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The role of NADPH oxidase in the pathogenesis of systemic sclerosis

Im Rahmen der vorliegenden Arbeit konnte nachgewiesen werden, dass die Rolle von NADPH-Oxidasen (Nox), speziell Nox4, bei der Fibroblastenaktivierung sowie der Fibrose der Haut eine Rolle spielt. Anhand von Expressionsanalysen auf RNA-und Proteinebene konnte gezeigt werden, dass der Nox4 komplex in gesunden dermalen Fibroblasten aber auch in dermalen Sklerodermie-Fibroblasten konstitutiv exprimiert wird. TGF-β1 unterdrückt die Nox4-Expression. Interessanterweise ist die TGF-β1-vermittelte Expression von Kollagen-Typ I Nox4-abhängig, da die pharmakologische Inhibition mit einem NADPH-Inhibitor (DPI) oder einer Nox4 siRNA die TGF-β1-Wirkung aufhebt. In Übereinstimmung damit hatte TGF-β1 in murinen Fibroblasten mit Nullmutation von Nox4 keinen Effekt auf die Expression von Kollagen. Im Bleomycin-Mausmodell der Sklerodermie unterdrückte eine pharmakologische Blockierung der Nox Aktivität durch DPI und Nox4 siRNA die Entwicklung einer Fibrose. Zusammengefasst deuten die Ergebnisse dieser Arbeit auf eine vielversprechende Strategie bei der zukünftigen Behandlung von fibrotischen Erkrankungen mit Nox4-Inhibitoren hin.

Titel: The role of NADPH oxidase in the pathogenesis of systemic sclerosis
Verfasser: Dosoki, Heba GND
Gutachter: Böhm, Markus
Organisation: FB 13: Biologie
FB 05: Medizinische Fakultät
Dokumenttyp: Dissertation/Habilitation
Medientyp: Text
Erscheinungsdatum: 2015
Publikation in MIAMI: 28.10.2016
Datum der letzten Änderung: 28.10.2016
Schlagwörter: NADPH-Oxidase; Fibroblasten; Systemische Sklerodermie; Fibrose; oxidativer Stress; Mausmodell der Sklerodermie; transforming growth factor (TGF-β1)
Fachgebiete: Biowissenschaften; Biologie
Sprache: Englisch
Format: PDF-Dokument
URN: urn:nbn:de:hbz:6-04239758120
Permalink: https://nbn-resolving.org/urn:nbn:de:hbz:6-04239758120
Onlinezugriff:
Inhalt:
Abstract ..................................................................................................................................... I
Acknowledgment ................................................................................................................... ..II
Dedication .............................................................................................................................. III
List of abbreviations ............................................................................................................... V
List of Figures ....................................................................................................................... XII
List of Tables ........................................................................................................................ XV
1. Introduction ......................................................................................................................... 1
1.1. Systemic sclerosis .......................................................................................................... 1
1.1.1. Definition and epidemiology ................................................................................... 1
1.1.2. The pathogenesis of systemic sclerosis ................................................................... 1
1.1.3. Skin fibrosis in SSc .................................................................................................. 2
1.1.4. Players of skin fibrosis in SSc ................................................................................. 3
1.1.5. Experimental model of systemic sclerosis............................................................... 8
1.2. Reactive oxygen species................................................................................................. 9
1.2.1. Sources of reactive oxygen species generation ....................................................... 9
1.2.2. ROS in cell signaling ............................................................................................. 10
1.2.3. Antioxidant studies in vitro, vivo and clinical trials .............................................. 11
1.3. NADPH oxidases (NOXs) ........................................................................................... 12
1.3.1. The family of NADPH oxidases ............................................................................ 12
1.3.4. Tissue distribution and subcellular localization .................................................... 14
1.4. NADPH oxidase 4 (Nox4) ........................................................................................... 15
1.4.1. Structure, function and regulation of Nox4 ........................................................... 15
1.4.2. Tissue distribution and subcellular localization .................................................... 17
1.4.3. Physiological and pathological role of Nox4 ........................................................ 18
1.4.4. Pathological role of Nox4 in fibrotic disorders ..................................................... 19
1.5. NADPH oxidase inhibition .......................................................................................... 20
2. Aim of the study ................................................................................................................ 23
3. Materials and Methods ..................................................................................................... 24
3.1. Materials ......................................................................................................................... 24
3.1.1. Chemicals .............................................................................................................. 24
3.1.2. Stimulants and pharmacological inhibitors ........................................................... 25
3.1.3. Media and Buffers ................................................................................................. 25
3.1.4. DNA ladder and protein markers ........................................................................... 27
3.1.5. Consumables and kits ............................................................................................ 28
3.1.6. Primers ................................................................................................................... 28
3.1.7. Antibodies .............................................................................................................. 30
3.1.8. Instruments and Laboratory equipment ................................................................. 31
3.1.9. Animals .................................................................................................................. 32
3.1.10. Software and computer programmes ................................................................... 32
3.1.11. Cells, cell lines and cell culture reagents ............................................................. 33
3.2. Methods ........................................................................................................................ 33
3.2.1. Cell biological methods ......................................................................................... 33
3.2.2. Molecular biological methods ............................................................................... 35
3.2.3. Biochemical methods ............................................................................................ 38
3.2.4. Immunological methods ........................................................................................ 41
3.2.5. Cytotoxicity assays ................................................................................................ 41
3.2.6. Confocal immunofluorescences microscopy ......................................................... 42
3.2.7. Assessment of dermal thickness. ........................................................................... 43
3.2.8. Determination of collagen protein content ex vivo. .............................................. 43
3.2.9. In silico promoter analysis ..................................................................................... 43
3.2.10. Measurement of NADPH oxidase activity assay ................................................ 43
3.2.11. Statistical analysis................................................................................................ 44
4. Results ................................................................................................................................ 45
4.1. Detection of Nox isoforms and adaptor proteins in HDFs ........................................... 45
4.1.1. Expression of Nox4 isoform and its adaptor proteins in normal HDFs ................ 45
4.1.2. Expression of Nox4 isoform and its adaptor proteins in affected skin and HDFs from SSc patients .............................................................................................................. 46
4.1.3. Expression of other Nox isoforms, cytosolic subunits and adaptor proteins in normal nHDFs .................................................................................................................. 48
4.1.4. Expression of additional Nox isoform and adaptor proteins in HDFs from SSc patients .............................................................................................................................. 49
4.2. Subcellular localization of Nox4 in HDFs ................................................................... 51
4.3. Induction of Nox4 expression and NADPH oxidase activity by TGF-β1 in HDFs ..... 53
4.3.1. TGF-β1-upregulates Nox4 expression at mRNA level ........................................... 53
4.3.2. TGF-β1 upregulates Nox4 expression at protein level........................................... 54
4.3.3. TGF-β1 enhances NADPH oxidase activity .......................................................... 56
4.4. Functional characterization of Nox4 in TGF-β1-mediated activation of HDFs ........... 57
4.4.1. Effect of the pharmacological inhibitor DPI on cell viability and metabolic activity in nHDFs.............................................................................................................. 57
4.4.2. Effect of the pharmacological inhibitor VAS2870 on cell viability in nHDFs .... 57
4.4.3. Pharmacological inhibition of NADPH oxidase by DPI reduces TGF-β1-mediated fibroblast activation .......................................................................................................... 58
4.4.4. VAS2870 does not reduce TGF-β1-mediated fibroblast activation ........................ 60
4.4.5. Genetic knockdown of Nox4 neutralizes TGF-β1-mediated fibroblast activation 61
4.5. Regulation of Nox4 expression by TGF-β1 in HDFs ................................................... 62
4.5.1. In silico promoter analysis of the human Nox4 gene ............................................ 62
4.5.2. Nox4 expression is depend on canonical smad3 signaling pathway in HDFs ...... 63
4.6. Pharmacological inhibition of NADPH oxidase activity by DPI reduces cutaneous fibrosis in the bleomycin mouse model of scleroderma ....................................................... 65
4.7. Nox4 is an emerging novel target for antifibrotic agents ............................................. 67
5. Discussion........................................................................................................................... 69
5.1. Nox4 is abundant in SSc HDFs .................................................................................... 69
5.2. Nox4 is colocalized with endoplasmic reticulum in HDFs .......................................... 71
5.3. Nox4 expression is regulated by TGF-β1 in HDFs ................................................... 73
5.4. Nox4 expression is regulated by the Smad2/3 pathway in HDFs ............................ 74
5.5. Nox4 inhibition prevented experimentally induced skin fibrosis in the bleomycin mouse model of scleroderma ............................................................................................ 75
5.6. α-MSH suppressed TGF-β1-mediated Nox4 gene expression in HDFs ................... 75
6. Conclusion & Future perspectives .................................................................................. 78
7. Pharmacological inhibition of Nox4 by GKT137831 reduces TGF-β1-mediated fibroblast activation .................................................................................................................................. 80
8. References .......................................................................................................................... 82
9. Appendix Figures...………………………………………….………………………….102
10. Curriculum Vita …………………………………………………................................104
XI