THE ROLE OF IRON IN PULMONARY HYPERTENSION A thesis presented for the degree of MD (Res) by Dr Geoffrey Watson Centre of Pharmacology and Therapeutics Imperial College London February 2018 1 I declare that this thesis was conducted and written by myself and the work included within is my own unless otherwise stated. The copyright of this thesis rests with the author. Unless otherwise indicated, its contents are licensed under a Creative Commons Attribution-Non Commercial 4.0 International Licence (CC BY-NC). Under this licence, you may copy and redistribute the material in any medium or format. You may also create and distribute modified versions of the work. This is on the condition that: you credit the author and do not use it, or any derivative works, for a commercial purpose. When reusing or sharing this work, ensure you make the licence terms clear to others by naming the licence and linking to the licence text. Where a work has been adapted, you should indicate that the work has been changed and describe those changes. Please seek permission from the copyright holder for uses of this work that are not included in this licence or permitted under UK Copyright Law. 2 Acknowledgements I would like to say an enormous thank you to my supervisors Dr. Luke Howard, Prof. Lan Zhao and Prof. Martin Wilkins. They have offered fantastic guidance throughout this project and have been incredibly patient with me. Dr Luke Howard has really gone out of his way to support me through this work for which I am eternally grateful. Special thanks also goes to Dr John Wharton and Dr Chris Rhodes. Both played vital roles in guiding me around the pitfalls of medical statistics and moulding me from clinician into a research fellow. I would like to thank the support of the cardio-pulmonary exercise test (CPET) laboratory, in particular Dr Kevin Murphy for both conducting the CPETs where data was accrued, as well as his invaluable real-time physiology teaching whilst I supervised these studies. He was pivotal in helping to analyse the CPET results, along with Dr Howard. I would like to thank the Hammersmith hospital cardiac catheter laboratory team, which without technical, nursing, and radiological support I would not have been able to conduct the invasive right and left cardiac catheterisations that has generated key data to support this thesis. I would like to thank Dr Chris Baker for his help in teaching me right heart catheterisation via the brachial approach which has changed the access route for research and clinical cases, as a default, locally and has allowed me to perform exercise catheterisations when needed, and in turn intra- oesphageal pressure measurements. Professor Michael Polkey was a key contributor towards the theoretical and technical aspects of measuring and interpreting intra-oesphagealpressure measurements for which I am extremely grateful. In turn, we have had an abstract accepted for the Eurpean Respiratory Society annual congress in September 2018 with our early findings presented in this thesis. I would like to thank Dr Ben Ariff for his expertise in helping to analyse cardiac MRI studies. I would like to thank Dr Souad Ali who was instrumental in helping me collect, process and collate research blood samples. I would like to thank the support of Imperial College Healthcare NHS Trust pathology laboratories in providing accurate patient data for iron status, haemoglobin, red cell distribution width, c-reactive protein, creatinine, B-type natriuretic peptide. I would like to thank Dr Mark Busbridge and his laboratory team at Charing Cross Hospital who provided soluble transferrin and hepcidin data. I would like to thank my wife, my parents and my brother. They have been monumentally supportive throughout the highs and lows. I could not have done it without them. I would like to thank my close friends for their support and solid base, and putting me up in a spare room at last minute during my research trips to London. 3 Abstract Introduction: Iron is a critical ion in the regulation of many cellular processes and iron deficiency (ID) has been shown to be a powerful predictor of survival in many diseases. It has been shown that ID, defined by increased soluble transferrin receptor (sTfR) levels impacts on outcomes in idiopathic pulmonary arterial hypertension (IPAH). sTfR is not widely available as an assay. It is also not known how common ID is in other forms of pulmonary hypertension (PH) and whether it impacts on function and survival. Aims: 1) To identify the best routinely-available biomarker of ID in patients with IPAH; 2) Assess the prevalence of ID in common forms of PH; 3) Explore the relationship between ID and function and survival in PH; 4) Run a double-blind, randomised, placebo-controlled trial of iv iron in IPAH. Methods: Data were collected historically from patients in the PH service at Hammersmith Hospital and analysed to provide the best clinical criteria to identify patients with ID. Subsequently, a larger cohort of patients with IPAH as well as other forms of PH was analysed to assess the prevalence of ID at diagnosis and the impact on functional status and survival. A crossover trial was run to assess the impact of iv iron on haemodynamics and exercise in IPAH. Results: ID can be best identified by using red cell distribution width (RDW) in IPAH. RDW is associated with exercise capacity in IPAH, but more so, is associated with functional changes in chronic thromboembolic PH. RDW was a strong marker of survival across all groups but independence could not be established from other markers. The clinical trial did not complete in time, but unblinded data suggest improvements in aerobic exercise, but no change in haemodynamics. Conclusion: ID is common in all forms of PH, and has functional and survival implications. Iron replacement in IPAH may improve exercise function but complete results from the trial are awaited. 4 Table of contents Thesis title page Declaration Acknowledgements Abstract Contents List of figures List of tables List of abbreviations List of Figures Chapter 1 Figure 1.1 Vascular remodelling in pulmonary arterial hypertension Figure 1.15 Regulation of dietary iron uptake and release from from iron-utilising cells by hepcidin. Figure 1.2 Regulation of hepcidin expression Figure 1.3. Regulation of phosphate homeostasis Figure 1.4 . Characterisation of iron status in patients with IPAH Figure 1.5. Soluble transferrin receptor (sTfR) levels in patients with IPAH 5 Chapter 2 Figure 2.1. Normalization of cardiac output data Figure 2.2. Characterization of Iron Status, Hb and inflammation in 153 patients with Idiopathic PAH Cohort 1 Figure 2.3. Relationship between sTfR and mortality Table 2.3. Iron deficiency and ID Figure 2.4. sTfR associations using standard iron markers and inflammation (IL-6) Figure 2.5. Scatter plot showing the relationship of RDW with sTfR Figure 2.6. ROC curves showing the ability of RDW and RDW corrected for IL-6 to predict sTfR> 28.1 Figure 2.7. Correlation between iron and Tsat in patients with IPAH Figure 2.8. Iron status and RDW in patients with IPAH , PAH-CTD , PAH-CHD and CTEPH Figure 2.9. Scatter plot demonstrating the relationship of age (years) with RDW Figure 2.10. The relationship of RDW with 6MWD Figure 2.105 ROC models for 3 year survival with 6MWD, RDW and cardiac output in IPAH. Figure 2.11. Kaplan-Meier curves stratified by median RDW (14.8%) in patients with Idiopathic PAH Figures 2.12 Kaplan-Meier estimates stratified by median iron (12.0 µmol/L) (A) and transferrin saturation (20%) (B) in patients with Idiopathic PAH. Figure 2.13. ROC models showing RDW, CRP and age as predictors of 3 year survival in PAH-CTD. Figure 2.14. Kaplan-Meier estimates stratified by median RDW (16%) and CRP (7 mg/L). Figure 2.15. Kaplan-Meier estimates stratified by median iron (10 µg/L) and transferrin saturation (16.0 %). Figure 2.16. Kaplan-Meier estimates stratified by median RDW (14.8%). Figure 2.17. ROC models for 3 year survival with 6MWD, RDW and cardiac output in CTEPH. Figure 2.18 Kaplan-Meier estimates stratified by median cut-off for RDW (14.9%). Figure 2.19 Kaplan-Meier estimates stratified by median cut-offs for iron Figure 2.20 Kaplan-Meier estimates stratified by median cut-offs for transferrin saturation (22.0%). 6 Chapter 3 Figure 3.1. Schematic overview of a 36-week, double-blind, randomized, placebo-controlled, crossover study Figure 3.2. Ferritin and TIBC in IPAH patients before and after the administration of iron or saline Figure 3.3. Ferritin levels in IPAH patients before and after the administration of iron Figure 3.4. Iron and transferrin saturation levels in IPAH patients before and after the administration of iron or saline Figure 3.5. Ferritin, iron and transferrin saturation levels in IPAH patients before and after the infusion of Ferinject or saline Figure 3.6. Haemoglobin levels in IPAH patients before and after the administration of saline or iron Figure 3.7. Haematocrit in IPAH patients before and after the administration of saline or iron Figure 3.8. MCV levels in IPAH patients before and after the administration of saline or iron Figure 3.9. RDW in IPAH patients before and after the administration of saline or iron Figure 3.10. Circulating phosphate levels in IPAH patients before and after the administration of iron or saline Figure 3.11. Changes in mean right atrial and pulmonary artery pressures, and subsequent pulmonary vascular resistance, 12 weeks after receiving either saline or iron infusion Figure 3.12 Changes in ventricular output measured by cardiac index and stroke volume, and SVO2 measured 12 weeks after receiving saline or iron infusions Figure 3.13.