibit unspecific anti-proliferative activity. ...54
Figure 4.25: Anti-fibrotic natural compounds contrarily influence para-communication with surrounding cells.. ...57
Figure 4.26: Anisomycin, Geldanamycin and Bufalin decrease autophagic flux. ...60
Figure 4.27: Gitoxigenin does not influence autophagic flux. ...61
Figure 4.28: miRNA profile analysis of HCFs treated with Anisomycin reveals three potential pro- and three potential anti-fibrotic miRNAs. ...62
Figure 4.29: miRNA profile analysis reveals three miRNAs which potentially exhibit diagnostic impact differentiating between HFpEF and HFrEF. ...63
Figure 4.30: miRNA-451a, -4783-5p and -663b validation revealed the opposite outcome of sequencing, therefore not sustaining diagnostic approach. ...64
11 Affidavit
Erklärung über die selbständige Anfertigung der Dissertation und die Nicht-Inanspruchnahme einer gewerblichen Promotionsvermittlung oder -beratung
Ich erkläre, dass ich die der Medizinischen Hochschule Hannover zur Promotion eingereichte Dissertation mit dem Titel
„Identification and functional characterization of anti-fibrotic natural compounds in vitro“
im Institut für Molekulare und Translationale Therapiestrategien (IMTTS), Medizinische Hochschule Hannover, unter Betreuung von Prof. Dr. Dr. T. Thum ohne sonstige Hilfe durch-geführt und bei der Abfassung der Dissertation keine anderen als die dort aufdurch-geführten Hilfsmittel benutzt habe.
Die Gelegenheit zum vorliegenden Promotionsverfahren ist mir nicht kommerziell vermittelt worden.
Insbesondere habe ich keine Organisation eingeschaltet, die gegen Entgelt Betreuerinnen und Betreuer für die Anfertigung von Dissertationen sucht oder die mir obliegenden Pflichten hinsichtlich der Prüfungsleistungen für mich ganz oder teilweise erledigt.
Ich habe diese Dissertation bisher an keiner in- oder ausländischen Hochschule zur Promo-tion eingereicht.
Weiterhin versichere ich, dass ich den beantragten Titel bisher noch nicht erworben habe.
Ergebnisse der Dissertation wurden/werden in folgendem Publikationsorgan Bibliothek Medizinische Hochschule Hannover veröffentlicht.
Hannover, den 4. Februar 2020
Acknowledgement 92
12 Acknowledgement
Many people contributed to the creation of this work directly and indirectly, and therefore I would like to sincerely thank you!
First of all, I would like to thank my supervisor Professor Dr. Dr. Thomas Thum, who gave me the opportunity to join the IMTTS group as a medical doctoral student, for his great guidance and support, for enabling me the possibility to open up new professional perspectives.
Dear Katharina and Jan, thank you very much for your guidance, our scientific interactions and trouble-shooting. Thank you for your splendid support and for always having “an open ear”. It was a big pleasure to be a part of your scientific team!
I am very grateful for the help of all of my colleagues of the IMTTS group.
In particular, I would like to thank the technicians Annette and Angelika who gave me intro-duction to all techniques in the lab and always helped in solving technical problem.
Furthermore, I would like to thank Dr. Shashi Gupta for introducing me into the field of trans-lational research and Dr. Ke Xiao for her bioinformatic assistance.
I also want to thank all the other technicians, PhD students and Post-Doc´s, whom I could always ask, if I had any scientific questions.
I am also grateful to the Hannover Biomedical Research School enabling the StrucMed Pro-gramme and Dr. Susanne Kruse for her support and care.
Furthermore, I would like to thank all the collaboration partners: A big thank you goes to Dr.
Robert Geffers for deep sequencing and to the group of Prof. Dr. Christine Falk for cytokine-measurements, every collaboration partner of the Fibrotargets consortium and the EU for financial support.
Dear Monika and Freya, thank you very much for listening to all the (little and big) problems which I had during my (our) StrucMed time - for sharing plenty of enjoyable early and late night hours together.
My special gratitude also goes to all my university friends, particularly Lisa and Samira:
Thank you for your care and support, your valuable friendships!
Dear Yannic, thank you very much for encouraging and strengthening whenever necessary, for always being there and believing in me – to be the best boyfriend to have.
Most importantly, I would like to thank my family for their support, patience and sympathy as well as for their encouragement to follow my way: my parents Daniela and Rolf and my sister Esther!