Effects of a modulation of the urokinase type plasminogen activator (u-PA) system in chronic hypoxia induced pulmonary vascular remodeling and right ventricular hypertrophy (RVH) in mice [Elektronische Ressource] / vorgelegt von Bhola Kumar Dahal

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• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2008
• Nombre de lectures: 20
• Poids de l'ouvrage: 2 Mo

Extrait

Effects of a modulation of the urokinase-type plasminogen
activator (u-PA) system in chronic hypoxia-induced pulmonary
vascular remodeling and right ventricular hypertrophy (RVH) in
mice




Inauguraldissertation
zur Erlangung des Grades eines Doktors der Humanbiologie
des Fachbereichs Medizin
der Justus-Liebig-Universität Giessen










vorgelegt von

Bhola Kumar Dahal
aus Inaruwa, Nepal




Giessen 2007



Aus dem Medizinischen Zentrum f ϋr Innere Medizin
Medizinische Klinik und Poliklinik II
Direktor: Prof. Dr. med. Werner Seeger
des Fachbereichs Medizin der Justus-Liebig-Universität Gie βen






Gutachter: Prof. Dr. Andreas Guenther
Gutachter: Prof. Dr. Klaus T. Preissner







Tag der Disputation: 11. März 2008


Urokinase-type plasminogen activator (u-PA) in pulmonary vascular remodeling 1

Index of contents
Index of contents……………………………………………………………………………...2
Index of figures.………………………………………………………………………………4
Index of tables.………………………………………………………………………………………...5
1 INTRODUCTION..............................................................................................................................6
1.1 Physiology of Pulmonary circulation ..................................................................................6
1.1.1 Functional anatomy of pulmonary circulation.................................................................6
1.1.2 Hypoxic pulmonary vasoconstriction (HPV) ..................................................................8
1.2 Pathophysiology of pulmonary vasculature.........................................................................9
1.2.1 Pulmonary vascular remodeling......................................................................................9
1.2.2 Right ventricular (RV) adaptation .................................................................................12
1.3 Pulmonary hypertension ....................................................................................................13
1.3.1 Definition and classification..........................................................................................13
1.3.2 Pulmonary arterial hypertension (PAH) ........................................................................17
1.3.3 Chronic thromboembolic pulmonary hypertension (CTEPH).......................................19
1.4 Animal models of pulmonary hypertension/ vascular remodeling.....................................20
1.4.1 Monocrotaline-induced pulmonary hypertension..........................................................20
1.4.2 Hypoxia-induced pulmonary hypertension/vascular remodeling..................................21
1.5 Plasminogen activation system..........................................................................................23
1.5.1 Urokinase (u-PA) and its variants .................................................................................23
1.5.2 ed its receptor (uPAR)....................................................................25
1.5.3 Inhibitors of urokinase...................................................................................................27
1.5.4 Coagulation and fibrinolysis in pulmonary arterial hypertension .................................29
1.5.5 Urokinase system in pulmonary vascular remodeling...................................................32
1.6 Aim of the study..................................................................................................................35
2 MATERIALS...................................................................................................................................36
2.1 Chemicals, Reagents, Injecting solution and substances...................................................36
2.2 Consumables......................................................................................................................37
2.3 Systems, machines and softwares ......................................................................................38
2.4 Materials for histology.......................................................................................................38
2.5 Antibodies ..........................................................................................................................40
3 METHODS.......................................................................................................................................41
3.1 Animals ..............................................................................................................................41
3.2 Induction of pulmonary hypertension in mice by hypoxia .................................................42
3.3 Treatment of mice with u-PA and u-PA inhibitor ..............................................................42
3.4 Hemodynamic and right ventricular hypertrophy (RVH) measurement in mice ...............42
3.5 Histology and morphometric analysis of murine lungs .....................................................43
3.6 Characterization of human lungs obtained from transplant programme ..........................46
3.7 Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis..46
3.8 Western blot analysis .........................................................................................................48
3.9 Casein gel zymography......................................................................................................49
Urokinase-type plasminogen activator (u-PA) in pulmonary vascular remodeling 2

3.10 Immunohistochemistry .......................................................................................................50
3.10.1 Cryo-preserved murine lung .....................................................................................50
3.10.2 Paraffin-embedded human lungs (donors, IPAH and CTEPH) ................................50
3.11 u-PA activity analysis in plasma and lung homogenates...................................................51
3.12 Data analysis .....................................................................................................................52
4 RESULTS.........................................................................................................................................53
4.1 Expression of u-PA and PAI-1 in the lungs of patients with CTEPH and IPAH as
compared to donor lungs ................................................................................................................53
4.1.1 u-PA and PAI-1 expression at mRNA level..................................................................53
4.1.2 Expression of u-PA and PAI-1 at protein level .............................................................54
4.1.3 u-PA activity in the lungs of CTEPH and IPAH patients as compared to donor lungs.55
4.1.4 Localization of u-PA and PAI-1 in the lungs from IPAH, CTEPH and donors ............57
4.2 Expression of u-PA and PAI-1 in chronically hypoxic mouse lungs .................................58
4.2.1 u-PA and PAI-1 gene expression under hypoxia...........................................................58
4.2.2 u-PA and PAI-1 protein level under hypoxia ................................................................59
4.2.3 Influence of hypoxia on lung u-PA activity ..................................................................60
4.2.4 Localization of u-PA and PAI-1 under hypoxia............................................................62
4.3 Hypoxia-induced right ventricular hypertrophy (RVH) in wild type, u-PA and PAI-1
deficient mice as well as in u-PA inhibitor (CJ463) or u-PA treated mice.....................................62
4.4 Hypoxia-induced muscularization in wild type, u-PA and PAI-1 deficient mice as well as
in CJ463 and u-PA treated mice.....................................................................................................68
4.5 Attenuation of hypoxia-induced loss of pulmonary vessels upon u-PA infusion ...............70
5 DISCUSSION...................................................................................................................................71
5.1 Regulation of u-PA and PAI-1 in the lungs of patients with pulmonary hypertension and
in murine lungs in response to chronic hypoxia .............................................................................71
5.1.1 u-PA and PAI-1 in the lungs from patients with IPAH and CTEPH.............................71
5.1.2 he murine lungs in response to chronic hypoxia ...........................74
5.2 Development of hypoxia-induced pulmonary vascular remodeling and RVH in wild type,
u-PA and PAI-1 knockout and u-PA inhibitor (CJ463) treated mice .............................................76
5.3 Attenuation of hypoxia-induced pulmonary vascular remodeling and RVH in u-PA treated
mice 79
6 SUMMARY.....................................................................................................................................83
7 ZUSAMMENFASSUNG.......................