Identification of Candidate G protein–coupled receptors: Role of purinergic receptor P2Y2 in Pulmonary Arterial Hypertension 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 Viswanathan, Gayathri of (Tuticorin, India) Giessen 2017 From the Department of Internal Medicine II and Excellence Cluster Cardio-Pulmonary System (ECCPS) of the Faculty of Medicine of the Justus Liebig University Giessen Director / Chairman: Prof. Dr. Werner Seeger First Supervisor and Committee Member: Prof. Dr. Ralph Schermuly Committee Member (Chair): Prof. Dr. Klaus-Dieter Schlüter Second Supervisor and Committee Member: Prof. Dr. Reinhard Dammann Committee Member: Prof. Dr. Christian Mühlfeld (Hannover) Date of Doctoral Defense: 21.07.2017 2 The family is one of nature's masterpieces. Dedicated to My parent: Viswanathan Kumarasamy and Malathi Viswanathan My Grandmother: Saradhambal who always picked me up on time and encouraged me to go on every adventure, especially this one. 3 TABLE OF CONTENTS Table of Contents 1 INTRODUCTION ........................................................................................ 8 1.1 Pulmonary arterial hypertension (PAH) ............................................................ 8 1.1.1 Definition of PAH .......................................................................................................... 8 1.1.2 Idiopathic PAH (IPAH) .................................................................................................. 8 1.1.3 Epidemiology ................................................................................................................ 8 1.1.4 Updated clinical classification of PH ............................................................................. 9 1.1.5 Histology of PAH ......................................................................................................... 10 1.1.6 Pathogenesis and pathophysiology of PAH ............................................................... 11 1.1.7 Imbalance of vascular effectors .................................................................................. 13 1.1.8 Environmental and medical factors ............................................................................ 14 1.1.9 Therapeutic approaches for PAH ............................................................................... 15 1.2 G protein-coupled receptors (GPCRs) ............................................................ 17 1.2.1 Canonical signaling of GPCRs ................................................................................... 18 1.2.2 GPCRs and effectors in membrane trafficking ........................................................... 20 1.2.3 Non-canonical signaling of GPCRs ............................................................................ 20 1.3 GCPRs as a therapeutic target in PAH ........................................................... 21 1.3.1 Endothelin signaling pathway ..................................................................................... 22 1.3.2 Prostanoid signaling pathway ..................................................................................... 22 1.3.3 Serotonin (5-HT) signaling pathway ........................................................................... 23 1.3.4 Angiotensin signaling pathway ................................................................................... 23 1.3.5 Apelin (APJ) signaling pathway .................................................................................. 23 1.3.6 β-chemokines (CC chemokines) and the C-C chemokine receptor type 7 (CCR7) signaling pathway ...................................................................................................................... 24 1.4 Purinoceptor 2 (P2Y2) mediated signaling pathways .................................... 25 1.4.1 P2Y2 receptor oligomerization ................................................................................... 25 1.4.2 P2Y2 receptor desensitization and trafficking ............................................................ 25 1.4.3 P2Y2 receptor coupling to receptor and non-receptor tyrosine kinases .................... 26 2 AIM OF STUDY ........................................................................................ 27 3 MATERIALS AND METHODS ................................................................. 28 3.1 Materials ........................................................................................................... 28 3.1.1 Chemicals, reagents and kits ..................................................................................... 28 3.1.2 Cell culture medium and reagents .............................................................................. 29 3.1.3 Primers used for high-throughput screening of GPCRs ............................................. 30 4 3.1.4 Candidate GPCR primers for quantitative real-time PCR in PASMC ......................... 30 3.1.5 Small interfering RNA (siRNA) ................................................................................... 31 3.1.6 Antibodies ................................................................................................................... 31 3.1.7 Equipment ................................................................................................................... 31 3.1.8 Other materials ........................................................................................................... 32 3.1.9 Software ...................................................................................................................... 33 3.2 Methods ............................................................................................................ 33 3.2.1 Patients’ characteristics .............................................................................................. 33 3.2.2 Microdissection of pulmonary vessels and cDNA synthesis ...................................... 33 3.2.3 High-throughput (HT) screening of GPCRs ................................................................ 35 3.2.4 Human pulmonary arterial smooth muscle cells (PASMCs) isolation and cell culture 36 3.2.5 Polymerase chain reaction (PCR) .............................................................................. 37 3.2.6 RNA interference by siRNA (GPCRs) ........................................................................ 39 3.2.7 BrdU cell proliferation assay ....................................................................................... 39 3.2.8 Wound healing - in vitro scratch assay ....................................................................... 40 3.2.9 Transwell migration assay .......................................................................................... 40 3.2.10 cAMP enzyme immunoassay (EIA) ............................................................................ 41 3.2.11 Western blotting .......................................................................................................... 42 3.2.12 Zymography ................................................................................................................ 44 3.2.13 P2Y2 ligand ................................................................................................................ 46 3.2.14 Statistical analysis ...................................................................................................... 46 4 RESULTS ................................................................................................. 47 4.1 Gene expression profiling of GPCRs in human pulmonary vessels ............ 47 4.1.1 High-throughput screening of 408 GPCRs with first set of primers ............................ 47 4.1.2 Validation of dysregulated GPCR expression by alternative sets of primers ............. 49 4.2 Screening for candidate GPCRs in the PASMCs of donors and IPAH patients via real-time PCR ....................................................................................................... 51 4.2.1 Expression of the upregulated candidate GPCRs in the PASMCs of donors and IPAH patients……… ........................................................................................................................... 51 4.2.2 Expression of the downregulated candidate GPCRs in the PASMCs of donors and IPAH patients ............................................................................................................................ 52 4.3 si-RNA knockdown of GPCRs in PASMCs ..................................................... 53 4.4 Proliferation of PASMCs after the siRNA knockdown of GPCRs ................. 54 4.5 Functional role of the purinergic receptor (P2Y2) in human PASMCs ......... 55 4.5.1 Effect of a P2Y2 agonist (MRS2768) on the basal proliferation of PASMCs ............. 56 4.5.2 The P2Y2 ligand MRS2768 inhibited PASMC proliferation induced by serum and PDGF…………. ......................................................................................................................... 56 5 4.5.3 The P2Y2 ligand MRS2768 inhibited PASMC migration induced by serum and PDGF…. .................................................................................................................................... 57 4.5.4 P2Y2 ligand MRS2768 inhibited matrix metalloproteinases 2 (MMP-2) in PASMCs . 61 4.5.5 P2Y2 ligand MRS2768 increased intracellular cAMP in PASMCs ............................. 62 4.6 Downstream signaling pathways of P2Y2 ...................................................... 63 4.6.1 MRS2768 increased exchange protein directly activated by cAMP (EPAC) in PASMCs .................................................................................................................................... 63 4.6.2 MRS2768 attenuated ROCK1 and myosin light chain 1 (MLC1) phosphorylation in PASMCs .................................................................................................................................... 64 5 DISCUSSION ........................................................................................... 66 5.1 GPCR expression profile in the pulmonary vasculature of IPAH patients ... 66 5.1.1 Micro-dissection of pulmonary arteries from donors and IPAH patients .................... 67 5.1.2 Upregulated GPCRs in the pulmonary vasculature of IPAH patients ........................ 67 5.1.3 Downregulated GPCRs in the pulmonary vasculature of IPAH patients .................... 72 5.2 Effect of GPCRs on human PASMC proliferation .......................................... 73 5.3 Role of purinergic receptor P2Y2 in PAH ....................................................... 74 5.3.1 Effect of P2Y2 agonist MRS2768 on PASMC proliferation ........................................ 75 5.3.2 Effect of P2Y2 agonist MRS2768 on PASMC migration ............................................ 76 5.3.3 MRS2768 regulates MMP2 in human PASMCs via the P2Y2 receptor ..................... 77 5.3.4 P2Y2 agonist MRS2768 increases cAMP production in human PASMCs ................. 78 5.3.5 MRS2768 increases EPAC expression in human PASMCs ...................................... 78 5.3.6 MRS2768 inhibits ROCK and decreases MLC phosphorylation in human PASMCs 79 6 CONCLUSION ......................................................................................... 81 7 OUTLOOK FOR THE FUTURE ............................................................... 82 8 SUMMARY ............................................................................................... 83 9 ZUSAMMENFASSUNG ........................................................................... 85 10 APPENDIX ............................................................................................... 87 11 LIST OF ABBREVIATIONS ................................................................... 101 12 LIST OF FIGURES ................................................................................. 105 13 LIST OF TABLES .................................................................................. 107 14 BIBLIOGRAPHY .................................................................................... 109 15 DECLARATION ..................................................................................... 124 6 16 ACKNOWLEDGMENT ........................................................................... 125 7 INTRODUCTION 1 INTRODUCTION 1.1 Pulmonary arterial hypertension (PAH) Pulmonary arterial hypertension is characterized by increased pulmonary vascular resistance (PVR) resulting in elevated pulmonary artery pressure (PAP) and leading to right- heart failure, and ultimately death if untreated [1, 2]. Pulmonary arterial hypertension is no longer an orphan disease [3], and 4 classes of drugs have been developed in the last 15 years [4, 5]. Breathlessness, fatigue, weakness, angina, and syncope are the symptoms of PAH [6]. The pathogenesis of PAH is characterized by pulmonary vasoconstriction, abnormal pulmonary vascular remodeling, and in-situ thrombosis [7]. 1.1.1 Definition of PAH Pulmonary arterial hypertension is defined by a mean PAP (mPAP) ≥ 25 mmHg at rest with a PVR > 3 wood units and an end-expiratory pulmonary artery wedge pressure (PAWP) ≤ 15 mmHg at the time of right-heart catheterization [8]. 1.1.2 Idiopathic PAH (IPAH) The emergence of PAH without a known risk factor or family context is known as IPAH. For patients with this sporadic disease, diagnosis is usually late due to the lack of a reliable screening program [9-11]. Idiopathic PAH is a progressive, nearly fatal condition with very few treatment options. Deregulated proliferation of pulmonary intimal endothelial cells and pulmonary arterial smooth muscle cells (PASMCs) results in progressive pulmonary vascular remodeling and subsequently elevated PAP during the IPAH progression. Specific targeted therapies have been developed using prostacyclin, endothelin-receptor antagonists, and phosphodiesterase 5 inhibitors. However, scientists are exploring new therapeutic modalities because of the insufficient efficacy and poor tolerability of these agents [12]. 1.1.3 Epidemiology Accurate diagnosis of PAH is difficult, and access to care is limited in many countries, which makes the global prevalence of PAH challenging to estimate. The global burden of PAH is likely greater than currently recognized, because risk factors, such as HIV, schistosomiasis, and sickle cell disease, are more prevalent in the developing world [13]. In developed countries, PAH cases will also likely increase as newer associations with PAH emerge, and 8 INTRODUCTION these include dialysis [14] and metabolic syndrome [15]. In addition, broad access to echocardiography results in earlier PAH diagnoses [16]. The epidemiology of PAH has been described by several registries [17-19]. In Europe, PAH occurs in 15 to 60 subjects per million of the population, resulting in 5 to 10 cases per million each year [18]. In sum, IPAH, heritable PAH, and drug-induced PAH comprise approximately half of registered PAH patients [17]. 1.1.4 Updated clinical classification of PH The first classification of pulmonary hypertension (PH) was proposed in 1973 [20]. A clinical taxonomy was established at the second World Symposium on PH (Evian, 1998) to categorize different classes of PH sharing similar pathological findings, hemodynamic characteristics, and management approaches (2). There are five categories of PH: (1) PAH, (2) PH related to left-heart disease, (3) PH due to lung disease/hypoxia, (4) thromboembolic PH, and (5) miscellaneous variants. In the consecutive meetings of that global body, a series of changes were carried out. However, the general architecture and the philosophy of the clinical classification remained unchanged. According to new data published in recent years, certain modifications and updates, especially for the first type of PH, were proposed during the Fifth World Symposium in 2013 (Table 1) [16, 21]. 9 INTRODUCTION Table 1. Updated classification of PH (Simonneau, 2013) [22] 1. PAH 1.1. Idiopathic PAH 1.2. Heritable PAH 1.2.1. Bone morphogenetic protein receptor type II (BMPR2) 1.2.2. Activin receptor-like kinase (ALK-1), Endoglin (ENG), SMAD Family Member 9 (SMAD9), Caveolin-1 (CAV1), Potassium Two Pore Domain Channel Subfamily K Member 3 (KCNK3) 1.2.3. Unknown 1.3. Drug- and toxin-induced 1.4. Associated with: 1.4.1. Connective tissue disease 1.4.2. HIV infection 1.4.3. Portal hypertension 1.4.4. Congenital heart diseases 1.4.5. Schistosomiasis 1′. Pulmonary veno-occlusive disease and/or pulmonary capillary hemangiomatosis 1′′. Persistent PH of the newborn (PPHN) 2. Pulmonary hypertension due to left-heart disease 2.1. Left ventricular systolic dysfunction 2.2. Left ventricular diastolic dysfunction 2.3. Valvular disease 2.4. Congenital/acquired left-heart inflow/outflow tract obstruction and congenital cardiomyopathies 3. PH due to lung disease and/or hypoxia 3.1. Chronic obstructive pulmonary disease 3.2. Interstitial lung disease 3.3. Other pulmonary diseases with mixed restrictive and obstructive pattern 3.4. Sleep-disordered breathing 3.5. Alveolar hypoventilation disorders 3.6. Chronic exposure to high altitude 3.7. Developmental lung diseases 4. Chronic thromboembolic pulmonary hypertension (CTEPH) 5. Pulmonary hypertension with unclear multifactorial mechanisms 5.1. Hematologic disorders: chronic hemolytic anemia, myeloproliferative disorders, splenectomy 5.2. Systemic disorders: sarcoidosis, pulmonary histiocytosis, lymphangioleiomyomatosis 5.3. Metabolic disorders: glycogen storage disease, Gaucher disease, thyroid disorders 5.4. Others: tumoral obstruction, fibrosing mediastinitis, chronic renal failure, segmental PH 1.1.5 Histology of PAH Intimal hyperplasia, medial hypertrophy, occlusion of small arteries, in-situ thrombosis, adventitial proliferation or fibrosis, infiltration of inflammatory or progenitor cells, and angio- proliferative “plexiform” lesions are the histological features of PAH (Figure 1). In PAH, the distribution and prevalence of those histological abnormalities are heterogeneous within the lung. Of note, plexiform lesions, which are often located downstream from occluded arteries, 10