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Role of Cgmp Phosphodiesterases in Idiopathic Pulmonary Fibrosis

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Role of Cgmp Phosphodiesterases in Idiopathic Pulmonary Fibrosis Role of cGMP Phosphodiesterases in Idiopathic Pulmonary Fibrosis Inaugural Dissertation submitted to the Faculty of Medicine in partial fulfillment of the requirements for the PhD-Degree of the Faculties of Veterinary Medicine and Medicine of the Justus Liebig University Giessen by Nikolova, Sevdalina Vaskova of Varna, Bulgaria Giessen 2009 From the Department of Medicine Director / Chairman: Prof. Dr. Werner Seeger of Medicine of the Justus Liebig University Giessen First Supervisor and Committee Member: Prof. Dr. Ralph Schermuly Second Supervisor and Committee Member: Priv.-Doz. Dr. Stephan Rosenkranz Committee members: Prof. Dr. Ernst Petzinger and Prof. Dr. Susanne Rohrbach Date of Doctoral Defense: 16th of March 2009 Table of content Table of content Table of content··················································································· I List of figures·····················································································IV List of tables ·······················································································V List of abbreviations··········································································VI Summary ·····························································································X Zusammenfassung ············································································XI I. Introduction······················································································ 1 1. Idiopathic pulmonary fibrosis (IPF). ·················································· 2 1.1. Classification.···································································································· 3 1.2. Definition.·········································································································· 4 1.3. Histopathology.································································································· 4 1.4. Pathogenesis.··································································································· 5 1.5. Role of AECs in IPF pathogenesis.································································· 10 1.5.1. Morphological characteristics of the alveolar epithelium.························· 10 1.5.2. ATII cells: stem cells of the alveolar epithelium. ······································ 10 1.5.3. ATII cells and alveolar re-epithealization. ················································ 12 1.5.4. Growth factor control of ATII cell proliferation.········································· 13 1.6. Diagnosis and evaluation.··············································································· 14 1.6.1. Clinical presentation. ··············································································· 14 1.6.2. Diagnosis.································································································ 14 1.7. Treatment. ······································································································ 15 1.7.1. Phosphodiesterase (PDE) inhibitors.······················································· 15 2. PDEs. ····························································································· 15 2.1. Cyclic nucleotide specificity. ··········································································· 16 2.2. cGMP.············································································································· 17 2.3. cGMP PDEs.··································································································· 19 2.4. cGMP PDEs in the lung. ················································································· 21 2.5. PDE6. ············································································································· 21 2.5.1. Phototransduction.··················································································· 22 2.5.2. The PDE6D subunit.················································································ 24 II. Aims of the study.········································································· 25 I Table of content III. Materials and Methods. ······························································· 27 3.1. Materials. ····················································································· 28 3.1.1. Instruments, consumables, chemicals and enzymes.······························ 28 3.1.2. Cloning reagents. ···················································································· 31 3.1.3. Cell culture materials. ·············································································· 32 3.1.4. Antibodies, blocking peptides and siRNA targeting sequences.·············· 33 3.1.5. Kits. ········································································································· 34 3.1.6. Buffers and solutions. ·············································································· 34 3.2. Methods.······················································································ 36 3.2.1. Human lung tissue.·················································································· 36 3.2.2. RNA isolation.·························································································· 36 3.2.3. cDNA synthesis. ······················································································ 37 3.2.4. PCR.········································································································ 38 3.2.5. RT-PCR.·································································································· 38 3.2.6. Agarose gel electrophoresis and PCR product purification. ···················· 39 3.2.7. Western blotting.······················································································ 40 3.2.8. Densitometry. ·························································································· 41 3.2.9. Immunohistochemical staining.································································ 41 3.2.10. Cloning. ································································································· 42 3.2.10.1. PCR amplification of complete gene sequence.························· 42 3.2.10.2. Generating ‘A-tailing’ to blunt-ended PCR fragments.················ 43 3.2.10.3. Ligation of A-tailed DNA Fragment into PGEMT-easy vector.···· 44 3.2.10.4. Heat shock transformation. ························································ 44 3.2.10.5. pGEMT easy recombinant clone selection.································ 45 3.2.10.6. Small scale plasmid DNA purification.········································ 45 3.2.10.7. Directional TOPO cloning in pcDNA 3.1 vector.························· 45 3.2.10.8. TOPO cloning reaction.······························································ 46 3.2.10.9. pcDNA 3.1 clone analysis. ························································· 46 3.2.10.10. Large scale endotoxin free plasmid extraction. ························ 46 3.2.10.11. Glycerol stock.·········································································· 47 3.2.11. siRNA ···································································································· 47 3.2.12. Cell culture.···························································································· 47 3.2.12.1. Cryopreservation and thawing of cell cultures.··························· 48 3.2.13. Transient transfection. ··········································································· 49 II Table of content 3.2.13.1. Transient transfection efficiency assessment.···························· 49 3.2.14. Measurement of cell proliferation.·························································· 49 3.2.14.1. MTT cell proliferation assays.····················································· 50 3.2.14.2. (3H)-Thymidine incorporation assay. ·········································· 50 3.2.15. Statistical analysis. ················································································ 50 IV. Results. ························································································ 51 4.1. mRNA expression profile of cGMP PDEs in IPF lungs. ·································· 52 4.2. mRNA detection of the PDE6 enzyme subunits in lung tissue samples of donors and IPF patients.························································································ 54 4.3. Protein expression of the PDE6 enzyme subunits in lung tissue samples of donors and IPF patients.························································································ 56 4.4. Cellular localization of the PDE6 enzyme subunits in lung tissue samples of donors and IPF patients.························································································ 57 4.5. The PDE6 enzyme subunits are expressed in human ATII cells. ··················· 58 4.6. Effects of PDE6D modulations on A549 cell proliferation. ······························ 60 4.7. PDE6D knockdown inhibits ERK phosphorylation. ········································· 62 V. Discussion ···················································································· 64 5.1. mRNA profile of cGMP PDEs in lung tissue samples of donors and IPF patients. ················································································································· 65 5.2. Detection of PDE6 enzyme subunits in lung tissue samples of donors and IPF patients. ·················································································································
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