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Validation of Phosphodiesterase Isozymes As Targets for Pulmonary Hypertension

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Validation of Phosphodiesterase Isozymes As Targets for Pulmonary Hypertension Validation of phosphodiesterase isozymes as targets for pulmonary hypertension Sarah Louise Trinder University College London Division of Medicine Pharmacology Page | 1 Declaration I, Sarah Louise Trinder confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Page | 2 Abstract Pulmonary hypertension (PH) is a debilitating disease with a very poor prognosis. Increasing lung cyclic GMP (cGMP) levels is clinically effective in treating the disease, but since many endogenous pathways regulate cGMP synthesis and metabolism, there exists considerable scope for further optimising cGMP-based therapy. In this thesis I have explored this possibility focusing on natriuretic peptides, vasoactive mediators that stimulate cGMP synthesis via a membrane- bound guanylate cyclase, and phosphodiesterases (PDE) which inactivate cGMP. Using organ bath pharmacology, I assessed the functional reactivity of systemic (aorta) and pulmonary vessels in response to atrial natriuretic peptide (ANP) and nitric oxide (NO; in the form of an NO-donor) in the absence and presence of isoform-selective PDE inhibitors. This was conducted in tissues from normoxic rats and animals exposed to 2 weeks hypoxia (10% O2). In addition, I undertook RT-PCR studies to determine if expression of specific PDEs changed in these vessels under a hypoxic environment. My data suggest that PDE 2 and PDE 5 are the principal PDE isoforms regulating ANP-dependent vasorelaxation in the pulmonary vasculature (from a functional and expressional standpoint) and that inhibition of these PDEs should bring about a selective pulmonary dilatation, particularly under hypoxic conditions. Using a model of hypoxia-induced PH, I demonstrated that the PDE2 inhibitor, BAY 60- 7550, prevents the pulmonary hypertension, pulmonary vascular remodelling and right ventricular hypertrophy characteristic of the disease. Moreover, combination of BAY 60-7550 with the neutral endopeptidase inhibitor ecadotril (augments circulating natriuretic peptide levels) produces an additive, if not synergistic, benefit against disease severity. Finally, I built on published work from our laboratory by investigating the effect of the PDE5 inhibitor sildenafil, in combination with ecadotril, in experimental models of PH and pulmonary fibrosis. Again, my data suggest that targeting PDE5 is also an effective therapeutic approach in these lung disorders. Page | 3 Table of Contents Declaration ................................................................................................................... 2 Abstract ........................................................................................................................ 3 Table of Contents ......................................................................................................... 4 List of Figures .............................................................................................................. 8 List of Tables.............................................................................................................. 10 List of Abbreviation ................................................................................................... 11 Publications ................................................................................................................ 16 Acknowledgements .................................................................................................... 17 Chapter 1: Introduction .............................................................................................. 18 1.1 Pulmonary Hypertension ...................................................................................... 18 1.1.1 Group 1 – Pulmonary arterial hypertension .................................................. 18 1.1.2 Group 2 – Pulmonary hypertension due to left heart disease ........................ 19 1.1.3 Group 3 – Pulmonary hypertension due lung disease and/or hypoxia .......... 20 1.1.4 Group 4 – Chronic thromboembolic pulmonary hypertension ..................... 20 1.1.5 Group 5 – Pulmonary hypertension with unclear multi-factorial mechanisms ................................................................................................................................ 21 1.1.6 Diagnosis ....................................................................................................... 23 1.1.7 Pathology and aetiology ................................................................................ 24 1.2 Endothelial dysfunction in pulmonary hypertension ........................................... 26 1.2.1 Nitric oxide and nitric oxide synthase ........................................................... 26 1.2.1.1 Nitric oxide, nitric oxide synthase and pulmonary hypertension ........... 27 1.2.2 Soluble guanylate cyclase ............................................................................. 28 1.2.2.1 Soluble guanylate cyclase and pulmonary hypertension ........................ 31 1.2.3 Cyclic 3’,5’-guanosine monophosphate ........................................................ 33 1.2.3.1 Cyclic 3’, 5’-guanosine monophosphate and pulmonary hypertension .. 35 1.2.4 Endothelin-1 .................................................................................................. 35 1.2.4.1 Endothelin-1 and pulmonary hypertension ............................................. 36 1.2.5 Prostacyclin ................................................................................................... 36 1.2.5.1 Prostacyclin and pulmonary hypertension .............................................. 37 1.2.6 Cyclic 3’, 5’-adenosine monophosphate ....................................................... 37 Page | 4 1.2.7 Serotonin ....................................................................................................... 38 1.2.7.1 Serotonin and pulmonary hypertension .................................................. 38 1.3 Natriuretic peptides .............................................................................................. 40 1.3.1.1 Atrial natriuretic peptide ......................................................................... 40 1.3.1.2 B-type natriuretic peptide ....................................................................... 42 1.3.1.3 C-type natriuretic peptide ....................................................................... 42 1.3.2 Natriuretic peptide receptors ......................................................................... 42 1.3.2.1 Natriuretic peptide receptor-A ................................................................ 43 1.3.2.2 Natriuretic peptide receptor-B ................................................................ 44 1.3.2.3 Natriuretic peptide receptor-C ................................................................ 44 1.3.3 Neutral endopeptidase ................................................................................... 45 1.4 Phosphodiesterases ............................................................................................... 46 1.4.1 PDE1: ‘Calcium/calmodulin-stimulated’ ...................................................... 46 1.4.2 PDE2: ‘cGMP-stimulated’ ............................................................................ 47 1.4.3 PDE3: ‘cGMP-inhibited’ ............................................................................... 47 1.4.4 PDE4 ............................................................................................................. 48 1.4.5 PDE5 ............................................................................................................. 50 1.4.6 PDE6 ............................................................................................................. 50 1.4.7 Manipulating cGMP-PDE activity in pulmonary hypertension .................... 50 1.5 Treatment of Pulmonary Hypertension ................................................................ 52 1.5.1 Calcium channel blockers ............................................................................. 52 1.5.2 Prostacyclin analogues .................................................................................. 53 1.5.3 Endothelin receptor antagonists .................................................................... 53 1.5.4 Inhaled nitric oxide ........................................................................................ 54 1.5.5 PDE5 inhibitors ............................................................................................. 54 1.5.6 Haem-dependent and -independent sGC activators ...................................... 55 1.5.7 Combination therapies for pulmonary hypertension ..................................... 56 1.6 Pulmonary Fibrosis .............................................................................................. 58 1.6.1 Phosphodiesterase 5 and pulmonary fibrosis ................................................ 59 1.7 Animal models of pulmonary hypertension ......................................................... 60 1.7.1 Chronic hypoxia ............................................................................................ 60 1.7.2 Sugen (SU-5416) and hypoxia ...................................................................... 61 1.7.3 Monocrotaline ............................................................................................... 62 Page | 5 1.7.4 Bleomycin ....................................................................................................
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