Health and Social Development, Faculty of (Okanagan)
Health and Exercise Sciences, School of (Okanagan)
DSpace
UBCO
Patrician, Alexander M. D.
Doctor of Philosophy - PhD
University of British Columbia
With ascent to high altitude, the reduction in the partial pressure of oxygen (PO₂) drives the stabilization of the hypoxia-inducible factor (HIF) family. As the key cellular oxygen sensor, HIF up-regulates the expression of numerous genes designed to increase and preserve O₂ tissue delivery. Since iron plays an inverse and constituent role in HIF stabilization, the overall objectives of the current thesis were: 1) determine the role of iron status manipulation on pulmonary, peripheral and cerebral responses to high-altitude; 2) and examine if indigenous high-altitude populations (i.e. Sherpa and Andeans), with ancestral adaptation to their hypoxic environment, exhibit iron-regulatory advantages. Study 1 investigated the effects of ascent and prolonged stay at 5050 m on iron status and hypoxic pulmonary vasoconstriction. The primary findings were that 1) during ascent, reductions in iron bioavailability were more prevalent in lowlanders, compared to Sherpa; 2) following partial acclimatization to 5050 m, iron infusion (HIF-down regulation) attenuated pulmonary artery systolic pressure (PASP) in both lowlanders and Sherpa while deferoxamine infusion (HIF-up regulation) had little influence. Study 2, in a block-randomized and placebo-controlled design, examined whether the pulmonary vasculature of lowlanders and adapted Andeans at 4300 m are responsive to iron infusion. The findings revealed that, unlike the placebo condition, iron infusion reduced PASP in both lowlanders and Andeans. Study 3 then evaluated the role of iron infusion on peripheral vascular function in the same volunteers at 4300 m. Here, iron infusion (but not saline) increased brachial blood flow and increased microvascular function across the 4-hours, in both lowlanders and Andeans. Upon pooling data (n=83), across both lowlanders and highlanders, Study 4 explored whether the same iron manipulations can modulate hypoxic cerebral vasodilation. It was found that baseline iron levels were related to the variability in magnitude of the hypoxic cerebral vasodilation at high-altitude. In summary, new evidence is provided that support iron status may influence blood flow regulation in the pulmonary, peripheral and cerebral circulations during exposure to high-altitude. Future studies are needed to explore the mechanisms and implications of these observations in high-altitude physiology and pathology.
i Physiological aspects of iron status and manipulation in lowlander and adapted highlanders at high altitude by Alexander M. D. Patrician M.Sc, Mid Sweden University 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctorate of Philosophy In THE COLLEGE OF GRADUATE STUDIES (Interdisciplinary Studies) (Health and Exercise Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) March 2022 © Alexander M. D. Patrician, 2022 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Physiological aspects of iron status and manipulation in lowlander and adapted highlanders at high altitude submitted by Alexander M. D. Patrician in partial fulfillment of the requirements of the degree of Doctorate of Philosophy. Prof. Philip Ainslie, School of Health & Exercise Sciences Supervisor Prof. Neil Eves, School of Health & Exercise Sciences Supervisory Committee Member Prof. Jonathan Little, School of Health & Exercise Sciences Supervisory Committee Member Prof. William Sheel, School of Kinesiology University Examiner Dr. Nisha Charkoudian, United States Army Research Institute of Environmental Medicine External Examiner iii Abstract With ascent to high altitude, the reduction in the partial pressure of oxygen (PO2) drives the stabilization of the hypoxia-inducible factor (HIF) family. As the key cellular oxygen sensor, HIF up-regulates the expression of numerous genes designed to increase and preserve O2 tissue delivery. Since iron plays an inverse and constituent role in HIF stabilization, the overall objectives of the current thesis were: 1) determine the role of iron status manipulation on pulmonary, peripheral and cerebral responses to high-altitude; 2) and examine if indigenous high-altitude populations (i.e. Sherpa and Andeans), with ancestral adaptation to their hypoxic environment, exhibit iron-regulatory advantages. Study 1 investigated the effects of ascent and prolonged stay at 5050 m on iron status and hypoxic pulmonary vasoconstriction. The primary findings were that 1) during ascent, reductions in iron bioavailability were more prevalent in lowlanders, compared to Sherpa; 2) following partial acclimatization to 5050 m, iron infusion (HIF-down regulation) attenuated pulmonary artery systolic pressure (PASP) in both lowlanders and Sherpa while deferoxamine infusion (HIF-up regulation) had little influence. Study 2, in a block-randomized and placebo-controlled design, examined whether the pulmonary vasculature of lowlanders and adapted Andeans at 4300 m are responsive to iron infusion. The findings revealed that, unlike the placebo condition, iron infusion reduced PASP in both lowlanders and Andeans. Study 3 then evaluated the role of iron infusion on peripheral vascular function in the same volunteers at 4300 m. Here, iron infusion (but not saline) increased brachial blood flow and increased microvascular function across the 4-hours, in both lowlanders and Andeans. Upon pooling data (n=83), across both lowlanders and highlanders, Study 4 explored whether the same iron manipulations can modulate hypoxic cerebral vasodilation. It was found that baseline iron levels were related to the variability in magnitude of the hypoxic cerebral vasodilation at high-altitude. In summary, new evidence is provided that support iron status may influence blood flow regulation in the pulmonary, peripheral and cerebral circulations during exposure to high-altitude. Future studies are needed to explore the mechanisms and implications of these observations in high-altitude physiology and pathology. iv Lay Summary Iron is an essential element that all mammals require for life. In low oxygen conditions (e.g. at high altitude), the demand for iron increases and consequently, iron levels within the body decline. It is unknown how reduced iron stores influence blood flow across different organs (i.e. lungs, brain and periphery) at high altitude, and whether high altitude populations (who have lived a high altitude for generations) exhibit more advanced strategies for using iron. The current thesis consists of four studies conducted at high altitude, in sea level residents and two different populations who have adapted to high altitude: Sherpa and Andeans. We provide new evidence that iron status may influence blood flow regulation at high-altitude – however, further studies are needed to explore the implications of these observations. v Preface The University of British Columbia Clinical Research Ethics Board approved all experiment chapter in this thesis (CREB IDs: H16-01028, H15-03358, H17-02687 and H18-01404). Chapter 1. This chapter was written by Alexander Patrician, and edited by Prof. Philip Ainslie. Chapter 2. This chapter was written by Alexander Patrician, and edited by Prof. Philip Ainslie. Chapter 3. This chapter was published in the Journal of Physiology: Willie CK, Patrician A, Hoiland RL, Williams AM, Gasho C, Subedi P, Anholm J, Drane A, Tymko MM, Nowak-Flück D, Plato S, McBride E, Varoli G, Binsted G, Eller LK, Reimer RA, MacLeod DB, Stembridge M, Ainslie PN. Influence of iron manipulation on hypoxic pulmonary vasoconstriction and pulmonary reactivity during ascent and acclimatization to 5050 m. J Physiol. 2021 Mar;599(5):1685-1708. Christopher Willie conceptualized and coordinated this study. Alexander Patrician was responsible for all data analyses. Alexander Patrician and Prof. Philip Ainslie drafted the manuscript. Alexander Patrician is the corresponding author. Co-authors edited and approved the final version of the manuscript. CREB IDs: H16-01028, H15-03358 Chapter 4. This chapter was published in Chest: Patrician A, Dawkins T, Coombs GB, Stacey B, Gasho C, Gibbons T, Howe CA, Tremblay JC, Stone R, Tymko K, Tymko C, Akins JD, Hoiland RL, Vizcardo-Galindo GA, Figueroa-Mujíca R, Villafuerte FC, Bailey DM, Stembridge M, Anholm JD, Tymko MM, Ainslie PN. Global Research Expedition on Altitude-related Chronic Health 2018 Iron Infusion at High Altitude Reduces Hypoxic Pulmonary Vasoconstriction Equally in Both Lowlanders and Healthy Andean Highlanders. Chest. 2021 Sep 8:S0012-3692(21)03848-4. Alexander Patrician and Prof. Philip Ainslie designed the experimental protocols in this chapter. Alexander Patrician was responsible for all data analyses, drafted the manuscript and is the corresponding author. Co-authors edited and approved the final version of the manuscript. CREB IDs: H17-02687 and H18-01404. vi Chapter 5. This chapter is currently under review at the American Journal of Physiology-Regulatory, Integrative and Comparative Physiology (ID: R-00016-2022R1): Patrician A, Coombs GB, Stacey B, Tremblay JC, Iannetelli A, Gasho C, Hoiland RL, Dawkins T, Vizcardo-Galindo GA, Figueroa-Mujíca R, Villafuerte FC, Stembridge M, Anholm JD, Tymko MM, Bailey DM, Ainslie PN. Global REACH 2018: Iron infusion improves peripheral blood flow and microvascular function in lowlanders and healthy Andeans at high altitude. Am J Physiol Regul Integr Comp Physiol. Under Review. Alexander Patrician and Prof. Philip Ainslie designed the experimental protocols in this chapter. Alexander Patrician was responsible for all data analyses, drafted the manuscript and is the corresponding author. Co-authors edited and approved the final version of the manuscript. CREB IDs: H17-02687 and H18-01404 Chapter 6. This chapter is currently under review at the Journal of Cerebral Blood Flow and Metabolism (ID: JCBFM-0017-22-ORIG): Patrician A, Willie C, Hoiland RL, Gasho C, Subedi P, Anholm JD, Tymko MM, Philip PN. Iron status and manipulation on cerebral blood flow at high altitude in lowlanders and adapted highlanders. J Cereb Blood Flow Metab. Under Review. Alexander Patrician, Christopher Willie and Prof. Philip Ainslie designed the experimental protocols in this chapter. Alexander Patrician was responsible for all data analyses, drafted the manuscript and is the corresponding author. Co-authors edited and approved the final version of the manuscript. CREB IDs: H16-01028, H15-03358, H17-02687 and H18-01404. Chapter 7. This chapter was written by Alexander Patrician, and edited by Prof. Philip Ainslie. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface ............................................................................................................................................ v Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Equations ........................................................................................................................ xiv Glossary ....................................................................................................................................... xv Acknowledgements .................................................................................................................... xvi Chapter 1. Research Objectives, Aims, and Hypotheses .................................................... 17 1.1. Study 1 (Chapter 3) ....................................................................................................... 17 1.1.1. Aim ........................................................................................................................... 17 1.1.2. Hypotheses ................................................................................................................ 17 1.2. Study 2 (Chapter 4) ....................................................................................................... 17 1.2.1. Aim ........................................................................................................................... 17 1.2.2. Hypotheses ................................................................................................................ 17 1.3. Study 3 (Chapter 5) ....................................................................................................... 17 1.3.1. Aim ........................................................................................................................... 17 1.3.2. Hypotheses ................................................................................................................ 18 1.4. Study 4 (Chapter 6) ....................................................................................................... 18 1.4.1. Aim ........................................................................................................................... 18 1.4.2. Hypotheses ................................................................................................................ 18 Chapter 2. Literature Review ............................................................................................... 19 2.1. What is iron and why is it important in physiology? .................................................... 19 2.2. Uptake and metabolism of iron in humans ................................................................... 21 2.3. Clinical implications of iron dysregulation................................................................... 23 2.4. Why is iron relevant to high altitude physiology? ........................................................ 26 2.5. The integrated vascular regulation of the pulmonary, peripheral and cerebral systems at high altitude ................................................................................................................................ 33 2.5.1. Pulmonary circulation ............................................................................................... 33 2.5.2. Peripheral blood flow ................................................................................................ 40 2.5.3. Cerebral blood flow .................................................................................................. 42 2.6. Iron metabolism in high altitude adaptation ................................................................. 45 2.7. Literature review synopsis ............................................................................................ 48 viii Chapter 3. Influence of iron manipulation on hypoxic pulmonary vasoconstriction and pulmonary reactivity during ascent and acclimatization to 5050 m ...................................... 49 3.1. Background ................................................................................................................... 49 3.2. Materials and methods .................................................................................................. 51 3.2.1. Ethical approval ........................................................................................................ 51 3.2.2. Experimental design.................................................................................................. 51 3.2.2.1. Study 1 – Ascent to 5050 m .................................................................................. 51 3.2.2.2. Study 2 – Iron or DFO infusion following partial acclimatization to 5050 m ...... 52 3.2.2.2.1. Changes in PIO2 ......................................................................................... 53 3.2.2.2.2. Changes in cardiac output .......................................................................... 53 3.2.2.2.3. Infusion ...................................................................................................... 53 3.2.3. Experimental Measures ............................................................................................. 54 3.2.3.1. Echocardiography ................................................................................................. 54 3.2.3.2. Blood sampling ..................................................................................................... 55 3.2.3.3. Biochemical analysis ............................................................................................ 55 3.2.4. Indexes of iron stores ................................................................................................ 56 3.2.4.1. Body store index 1 ................................................................................................ 56 3.2.4.2. Body store index 2 ................................................................................................ 56 3.2.4.3. Iron recruitment index........................................................................................... 57 3.2.5. Statistical Analyses ................................................................................................... 57 3.3. Results ........................................................................................................................... 58 3.3.1. Study 1: Progressive reductions in serum iron and comparable pulmonary pressures in both lowlanders and Sherpa upon ascent to high altitude .................................................... 58 3.3.1.1. Iron homeostasis during ascent ............................................................................. 58 3.3.1.2. Cardiopulmonary status during ascent .................................................................. 59 3.3.2. Study 2a: Comparable pulmonary vascular reactivities to changes in PIO2 – but not during submaximal exercise at 50% Wpeak – in lowlanders and Sherpa following partial acclimatization. ........................................................................................................................ 63 3.3.3. Study 2b: Manipulating iron bioavailability led to similar pulmonary vascular responses in lowlanders and Sherpa to changing PIO2 at rest, but partially divergent reactivities at 25% Wpeak. ......................................................................................................... 64 3.3.3.1. Sub-maximal recumbent exercise at 25% Wpeak ................................................... 65 3.3.4. Study 2c: Inflammatory status is not different between lowlanders and Sherpa and is unaffected by iron manipulation. ............................................................................................. 66 3.4. Discussion ..................................................................................................................... 79 3.4.1. Dissociation of iron status and PASP during ascent to high altitude? ...................... 79 3.4.2. Divergent HPV between lowlanders and Sherpa at altitude? ................................... 80 3.4.3. Iron and the pulmonary vascular reactivity to changing inspired PO2 and submaximal exercise ................................................................................................................ 81 3.4.4. Chemoreflex, pulmonary responsiveness and iron ................................................... 82 3.4.5. Inflammation and iron homeostasis .......................................................................... 83 3.4.6. Is iron supplementation useful at high altitude? ....................................................... 83 3.4.7. Experimental considerations ..................................................................................... 84 3.5. Conclusion .................................................................................................................... 85 ix Chapter 4. GLOBAL REACH 2018: Iron infusion at high altitude reduces hypoxic pulmonary vasoconstriction equally in both lowlanders and healthy Andean highlanders 86 4.1. Background ................................................................................................................... 86 4.2. Methods......................................................................................................................... 87 4.2.1. Ethical Approval ....................................................................................................... 87 4.2.2. Participants ................................................................................................................ 87 4.2.3. Experimental Design ................................................................................................. 88 4.2.3.1. Infusion ................................................................................................................. 88 4.2.4. Experimental Measures ............................................................................................. 89 4.2.4.1. Echocardiography ................................................................................................. 89 4.2.4.2. Biochemical analysis ............................................................................................ 90 4.2.5. Statistical analysis ..................................................................................................... 90 4.3. Results ........................................................................................................................... 90 4.3.1. Comparison of lowlanders and healthy Andeans at baseline .................................... 90 4.3.1.1. Iron metrics ........................................................................................................... 90 4.3.1.2. Hypoxic pulmonary vasoconstriction ................................................................... 91 4.3.2. Effects of intravenous iron ........................................................................................ 92 4.3.2.1. Iron metrics ........................................................................................................... 92 4.3.2.2. Time course over 4 hours at high altitude ............................................................. 94 4.3.2.3. Hypoxic pulmonary vasoconstriction ................................................................... 94 4.4. Discussion ................................................................................................................... 102 4.4.1. Pulmonary vascular responsiveness to hypoxia and iron ....................................... 102 4.4.2. Iron metabolism – higher ferritin levels in Andeans .............................................. 103 4.4.3. Considerations of iron status during prolonged stay at high altitude...................... 104 4.5. Interpretation ............................................................................................................... 105 Chapter 5. Global REACH 2018: Iron infusion improves peripheral blood flow and microvascular function in lowlanders and healthy Andeans at high altitude ..................... 106 5.1. Background ................................................................................................................. 106 5.2. Material and Methods ................................................................................................. 107 5.2.1. Ethical Approval ..................................................................................................... 107 5.2.2. Participants .............................................................................................................. 107 5.2.3. Experimental Design ............................................................................................... 108 5.2.3.1. Infusion ............................................................................................................... 108 5.2.4. Experimental measures ........................................................................................... 109 5.2.4.1. Flow-mediated dilation and reactive hyperemia ................................................. 109 5.2.4.2. Blood pressure, heart rate, and pulse oximetry ................................................... 109 5.2.4.3. Nitric oxide sampling and analysis ..................................................................... 109 5.2.4.4. Iron status ............................................................................................................ 110 5.2.4.5. Viscosity, Hb and Hct ......................................................................................... 110 5.2.5. Data analyses .......................................................................................................... 110 5.2.6. Statistical analyses .................................................................................................. 111 5.3. Results ......................................................................................................................... 111 5.3.1. Pre-infusion (baseline) ............................................................................................ 111 x 5.3.2. Post-infusion (saline versus iron)............................................................................ 112 5.4. Discussion ................................................................................................................... 120 5.4.1. Iron and peripheral tissue perfusion ........................................................................ 121 5.4.2. Iron and NO ............................................................................................................ 121 5.4.3. Clinical basis of iron infusion on endothelial dysfunction ..................................... 122 5.4.4. Experimental limitations and practical implications............................................... 123 5.5. Perspectives and significance ..................................................................................... 124 Chapter 6. Iron status and manipulation on cerebral blood flow at high altitude in lowlanders and adapted highlanders ...................................................................................... 125 6.1. Background ................................................................................................................. 125 6.2. Materials and Methods ................................................................................................ 127 6.2.1. Ethical Approval ..................................................................................................... 127 6.2.2. Experimental Design ............................................................................................... 127 6.2.3. Participants .............................................................................................................. 128 6.2.3.1. Study 1 – Lowlanders and Sherpa at 5,050 m .................................................... 128 6.2.3.2. Study 2 – Lowlanders and Andeans at 4300 m................................................... 128 6.2.3.3. Infusion randomization ....................................................................................... 129 6.2.4. Changes in PIO2 ...................................................................................................... 129 6.2.5. Experimental measures ........................................................................................... 129 6.2.5.1. Cerebral blood flow ............................................................................................ 129 6.2.5.2. Iron status ............................................................................................................ 130 6.2.6. Data analysis ........................................................................................................... 130 6.2.7. Statistical analysis ................................................................................................... 130 6.3. Results ......................................................................................................................... 131 6.3.1. Baseline iron status on global cerebral blood flow ................................................. 131 6.3.2. Between study infusion comparability.................................................................... 131 6.3.3. Study 1: DFO and iron at 5,050 m .......................................................................... 132 6.3.4. Study 2: Saline and iron at 4,300 m ........................................................................ 133 6.4. Discussion ................................................................................................................... 139 6.4.1. Cerebral blood flow, HIF expression, and iron status at high altitude ................... 139 6.4.2. Exaggerated vertebral artery blood flow during hypoxia ....................................... 141 6.4.3. Role of high-altitude ancestry ................................................................................. 141 6.5. Conclusion .................................................................................................................. 142 Chapter 7. Conclusion ......................................................................................................... 143 7.1. Overview of research objectives and study findings .................................................. 143 7.2. Overall interpretation – Is iron supplementation useful? ............................................ 144 7.3. Overall interpretation – Is there a facet of iron metabolism that can be considered to be an attribute of successful high-altitude adaptation? ................................................................. 147 7.3.1. Potentially confounding influence of heavy metal pollutants at high altitude ........ 148 7.4. Field research at high altitude and the challenges ...................................................... 149 7.4.1. Temporal assessments of physiological outcomes ................................................. 149 xi 7.4.2. Ethiopia ................................................................................................................... 150 7.5. Future directions ......................................................................................................... 150 7.5.1. Role of sex in iron metabolism at high altitude ...................................................... 150 7.5.2. Microgravity and space ........................................................................................... 152 7.6. Conclusion .................................................................................................................. 153 Bibliography .............................................................................................................................. 154 xii List of Tables Table 2.1. Laboratory data of normal and progressive stages of iron deficiency. ........................ 25 Table 3.1. Sherpa demographics and time at altitude. .................................................................. 67 Table 3.2. Iron homeostasis during ascent to altitude in lowlanders and Sherpa. ........................ 68 Table 3.3. Cardiopulmonary variables during ascent to altitude in lowlanders and Sherpa. ........ 69 Table 3.4. Pulmonary vascular reactivities following partial acclimatization at 5050 m. ............ 70 Table 3.5. Iron homeostasis and hematological measures prior to and following iron or DFO infusion. ........................................................................................................................................ 72 Table 3.6. Cardiopulmonary variables prior to and following iron or DFO infusion. .................. 73 Table 3.7. Cardiopulmonary responses to submaximal exercise in lowlanders and Sherpa, before and after either infusion of iron or DFO. ...................................................................................... 75 Table 3.8. Inflammatory markers in lowlander and Sherpa at 5050 m......................................... 77 Table 3.9. Impact of iron manipulation on inflammatory markers at 5050 m .............................. 78 Table 4.1. Baseline summary of pooled lowlanders and pooled Andeans. .................................. 96 Table 4.2. Cardiopulmonary measures in pooled lowlanders and Andeans (prior to infusion) during room air breathing and reduced PIO2................................................................................. 97 Table 4.3. Resting cardiopulmonary measures at baseline and following the infusion of saline at 4300 m. ......................................................................................................................................... 98 Table 4.4. Resting cardiopulmonary measures at baseline and following the infusion of iron at 4300 m. ......................................................................................................................................... 99 Table 4.5. Cardiopulmonary reactivities to hypoxia, prior-to and following the infusion of saline...................................................................................................................................................... 100 Table 4.6. Cardiopulmonary reactivities to hypoxia, prior-to and following the infusion of iron...................................................................................................................................................... 101 Table 5.1. Measurement summary of pooled lowlanders and healthy Andeans at baseline....... 116 Table 5.2. Hematological measures by infusion type. ................................................................ 117 Table 5.3. Peripheral vascular function in lowlander and Andeans receiving saline infusion. .. 118 Table 5.4. Peripheral vascular function in lowlander and Andeans receiving iron infusion. ..... 119 Table 6.1. During room air breathing (PIO2=87 mmHg) at pre- and post-infusion at 5,050 m in lowlanders and Sherpa. ............................................................................................................... 135 Table 6.2. Absolute change from room air (PIO2=87mmHg) to hypoxia (PIO2=67mmHg) in lowlanders and Sherpa. ............................................................................................................... 136 Table 6.3. During room air breathing (PIO2=96 mmHg) at pre- and post-infusion at 4,300 m in lowlanders and healthy Andeans. ............................................................................................... 137 Table 6.4. Absolute change from room air (PIO2=96 mmHg) to hypoxia (PIO2=73 mmHg) in lowlanders and healthy Andeans. ............................................................................................... 138 xiii List of Figures Figure 2.1. Paleontological significance of iron for life on Earth. ............................................... 20 Figure 2.2. An overview of iron absorption and metabolism. ...................................................... 22 Figure 2.3. Illustration of the role of prolyl hydroxylase (PHD) in stabilization of HIF.............. 27 Figure 2.4. Summary of high-altitude studies assessing iron status using serum iron and/or ferritin, during ascent to- and prolonged stay at specific altitude. ................................................ 28 Figure 2.5. Iron absorption and uptake into red blood cells (RBCs). ........................................... 30 Figure 2.6. Iron status markers during Operation Everest III. ...................................................... 31 Figure 2.7. Iron infusion and acute mountain sickness (AMS). ................................................... 33 Figure 2.8. Hypoxic pulmonary vasoconstriction (HPV) in humans............................................ 34 Figure 2.9. Time course of HPV in humans exposed to hypoxia. ................................................ 37 Figure 2.10. Iron (or saline) infusion in lowlanders before ascent to 5050 .................................. 39 Figure 2.11. Arteriogenesis via digital angiograms of a hindlimb ischemic model in rabbits. .... 41 Figure 2.12. Distribution of non-heme iron in the human brain. .................................................. 44 Figure 2.13. Relationship between pulmonary pressure and cardiac output in lowlanders and high-altitude natives. ..................................................................................................................... 47 Figure 3.1. Iron metric during ascent to 5050 m in lowlanders (black) and Sherpa (red). ........... 59 Figure 3.2. Serial cardiopulmonary measurements in 20 lowlanders (black) and 12 Sherpa (red) during ascent from Kathmandu (1400 m) to 5050 m at the Pyramid laboratory. ......................... 60 Figure 3.3. Indices of total iron stores in 20 lowlanders (black) and 11 Sherpa (red) during ascent to high altitude. ............................................................................................................................. 62 Figure 3.4. Pooled pulmonary vascular reactivities to changing inspired O2 and cardiac output in both lowlanders and Sherpa prior to infusion. .............................................................................. 63 Figure 3.5. Impact of iron infusion [via iron (III)-hydroxide sucrose] and iron chelation (via desferrioxamine) in lowlanders and Sherpa at 5050 m. ................................................................ 64 Figure 3.6. Individual and mean pulmonary reactivity to increasing cardiac output – coinciding to baseline, and 25 % peak wattage during recumbent cycling – in lowlanders and Sherpa. ....... 65 Figure 4.1. Impact of saline (grey bars) or iron infusion (red bars) on iron metrics in lowlanders and healthy Andeans. .................................................................................................................... 93 Figure 4.2. Impact of saline and iron infusion on the PASP in lowlanders and healthy Andeans........................................................................................................................................................ 94 Figure 4.3. Pulmonary vascular reactivity to hypoxia in lowlanders and Andeans receiving either saline or iron infusion. .................................................................................................................. 95 Figure 4.4. Baseline serum iron measurements in lowlanders and Andeans. ............................. 105 Figure 5.1. Impact of saline infusion. ......................................................................................... 113 Figure 5.2. Impact of iron infusion. ............................................................................................ 115 Figure 5.3. Summary of the impact of iron infusion on peripheral vascular function in hypoxia...................................................................................................................................................... 120 Figure 6.1. Influence of baseline iron status on gCBF in pooled lowlanders and pooled highlanders at high altitude. ........................................................................................................ 132 Figure 6.2. The change of blood flow to the posterior part of the brain (i.e., ∆QVA) from baseline to hypoxia). ................................................................................................................................. 134 Figure 6.3. Summary of global CBF during prolonged stay at high altitude.............................. 140 Figure 7.1. Summary of the role of iron (Fe) infusion on pulmonary, peripheral and cerebral responses at high-altitude. ........................................................................................................... 146 xiv List of Equations Equation 3.1. Stoke volume (SV). ................................................................................................ 54 Equation 3.2. Pulmonary artery systolic pressure (PASP). .......................................................... 54 Equation 3.3. Mean pulmonary artery pressure (mPAP). ............................................................. 54 Equation 3.4. Pulmonary vascular resistance (PVR). ................................................................... 54 Equation 3.5. Pulmonary vascular resistance proportional to hematocrit (PVRHct) ..................... 54 Equation 3.6. Transferrin saturation (TrF Sat).............................................................................. 55 Equation 3.7. Body iron. ............................................................................................................... 56 Equation 3.8. Soluble transferrin receptor (sTfR) index. .............................................................. 56 Equation 3.9. Iron recruitment index ............................................................................................ 57 Equation 4.1. Mosteller body surface area.................................................................................... 89 Equation 4.2. Total iron binding capacity (TIBC). ....................................................................... 90 Equation 5.1. Blood flow. ........................................................................................................... 110 Equation 6.1. PETCO2 calculation. .............................................................................................. 130 xv Glossary AMS – acute mountain sickness CBF – cerebral blood flow CMS – chronic mountain sickness CI – cardiac index CO – cardiac output DcytB – duodenal cytochrome B reductase DFO – desferrioxamine EPO – erythropoietin Fe2+ – ferrous iron Fe3+ – ferric iron FMD – flow mediated dilation HAPE – high altitude pulmonary edema Hb – hemoglobin Hct – hematocrit HIF – hypoxia-inducible factor HPV – hypoxic pulmonary vasoconstriction IRP – iron regulatory protein IRE – iron-responsive element MAP – mean arterial pressure mPAP – mean pulmonary artery pressure NO – nitric oxide NTBI – non-transferrin bound iron PASP – pulmonary artery systolic pressure Pb – lead PETCO2 – partial pressure of end-tidal CO2 PHD – prolyl hydroxylase-domain PIO2 – partial pressure of inspired O2 PVR – pulmonary vascular resistance RH – reactive hyperemia ROS – reactive oxygen species SpO2 – peripheral oxygen saturation sTfR – soluble transferrin receptor SV – Stoke volume TfR1 – transferrin receptor 1 TrF sat – transferrin saturation TIBC – Total iron-binding capacity VE – ventilation VEGF – higher vascular endothelial growth factor xvi Acknowledgements First, I would like to thank Prof. Philip Ainslie. As a mentor and friend, I am continually impressed with your ability to inspire those around you and make the most out of both science and life. It has been life-defining and a true highlight working with you. Drs. Jonathon Moore and Michael Stembridge, while you have both been sources of encouragement and support over the years, had it not been for our early conversations back in 2016, I would not have known about the 2016 UBC Expedition to Nepal or been introduced to such a great group. Dr. Christopher Willie, exploring iron regulation in hypoxia was your original idea. As lead of the 2016 UBC expedition to Nepal, I learned much from you, and this thesis relied greatly upon your efforts. While life was certainly too short, the footsteps you left behind will never be forgotten. I would like to give a special thanks to my committee members Profs. Neil Eves and Jonathan Little who have always been encouraging and supportive. Special thanks to Neil, who, on more than one occasion, provided me with valuable equipment and insight for pursuing diving research projects in Croatia. To my current and past lab mates, thank you for being a second family and donating countless hours along the way – this thesis would certainly not be possible without you. It has been a true pleasure to work and explore the world (and limits) with you. To my friends and family, especially my mom (Laura), thank you for your unconditional support. Lastly, and most importantly, to Melissa, thank you for always being there, your endless encouragement, and the many adventures along the way. 17 Chapter 1. Research Objectives, Aims, and Hypotheses 1.1. Study 1 (Chapter 3) 1.1.1. Aim To examine the adaptational role of iron bioavailability on the pulmonary vascular responses to acute and chronic hypobaric hypoxia during rest, exercise and acute changes in the partial pressure of inspired O2 (PIO2) in sea-level residents and high altitude adapted Sherpa. 1.1.2. Hypotheses The two primary hypotheses were: (1) during progressive ascent to high altitude (5050 m), the increase in hypoxic pulmonary vasoconstriction (HPV) will be attenuated in Sherpa compared to lowlanders due, in part, to Sherpa demonstrating an improved iron status [and a theoretically hypo-responsive hypoxia inducible factor (HIF) system]; and (2) following ∼2 weeks of partial acclimatization to 5050 m, iron infusion will attenuate, and iron chelation will amplify the HPV both at rest and during acute changes in PIO2, and submaximal exercise; however, these responses would again be blunted in Sherpa compared to lowlanders. 1.2. Study 2 (Chapter 4) 1.2.1. Aim To examine the role of iron bioavailability on the pulmonary vascular regulation to hypoxia in sea-level residents and healthy high-altitude adapted Andeans, with and without acute reductions in PIO2. 1.2.2. Hypotheses Healthy Andeans and lowlanders would demonstrate comparable pulmonary vascular responses to hypoxia, and both groups would be sensitive to iron infusion. 1.3. Study 3 (Chapter 5) 1.3.1. Aim To explore the iron-mediating role on HIF and the peripheral vasculature in humans during a HIF-up regulated state (i.e. high-altitude exposure, where the combined effects of hypoxia and iron store depletion are expected to be prevalent) 18 1.3.2. Hypotheses Iron infusion would lead to an increase in both peripheral blood flow and microvascular function; these elevations would be more apparent in lowlanders, compared to the high altitude adapted Andeans 1.4. Study 4 (Chapter 6) 1.4.1. Aim To explore the role of iron status on cerebral blood flow (CBF) at high altitude, in populations with varying adaptational status (i.e. sea-level residents, Sherpa and Andeans at high altitude) 1.4.2. Hypotheses Elevations in CBF would occur in response to acute iron depletion (i.e. increasing HIF activity) and repletion (i.e. decreasing HIF activity). Furthermore, we hypothesized that during exaggerated hypoxia at high altitude, the increase in CBF would be amplified by chelation and attenuated by iron infusion. Finally, by assessing iron status in high altitude populations with ancestral adaptation to hypoxic exposure, we sought to explore whether the genotypic advantages of Andeans and Sherpas would manifest through the modulation of CBF. 19 Chapter 2. Literature Review 2.1. What is iron and why is it important in physiology? Iron is Earth’s fourth-most common element, comprising 6.2% of the Earth’s crust (Aggett, 2012) and serves as a key constituent in Earths magnetosphere (NASA, 2021). Aside from planetary accretion and the good fortune of exhibiting unique physical and chemical conditions that facilitated optimal core-mantle segregation, the biological significance of iron dates back billions of years (Wade et al., 2021). Before the rise in atmospheric O2 levels, ~2.45 billion years ago (known as the Great Oxygenation Event (Holland, 2006)], ferrous iron (Fe2+) was found in high concentrations in the sea (Figure 2.1). At such time, Archean life existed in an anoxic environment with an abundance of soluble iron. For example, the proliferation of iron-sulfur clusters were catalytic cofactors crucial for biological electron transport (Johnson et al., 2005), from anaerobically active Archaea (Boyd et al., 2009). Although controversial, the synchronous deposition of banded iron formations with the Great Oxygenation Event suggests an interconnectedness between the propagation of life and iron regulation. For example, a simplified model proposed by Knoll & Nowak (2017), highlights such a transition using two likely ancient bacterial populations of the sea; anoxygenic photosynthetic bacteria (use Fe2+ as an electron donor) and cyanobacteria (used water to gain electrons). The dominance of anoxygenic photosynthetic bacteria would eventually be over taken by cyanobacteria, as atmospheric O2 levels rose and soluble iron was oxidized, forming banded iron formations. Over millions of years, a point of inflection was reached, where the growth rate of anoxygenic photosynthetic bacteria rapidly declined and cyanobacteria exponentially increased. This ultimately caused an explosion of cyanobacteria and, therefore, the Great Oxygenation Event (Knoll & Nowak, 2017). An interesting feature conserved across cyanobacteria, is that they are equipped with siderophore-synthesizing systems (Hider & Kong, 2010). Siderophores possess the necessary mechanisms to efficiently acquire insoluble Fe3+, which in a progressively ferrous-depleting environment, presents these cyanobacteria with an evolutionary advantage (Wade et al., 2021). Stratification of sea water was also expected to have occurred (surface photic layer would contain with less soluble Fe2+ compared to deeper layers); thus, such gradual conditions could have provided an additionally selective pressure for the survival of siderophorically-capable cyanobacteria (Williams & Rickaby, 2012; Wade et al., 2021). 20 Figure 2.1. Paleontological significance of iron for life on Earth. Before the great oxygenation event (GOE) at 2.45 Ga, soluble ferrous iron (Fe2+) was found in high concentrations in the sea. Eventually photosynthesizing bacteria led to the oxidation of Fe2+ to Fe3+ and precipitated as banded iron formations; this facilitated the surge in atmospheric oxygen (O2) and emergence of eukaryotic organisms. CO2, carbon dioxide. Adapted from (Williams, 2012) with permission. Since iron regulatory processes are conserved across pathogen, plant and animal kingdoms, it has prompted postulation that iron homeostasis may have even preceded oxygen homeostasis (Theil & Goss, 2009). For example, assimilation of iron is a crucial factor influencing infection and pathogen success (Cassat & Skaar, 2013). In plants, there exist mechanisms that enhance iron update from the soil and facilitate iron transport for respiration and photosynthesis (Kim & Guerinot, 2007). In the animal kingdom, iron fulfills a multitude of roles in our physiology as it dictate a multitude of redox reactions necessary for O2 and energy metabolism (Wessling-Resnick, 2012). 012345Total biomass (au)Billions of years ago (Ga)Unicellualr EukaryotesMulticellular eukaryotesProkaryotesLog [O2]Log [CO2] O2CO2GOEBanded iron formation (au)Earth formsLogFe(aq) 21 In animals, the most notable role of iron is demonstrated through its function in metalloproteins (i.e. proteins containing at least one iron ion), such as 1) heme-globins [e.g. hemoglobin (Hb), myoglobin], neuroglobin and ferroportin; 2) heme-enzymes involved with electron transfer of cytochrome function (e.g. cytochrome P450 oxidases) and oxidases important for protecting against oxidative stress (e.g. peroxidases and catalase); 3) iron-sulfur clusters involved in energy production (succinate, isocitrate and NAPH dehydrogenases, and aconitase) and oxidoreductase activities (e.g. xanthine oxidase); 4) iron-cofactor dependent activities (phenylalanine, tyrosine, and tryptophan hydroxylases, and proline and lysine hydroxylases) [reviewed in: (Aggett, 2012)]. Therefore, iron is an essential constituent of life. As such, any marked decline in iron bioavailability is associated with negative impacts to human health (Camaschella, 2019). The subsequent sections will cover how humans acquire iron and explore clinical pathological conditions and their consequences of iron dysregulation. Given the role of iron metabolism at high altitude, the integrated physiological responses to hypoxia will also be covered, with a focus on acclimatization and adaptation. 2.2. Uptake and metabolism of iron in humans In humans, iron [which biologically exists as either Fe2+ (ferrous) or ferric (Fe3+)] is required to come from exogenously sources. Thus, iron can be derived from Hb and myoglobin (via red meat, fish and poultry; referred to as heme iron), or from plants (e.g. beans, grains, vegetables; referred to as non-heme iron). The absorption rates of heme iron are ~15-35%, compared ~2-20% for non-heme iron (Wessling-Resnick, 2012). Upon digestion and passage into the duodenum, Fe3+ is converted to Fe2+ via duodenal cytochrome B reductase (DcytB), for absorption into enterocytes via divalent metal transporter 1 (DMT1; Figure 2.2). The exact transporter for the uptake of heme iron has yet to be elucidated, but upon entry into enterocytes, it is degraded by heme-oxygenase 1 (Ganz, 2013; Muckenthaler et al., 2017). The iron exporter protein ferroportin, exports Fe2+ into the extravascular space and circulation, where ceruloplasmin re-convers Fe2+ back into Fe3+ for binding to transferrin (via oxidation of hephaestrin). Transferrin accepts two iron atoms and limits the accumulation (and therefore the potential for pro-oxidant effects) of free iron (i.e., non-transferrin bound iron; NTBI). 22 Transferrin bound iron acts as a dynamic reservoir that facilitates the movement of iron to/from storage via ferritin (in the liver or other tissues) or utilization in erythropoiesis (with a daily cycling of 20-25 mg of iron per day) and in other cellular processes [e.g. metalloprotein function (Wessling-Resnick, 2012)]. Ferritin is a protein complex consisting of 24 protein subunits, that encapsulates thousands of iron atoms, and the amount measured in the blood serves as a clinical indicator of iron store status (Milman et al., 1983). However, according to a recent Cochrane review, serum ferritin [at a threshold of 30 ug/l] is only a reasonable indicator of iron deficiency, and there is a lack of robust evidence for serum ferritin to be able to diagnose iron-deficiency in healthy people (in the absence of disease) (Garcia-Casal et al., 2021). Figure 2.2. An overview of iron absorption and metabolism. Dietary iron (Fe) absorbed in enterocytes and exported by ferroportin (FPN) into circulation where iron binds to transferrin (Tf) and circulated throughout body. Iron encapsulated within in ferritin and stored predominantly in the liver and muscle tissue. Iron use ranges from erythropoiesis and heme-globin (HB) synthesis, serving as a constituent for energy production processes (EP), and within cofactor-dependent activities (CoF). Approximate iron stores (in mg) and distribution throughout the body are included. Iron is lost from menstruation, blood loss, cell shedding, urine and sweat. 23 The balance between the storage of iron versus utilization (termed iron homeostasis) is controlled by the coordination of a number of factors, namely: iron regulatory proteins [IRPs; controls the levels of iron uptake proteins such as transferrin receptor 1 (TfR1) and DMT1]; the iron storage protein ferritin; the iron exporter protein ferroportin (mediates the uptake of iron from duodenal enterocytes, and release of iron from the liver, spleen, and muscle tissues into the circulation); and hepcidin (produced in the liver which promotes iron storage and limits the release of iron into the circulation and its availability to peripheral tissues by internalizing ferroportin) [reviewed in: (Hirota, 2019; Lakhal-Littleton et al., 2019)]. During times of low iron levels, IRPs (i.e. IRP1 & IRP2) post-transcriptionally bind to iron-responsive elements (IREs) to promote cellular iron uptake by increasing transferrin receptor mRNA and prevent sequestration by inhibiting ferritin mRNA translation [reviewed in: (Smith et al., 2008b)]. 2.3. Clinical implications of iron dysregulation In men and women, the total iron body content is estimated to be 3.8 g and 2.3 g, respectively (Wessling-Resnick, 2012). Females typically have lower iron levels – a feature likely due to a combination of factors ranging from menstrual blood volume losses, reduced dietary intake/absorption and pregnancy. Consequently, this also means that females are more likely to be iron deficient (Lopez et al., 2016; Pasricha et al., 2021). For example, it is estimated that world-wide 33% of non-pregnant and 40% of pregnant women are iron-deficient anemic, with even more expected to be iron deficient non-anemic (WHO, 2020). In an individual with replete iron levels, the iron pools within the body are contained within circulating erythrocytes (1 mg/ml packed volume which equates to 1800-2500 mg), stored within the liver/spleen (mostly as ferritin; 300-1000 mg), utilized within bone marrow for erythropoiesis (100-300 mg), bound to transferrin (2-4 mg) and recycled by reticuloendothelial macrophages at a daily rate of 15-25 mg/day and absorbed/excreted (1-2 mg/day (Hentze et al., 2004; Hentze et al., 2010; Mole, 2010; Ganz, 2013; Muckenthaler et al., 2017). Upon normal conditions, erythrocytes are produced in the bone marrow at rate of 200x109 red blood cells/day, which equates to 6 g/day of Hb requiring 20 mg/day of iron (Muckenthaler et al., 2017). 24 During states of reduced dietary iron consumption or impairments to absorption and/or metabolism, iron status of the body falls outside the optimal range (Table 2.1) and leads to an array of clinical conditions/pathologies (Steinbicker & Muckenthaler, 2013). Iron deficiency can arise from a variety of stimuli, including low iron intake (e.g. vegans/vegetarians and malnutrition), decreased absorption of iron (e.g. celiac disease, gastrectomy), chronic blood loss (e.g. bleeding, donation), a co-existing pro-inflammatory state (e.g., chronic kidney disease, autoimmune disorders) and/or an increased demand for iron (e.g., children/adolescence, pregnant women, high altitude sojourn) (Camaschella & Girelli, 2020). The pre-existing condition, will also dictate the allocation absorbed iron. For example, using radioactive iron (59Fe) tracing (Callender, 1962), it has been demonstrated that iron replete individuals store ~30% of orally consumed iron and utilize ~70% for erythrocytosis over 10-14 days; while in iron-deficient individuals, 100% of consumed iron will be detectable in new red blood cells within a couple days (therefore, stores remain low); and in the condition of anemia, a greater portion of the absorbed iron will be stored, rather than be directed to erythrocytosis (Callender, 1962). Iron overload can also arise from a variety of situations, via the increased absorption of intestinal iron (from 2-4 mg/day; e.g. hereditary hemochromatosis), ineffective erythrocytosis (e.g. thalassemia), exaggerated hemolysis (e.g. sickle cell disease) and coincide with a pro-inflammatory state (e.g. liver disease) or transfusion overload (e.g. to treat hemolytic anemias) (Hollerer et al., 2017; Bacon & Kwiatkowski, 2021). It is interesting that both iron deficiency and overload can be attributable to underlying inflammatory-related pathology. For example, while ferritin, hepcidin and ceruloplasmin are involved in iron regulation, they are also all acute phase reactants of inflammation (Ross, 2017), and integrated within the activity derived from HIF family (Hirota, 2019). As such, elevated ferritin levels (e.g., >200-300 ng/ml) are indicative of a myriad of medical concerns [e.g., liver disease, hepatitis, infection, inflammation, hemochromatosis (Wallace & Subramaniam, 2009; Wessling-Resnick, 2010; Swenson et al., 2018)]. Therefore, there is substantial overlap and confounding influence between inflammatory responses and commonly utilized indices of iron regulation (Ross, 2017). 25 Table 2.1. Laboratory data of normal and progressive stages of iron deficiency. Iron deficiency with severe anemia Iron deficiency with mild anemia Iron deficiency without anemia Normal Iron overload (asymptomatic) Iron overload (symptomatic) Hemoglobin 6-7 g dl-1 9-12 g dl-1 Women: 12-15 g dl-1 Men: 14-17 g dl-1 Women: 12-15 g dl-1 Men: 14-17 g dl-1 - - Red blood cell size and appearance Decrease in MCV and decrease in MCHC slight decrease in MCHC Normal Normal - - Serum Ferritin <10 µg l-1 <20 µg l-1 <40 µg l-1 40 to 200 µg l-1 Women: 120-1000 µg l-1 Men: 150-1000 µg l-1 500-1000 µg l-1 Serum iron <40 µg dl-1 <60 µg dl-1 60 to 150 µg dl-1 60 to 150 µg dl-1 150-280 µg dl-1 180-300 µg dl-1 Total iron-binding capacity (TIBC; transferrin) >410 µg dl-1 350 to 400 µg dl-1 300 to 390 µg dl-1 300 to 360 µg dl-1 Transferrin saturation <10% <15% 20% 20-50 % 45-100% 80-100% Reticulocyte hemoglobin Data not available 14.8-34.0 pg 22.3-34.7 pg 30.6-35.4 pg - - Bone marrow iron stain Iron absent Iron absent Iron absent Adequate iron present - - Non-transferrin bound iron† - - - <1 µmol l-1 1-10 µmol l-1 Bold type indicated the progressive step toward iron deficiency and the test most likely to define such stage. MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume. Bone marrow iron staining is considered the gold standard, however is not routinely used in the evaluation or diagnosis of iron deficiency. †In conditions of congenital transferrin deficiencies, NTBI has been reported to be as high as 20 µmol l-1 [data from (Anderson, 1999)]. Iron deficiency and overload adapted from (Auerbach, 2021) and (Bacon et al., 2011; Bacon, 2021), respectively. 26 2.4. Why is iron relevant to high altitude physiology? With ascent to high altitude, there is a progressive reduction in the partial pressure of oxygen (PO2) and stabilization of the hypoxia-inducible factor (HIF) family. The HIF family, particularly HIF- has been established as the key cellular oxygen sensor (Semenza et al., 1998; Kaelin & Ratcliffe, 2008) that binds to hypoxia-responsive elements in gene promoters to up-regulate expression of >100 genes to coordinate the increase in O2 supply to hypoxic tissue. Iron – due to its constituent role in HIF- stabilization, via prolyl hydroxylase domain (PHD) activity [Figure 2.3 (Maxwell et al., 1999; Knowles et al., 2003)] – influences the coordination of HIF-mediated responses (Gassmann & Muckenthaler, 2015). One of the most notable HIF-mediated responses is erythropoiesis – which is the production of red blood cells to elevate the arterial carrying capacity of the blood. Following the transcription the erythropoietin (EPO) gene, and production of EPO from the kidney, circulating levels of EPO increase within hours of exposure to altitudes as low as 1700-2800 m [at 6 & 24 hrs; (Ge et al., 2002)] to 3000-4000 m [significant from 114 min and 84 min, respectively (Eckardt et al., 1989)]. These elevations in EPO persist for as long as the individual continues to ascend and have sufficient iron stores [up to 8000 m over 31 days (Robach et al., 2004)]. However, upon persistence at an altitude, EPO declines over a week, while hemoconcentration occurs (Robach et al., 2004; Siebenmann et al., 2017). Hb is a tetrameric molecule composed of two alpha- and two beta-globin heme pairs (each containing a Fe2+ iron atom), that in total, has the ability to bind to four molecules of O2 (equates to 1.34 ml of O2 g-1 of Hb). Notwithstanding the different Hb isoforms and genotypic differences across vertebral mammals, the importance of Hb for O2 transport and supporting aerobic metabolism, is highlighted in the feature that Hb allows blood to carry 50-70 times more O2 than plasma would alone (Mairbäurl & Weber, 2012). 27 Figure 2.3. Illustration of the role of prolyl hydroxylase (PHD) in stabilization of HIF. Illustration of the role of PHD in stabilization of HIF. Stimulatory actions are denoted by ↓ and inhibitory actions by ⊥. Factors that disrupt PHD activity [e.g., reactive oxygen species (ROS)] allow HIF stabilization. From (Kaelin & Ratcliffe, 2008) with permission. 28 Figure 2.4. Summary of high-altitude studies assessing iron status using serum iron and/or ferritin, during ascent to- and prolonged stay at specific altitude. Filled and open symbols denote serum iron and ferritin respectively. For each study, sex of participants are also denoted (m, male; f, female; n/a, if not provided). Sea level (SL) altitudes were at <500 m. Negative days indicate ascent to high altitude, while zero and positive days indicates the arrival and duration of residence at high altitude, respectively. The altitudes of studies included are from 3450 m (Klein et al., 2021), 4300 m (Robach et al., 2004; Smith et al., 2009; Talbot et al., 2012), 4559 m (Mairbäurl et al., 1990; Robach et al., 2007; Goetze et al., 2013; Altamura et al., 2015), 5050 m (Bonfichi et al., 2000; Holdsworth et al., 2020), 5260 m (Ryan et al., 2014) and 5400 m (Piperno et al., 2011). At high altitude, because of the combined increase in the requirement for iron (i.e. erythropoiesis), but decrease in appetite and food consumption (particularly of dietary iron), there is a general decline in iron bioavailability [Figure 2.4 (Mairbäurl et al., 1990; Bonfichi et al., 2000; Robach et al., 2004; Robach et al., 2007; Smith et al., 2009; Piperno et al., 2011; Talbot et al., 2012; Goetze et al., 2013; Ryan et al., 2014; Altamura et al., 2015; Holdsworth et al., 2020)]. The importance of iron in high altitude research was appreciated as early as the 1950s. Initially recognized during the sojourn of patients with tuberculosis to 1800 m, it was postulated that low iron stores compromised the ability to acclimatize (Wanner & Jasinski, 1953). In 1954, Cruscuolo and colleagues assessed rats at ~6100 m for 6 weeks and demonstrated that, while survival was comparable between rats fed a low iron diet (compared to those on a high iron diet), the rise in Hb was only significant in the high-iron group (Criscuolo et al., 1955). In humans, Reynafarje and 29 colleagues assessed iron absorption and metabolism into Hb using 59Fe, and highlighted the 3-fold increase in iron absorption at high altitude, compared to sea level (Reynafarje et al., 1959). During the Swiss Dhaulagiri expedition of 1958, Winterhalter appeared to demonstrate that oral ferrous gluconate enhanced hemoconcentration [(Winterhalter, 1960) as translated by (Hornbein, 1962)]. However, when Hornbein and colleagues performed an iron supplementation study during the 1960 American Pakistan Karakoram Expedition to Masherbrum (~7800 m) participants in the iron group (who received 500 mg iron as iron-dextran injected intramuscularly 4 weeks before departure and oral 1.0-1.5 g ferrous gluconate daily from days 8 to 52), demonstrated similar increases in Hb as a control group (who did not receive injection and oral iron). It is noteworthy though, that both groups received an estimated daily intake of 10-15 mg elemental iron from their high altitude rations) (Hornbein, 1962). It was later shown that intestinal iron absorption (quantified as the percent of iron reaching red blood cells via 59Fe tracing) increases ~5-fold within a few days at 4540 m [Figure 2.5; Morochocha, Peru; 3.6±1.27 % at sea level to 14.3±3.78 % on day 2 and 16.3±2.34 % on day 8; (Reynafarje & Ramos, 1961)]; therefore, these findings may partly explain why there were no differences between the iron and placebo groups in the study by Hornbein (1962). 30 Figure 2.5. Iron absorption and uptake into red blood cells (RBCs). Iron incorporated into RBCs by 59Fe radiolabeling of ferrous sulfate (100 mg). Each measurement at 4540 m consisted of n=5. Adapted from (Reynafarje & Ramos, 1961) with permission. During Operation Everest II [a 38-day hypobaric chamber study to the summit of Mt. Everest (Houston et al., 1987)], efforts were made to limit iron deficiency by standardizing the daily iron intake in male participant meals to 26.5±16.0 mg [(Rose et al., 1988); the recommended daily allowance of iron today in adult males and premenopausal women are 8 g and 18 mg/day, respectively (Institute of Medicine, 2001)]. While serum iron or ferritin data were not collected, it is reasonable to expect that there were reductions in iron status as the study by Rose and colleagues did show a loss of appetite and decrease in food intake, especially during prolonged exposure to the highest altitudes (Rose et al., 1988). As such, in Operation Everest III [a 31-day hypobaric chamber study to summit of Mt. Everest (Richalet, 2010)] there were coinciding reductions in iron status (Figure 2.6), energy intake, body mass and appetite (Westerterp-Plantenga et al., 1999; Robach et al., 2004). 02468101214161820BL(Lima)2 8 30Fe appearing in RBC (%)Days after arriving at 4540 mASCENT 31 Figure 2.6. Iron status markers during Operation Everest III. Consisting of two separate experiments: Experiment A (n=8, with n=7 at 8000 m) emulated ascent from sea level (SL) to 8000 m, and Experiment B (n=10) replicated the prolonged stay at 4350 m. Measurement timepoints during Experiment B were at 3, 5 and 7 days at 4350 m (i.e. H3, H5 and H7 respectively) and days 1 and 2 upon return to SL (i.e. RSL1 and RSL2, respectively). *P<0.05 from SL. Soluble transferrin receptor, sTfR; Erythropoietin, EPO. From (Robach et al., 2004) with permission. 32 Since progressive reductions in iron status are demonstrated to occur during high-altitude sojourn (Figure 2.4), impairments to the brevity of iron-dependent regulatory processes (e.g., erythropoiesis, angiogenesis) will inevitably occur. The question arises: Does this decline in iron status influences other high-altitude physiological processes? Although there is a paucity of data exploring such relevant questions, a series of studies from the University of Oxford, led by Prof. Peter Robbins, were designed and conducted to explore this question. A classical and widely used metric of high altitude tolerance is the acute mountain sickness (AMS) score; this score categories altitude-related symptoms of headache, gastro-intestinal distress, fatigue/weakness, dizziness/light-headedness, clinical symptoms and sleep quality (Roach et al., 1993), albeit recently updated to exclude sleep symptoms (Roach et al., 2018). In terms of lowlanders ascending to high altitude, AMS is characteristically worse 6-12 hours after ascent, and slowly dissipates over ~3 days if no ascent occurs and one acclimatizes to the altitude (Hackett & Roach, 2001). Interestingly, an infusion of iron [iron (III)-hydroxide sucrose (200 mg)] prior to ascent (compared to saline), has been shown to attenuate AMS severity on the first morning at 4300 m [Figure 2.7; (Talbot et al., 2011)]. Additionally, an iron infusion prior to departure (Holdsworth et al., 2020) or shortly upon shortly upon arrival (Smith et al., 2009). have been shown to proffer pulmonary vascular benefits for the trekker/mountaineer. While there are limited data in other vascular beds, it appears that ensuring a replete iron status (verses being iron deficit) would seem advantageous upon ascent and during prolonged stay at high altitude. The following section discussion summarizes different vascular beds within the body and provides an overview of available iron-related literature. 33 Figure 2.7. Iron infusion and acute mountain sickness (AMS). Effect of infusion of iron [iron (III)-hydroxide sucrose (200 mg) or saline] in sea level residents who rapidly ascended to 4300 m. The data (n=12 in each group, with mean ± standard error of the mean) illustrate the absolute increase in AMS scores from sea level (Lima) to the first morning at 4300 m (Cerro de Pasco). It should be noted that an AMS score of ≥3, in the presence of headache, is the cut-off for detection of AMS. From (Talbot et al., 2011) with permission. 2.5. The integrated vascular regulation of the pulmonary, peripheral and cerebral systems at high altitude 2.5.1. Pulmonary circulation The pulmonary circulation is a key step for oxygen transfer. It forms a low pressure-high blood flow system which is essential in facilitating efficient gas exchange via a thin alveolar-capillary interface (Weibel, 1973). However, with exposure to hypoxia and/or ascent to high altitude (and thus a reduced partial pressure of alveolar oxygen), individuals typically exhibit a rise in pulmonary artery pressure – a phenomena referred to HPV (hypoxic pulmonary vasoconstriction) [reviewed in (Reeves et al., 1979; Swenson, 2013)]. Historically, HPV was first linked to alveolar hypoxia by Plumier, who exposed spontaneously breathing dogs to hydrogen (i.e. an unobstructed model of asphyxia) and showed a rise in pulmonary artery pressure (Plumier, 1904). This link was not established until von Euler and Liljestrand experimentally manipulated alveolar gases in anaesthetized open-chested cats, and demonstrated the constrictive effect of alveolar hypoxia (i.e. 34 fractional of inspired O2; FiO2, 0.105) on pulmonary arteries (von Euler & Liljestrand, 1946). HPV was subsequently established in humans [Figure 2.8; (Motley et al., 1947)], and then isolated lung preparations confirmed that the vasoconstrictive response was locally mediated, and not dependent upon neural or humoral signaling (Nisell, 1948). Figure 2.8. Hypoxic pulmonary vasoconstriction (HPV) in humans. Utilizing invasive right heart catheterization, pressures within the pulmonary artery (PA) were shown to increase upon exposure to hypoxic air (FiO2, 0.1). Note the 4-fold increase in pulmonary vascular resistance (PVR) from room air to hypoxia. From (Motley et al., 1947) with permission. From the 1950’s onwards, the complexities and nuances of HPV have been extensively studied and comprehensively reviewed (Sylvester et al., 2012; Swenson, 2013; Lumb & Slinger, 2015; Dunham-Snary et al., 2017). The role of HPV likely stems from foetal life, as a mechanism for the fetal lungs to shunt blood away unventilated lungs. In air breathing individuals, HPV can optimize pulmonary gas exchange by matching perfusion to ventilation (VE/Q), during conditions of regional-specific situations of poor VE/Q mismatch (e.g. atelectasis) or pulmonary artery recruitment during reductions in barometric pressure. However, even though HPV is conserved across the mammalian kingdom (e.g. cow, rat, cat, sheep, dog, llama, pig), the response can be undesirable. For example, in the acute setting, excessive HPV can occur and cause a heterogeneric blood flow pattern (Dehnert et al., 2006; Hopkins & Levin, 2006) that can lead to high altitude pulmonary edema [HAPE (Swenson, 2020)]. Moreover, in the setting of chronic hypoxia, when 35 HPV persists, it can facilitate remodeling of the small distal pulmonary arteriole branches (Arias-Stella & Saldana, 1963) and lead to chronic hypoxic pulmonary hypertension (Rotta et al., 1956; Grover, 1990; Penaloza & Arias-Stella, 2007). With ascent and prolonged stay at high altitude, there are a number of factors which play a potent modulatory role on HPV. These factors include the stabilization of HIF (Frise & Robbins, 2015; Gassmann & Muckenthaler, 2015; Prabhakar & Semenza, 2015; Hirota, 2019) and upstream iron regulatory activity [e.g. ferroportin-hepcidin axis; (Lakhal-Littleton et al., 2019)]. Additionally factors that can influence HPV include changes in ventilation [particularly alveolar PO2, which accounts for ~2/3rd of HPV response (Balanos et al., 2003)]; acid-base status [respiratory alkalosis has a blunting effect on HPV (Swenson, 2016)]; viscosity/hematocrit [as an inversely relationship (Manier et al., 1988; Vanderpool & Naeije, 2018)]; heredity [at even a pre-pubertal age (Kriemler et al., 2008)], exercise [affecting the slope in an hypoxic stimulus-dependent manner (Groves et al., 1987)], and age [greater magnitude of change in older adults (Balanos et al., 2015)], and at least in animals, some evidence (pertaining to estradiol or pregnancy) for a role of biological sex [reviewed in (Sylvester et al., 2012). The link between iron bioavailability and HIF on cardiopulmonary function stems from the discovery of a family of isoenzymes in 2001, termed PHD (prolyl hydroxylase-domain) proteins. These PHDs, were shown to facilitate the hydroxylation of a proline residue on HIF, and therefore HIF-von Hippel-Lindau binding (necessary for HIF stabilization) (Ivan et al., 2001; Jaakkola et al., 2001; Yu et al., 2001). Importantly, the dependency of PHD activity on O2, iron, ascorbate and 2-oxoglutarate were elucidated [reviewed in: (Smith et al., 2008b)]. These works contributed to a stream of publications by the Oxford group that had already time coursed HPV [Figure 2.9 (Dorrington et al., 1997)]. In 2002, Robbins and colleagues showed an acute infusion of an iron chelator [i.e. desferrioxamine (DFO) at a dose of 4 g/70 kg body weight] increased pulmonary artery systolic pressure (PASP) by ~4 mmHg at rest (Balanos et al., 2002). Then the role of iron status (via infusion of iron or a chelator) on of HPV was established – infusion of 200 mg iron sucrose attenuated the rise in PASP (PASP increased by 124±77 % with saline and by 59±67 % with iron) and infusion of DFO (4 g/70 kg over 8 hours) augmented HPV (PASP increased by 1±12 % with saline and by 36±26 % with (DFO) (Smith et al., 2008a). This led to a number of 36 follow up iron infusion studies at high altitude (Smith et al., 2009; Talbot et al., 2011; Holdsworth et al., 2020) and laboratory-based iron/chelator studies in humans (Talbot et al., 2012; Talbot et al., 2014; Bart et al., 2016; Frise et al., 2016; Bart et al., 2018; Cheng et al., 2019). Mechanistic animal models were then designed to examine the role of HIF regulation and iron metabolism (especially importance of the hepcidin-ferroportin axis) on cardiac/pulmonary/smooth muscle function (Lakhal-Littleton et al., 2015; Lakhal-Littleton et al., 2016; Lakhal-Littleton & Robbins, 2017; Lakhal-Littleton et al., 2019). 37 Figure 2.9. Time course of HPV in humans exposed to hypoxia. Using right heart catheterization, systolic (square), mean (circle) and diastolic (triangle) pulmonary artery pressure during 8 hours of isocapnic hypoxia [partial pressure of end-tidal O2 (PETO2) of 50 mmHg]. The unchanged pulmonary artery wedge pressure (an estimate of left atrial pressure) during hypoxia highlights the rise in pulmonary artery pressure is due to constriction occurring within the lung. From (Dorrington et al., 1997) with permission. There are two studies, in particular, that are directly relevant to this thesis in relationship to the role of iron on pulmonary vascular function at high altitude. In the study by Smith and colleagues (Smith et al., 2009) lowlanders (i.e. individuals who were second-generation born and raised at sea level) were transported from Lima by car in 8 hours to 4340 m (Cerro de Pasco). After 3 days at 4340 m in Peru, lowlanders were randomized in a blinded fashion to receive either intravenous iron (200 mg iron-sucrose) or saline (100 ml). Those who were administered iron showed a 38 significant reduction in PASP of ∼16% by 4 hr following infusion (Smith et al., 2009). Another feature of this study was that they also performed iron infusions in Andeans suffering from chronic mountain sickness (CMS; Hb ≥21 g/dl and Qinghai CMS questionnaire score >5). Their aim was to investigate the influence of iron on pulmonary remodeling and pulmonary hypertension, which are characteristically negative features of CMS (Penaloza & Arias-Stella, 2007). Following 4 days of venesection (500 ml/day; which decreased iron stores and increased PASP), the infusion of iron (i.v. 400 mg iron-sucrose administered over two days; 200 mg/day) was ineffective in altering PASP in Andeans with chronic mountain sickness. Pulmonary remodeling and pulmonary hypertension are characteristic feature of CMS (Penaloza & Arias-Stella, 2007); therefore, the authors postulated that the extent of pulmonary remodeling was indelible and/or the dose (and 5 days of wash-in) was insufficient in magnitude and time (Smith et al., 2009). More recently, Holdsworth and colleagues (2020) assessed pulmonary vascular function during ascent to 5100 m in lowlander participants who were randomized to receive a single infusion of iron (1 g ferric carboxymaltose) or saline (two-weeks before ascent). Both PASP and PVR were not statistically different between the iron and saline conditions across ascent. However, the authors concluded ”iron infusion appears to reduce PVR and improved right heart function” based on the iron group showing enhanced indices of stroke volume and right ventricular function (Figure 2.10) (Holdsworth et al., 2020). Ultimately, ensuring iron surplus (and not a deficit) would seem advantageous upon further ascent to high or extreme altitude. 39 Figure 2.10. Iron (or saline) infusion in lowlanders before ascent to 5050 Lowlanders ascending to 5100 m, having either received iron or saline prior to sojourn. CI, cardiac index; EPO, erythropoietin; LIMP and RIMP, left and right ventricular index of myocardial performance, respectively; PVR, pulmonary vascular resistance; RVSP, right ventricular systolic pressures (synonymous with PASP); sTfR, soluble transferrin receptor; SVI, stroke volume index; Tsat, transferrin saturation; TAPSE, tricuspid annular plane systolic excursion. From (Holdsworth et al., 2020) with permission. 40 2.5.2. Peripheral blood flow The vascular endothelium serves as a selectively permeable layer of cells that line all blood vessels of the circulatory system. This thin endothelial layer plays a critical role in vascular health, as any deterioration/dysfunction is a key contributor to the progression to vascular pathology (Cahill & Redmond, 2016). Within each blood vessel, the pulsatile blood flow continuously exerts a frictional force that is tangential to the endothelial surface (i.e. shear stress). These hemodynamic forces provide the necessary physiologic stimuli to promote expression of endothelial genes and endothelial-derived bioactive components that protect against atherosclerosis (Stone et al., 2003). At high altitude, there are many factors which can alter artery shear stress patterns, and thus affect endothelial function. For example, there are opposing increases in sympathetic nervous activity [stimulus for vasoconstriction (Hansen & Sander, 2003; Tymko et al., 2020b)] and metabolic-induced vasodilation (Marshall, 2015; Dinenno, 2016) that influence vascular tone. There are also increases in oxidative stress (Joanny et al., 2001; Faiss et al., 2013; Lewis et al., 2014) that can reduce nitric oxide (NO) bioavailability (Milstien & Katusic, 1999), and increases in Hb and blood viscosity that interact to drive shear rate and endothelium-dependent vasodilator effects on vascular resistance (Koller et al., 1993; Salazar Vázquez et al., 2010). As such, at high altitude, there exists an extensive breadth of studies exploring vascular function in sea-level and high-altitude residents [reviewed in: Tymko et al. (2019)]. While it is logical to postulate that HIF-down regulation could exert influence over the peripheral vasculature, there exists little literature exploring this relationship in vivo in humans. However, what is becoming evident from animal models – particular ischemic preparations – is that HIF activation triggers processes of angiogenesis (i.e. the sprouting of new capillary branches from pre-existing blood vessels) and arteriogensis (i.e. the remodelling of pre-existing blood vessels, also known as collaterals, by an increase in luminal diameter to accommodate an increase in flow). These two responses are driven via the influence of downstream propagation of angiogenic factors on endothelial cells [reviewed in: (Rey & Semenza, 2010)]. For example, in a rabbit ischemic hindlimb model, HIF activation (via an adenoviral vector) facilitated both angiogenesis and arteriogensis; Figure 2.11; (Patel et al., 2005). Likewise, in monocytes from coronary artery disease patients, those with higher vascular endothelial growth factor (VEGF) expression also 41 demonstrated more coronary artery collaterals, even after controlling for sex, smoking and pre-medical history (e.g. diabetes, hypertension, hypercholesteremia, family history) (Schultz et al., 1999). The prevalence of coronary artery collaterals has since been linked to HIF-1𝛼 (Song-ming et al., 2008). The exception to a lack in human data, is a notable study by Cowburn and colleagues (2013). By utilizing an extensive array of techniques in the skin of mice and humans, the authors revealed that 1) blood pressure was 14% higher in mice with HIF-1𝛼 knockout (and 18% lower in HIF-2𝛼 knockout), compared to wild-type controls, and 2) in human participants, the severity of arterial hypertension is negatively and positively associated with HIF-1𝛼 and HIF-2𝛼 expression, respectively (Cowburn et al., 2013). Figure 2.11. Arteriogenesis via digital angiograms of a hindlimb ischemic model in rabbits. The hindlimb ischemic model was performed utilizing an intravascular occlusion of femoral superficial artery in a rabbit. A) Baseline, prior to occlusion. B) Coils to occlude artery (highlighted by arrows) and needles for adenoviral injection of HIF-activator. C) Immediately after occlusion, arrows denote coil). D) 14 days post-occlusion and treatment with HIF-activator, showing collateral vessel development in the medial thigh. From (Patel et al., 2005) with permission. 42 While the influence of iron on vascular function at high altitude has not been considered, intravenous iron infusion is widely performed clinically [e.g. sickle cell anemia (Blum et al., 2005)] and there exists a myriad of studies exploring the negative (and lack there-of) influence of iron on a variety of pre-clinical and human (healthy and pathological populations) models of varying vascular dysfunction states [reviewed in: Koskenkorva-Frank et al. (2013)]. For example, at ~10 min following the infusion of ferric saccharate (100 mg), there was a transient reduction in endothelium-dependent flow mediated dilation (FMD) in healthy humans (Rooyakkers et al., 2002). Likewise, in a human experimental model of endothelial dysfunction (via oral methionine to induce homocysteine-induced endothelial dysfunction), infusion of iron-sucrose (100 mg) exaggerated the decline in FMD at 1-hour post-infusion, compared to placebo (Zheng et al., 2006). It is important to note that in both studies, the transient endothelial dysfunction was no longer different from the control condition at 4 hours (Rooyakkers et al., 2002; Zheng et al., 2006). Additionally, forearm blood flow in dialysis patients was unaltered at 2 hours after infusion of iron sucrose (300 mg) (Schaller et al., 2005). It is interesting that iron sucrose has a pronounced effect of increasing NTBI (non-transferrin bound iron) levels post-infusion which peaks at ~4 hours (Garbowski et al., 2020). This peak in NTBI aligns with the 5-fold increase in NTBI at 4 hours in the study by Zheng and colleagues (Zheng et al., 2006). However, the lack of decline in metrics of vascular function at rising/peak NTBI levels is surprising (Rooyakkers et al., 2002; Schaller et al., 2005; Zheng et al., 2006), since NTBI is capable of catalyzing reactive oxygen (ROS) species production (Kruszewski, 2003). 2.5.3. Cerebral blood flow While the brain requires a disproportionally high supply of O2 and nutrients, it has little capacity for substrate storage (Brown & Ransom, 2007). Therefore, there exists a series of overlapping regulatory pathways that aim to maintain precise control of CBF and oxygen delivery. This of course is important as any inadequacies, irrespective of their brevity, are swiftly reinforced by syncope, brain damage and even death (Smith et al., 2011a). These main regulators of CBF are arterial blood gases (particularly the partial pressure of arterial CO2; PaCO2), mean arterial pressure (MAP), cerebral metabolism and the autonomic nervous system [reviewed in (Willie et al., 2014b)]. While iron is not classically associated to exert a profound influence over cerebral vascular control, any instability to brain iron levels is associated with a range of clinical 43 pathologies [deficiency: restless leg syndrome, hypomyelination; overload: neurogeneration, amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease (Duck & Connor, 2016)]. Therefore, it is not surprising that many of the same factors that are important for systemic iron homeostasis (e.g. IRPs, TfR1, ferroprotein, ferritin) and also act to regulate brain iron metabolism and the transport of iron across the blood brain barrier [reviewed in (Duck & Connor, 2016)]. Transferrin transcytosis is the classical mode of transport of iron across the blood brain barrier – a phenomenon that was first shown using a mouse perfusion model, radiolabeled transferrin (I125-Tf) that crossed the blood brain barrier (Fishman et al., 1987). Since then, mechanisms by which iron can cross the blood brain barrier are established, including the endothelial-derived machinery (i.e. endocytosis) and transport via ferritin (Simpson et al., 2015; Duck & Connor, 2016)]. Non-heme iron was first discovered in the human brain by Zaleski in 1886, and shown by Spatz in 1922 to be irregularly distributed throughout the brain [(Zaleski, 1887; Spatz, 1922); translated in (Koeppen, 1995)]. Eventually with histochemical advancements, iron was confirmed to vary across intra-cranial regions and slowly accumulate with age until adulthood when it roughly plateaus (Figure 2.12; (Hallgren & Sourander, 1958; Drayer et al., 1986). From these early studies, iron within the brain is preferentially located in the basal ganglia (i.e. globus pallidus and putamen) but also appears, to a lesser extent in subcortical U fibers, front white matter and glial cells (Drayer et al., 1986; Morris et al., 1992). 44 Figure 2.12. Distribution of non-heme iron in the human brain. Data represents non-heme iron extracted from homogenized brain tissue samples, in 81 patients (varying in age) without cerebrovascular or neuropsychiatric disorders. Non-heme iron levels are highest in globus pallidus (Panel A) and putamen (Panel B), following by the thalamus (Panel C) and prefrontal cortex (Panel D). Adapted from (Hallgren & Sourander, 1958) with permission. The related CBF responses to hypobaric hypoxia have been well described (Severinghaus et al., 1966; Huang et al., 1987; Willie et al., 2015; Hoiland et al., 2019). For example, upon exposure to hypoxia, CBF increases in order to compensate for the initial reductions in arterial O2 content in order to maintain cerebral O2 delivery (Willie et al., 2014a; Hoiland et al., 2016). Eventually, the acute rise in CBF is attenuated, coinciding with an increase in arterial O2 content via erythropoiesis, ventilatory acclimatization and compensation of the initial respiratory alkalosis (Severinghaus et al., 1966; Ainslie & Subudhi, 2014; Swenson, 2016). Perhaps not surprisingly, the HIF expression within the human brain has not been quantified during hypoxia. However, from rodent models, cortical HIF- expression during hypoxia follows a similar trajectory to the CBF responses. For example, peak HIF-1 expression occurred with 6-12 hours of exposure to hypoxia (barometric pressure of 380 mmHg), which halved by day 7, and normalized within ~3 weeks (Chávez et al., 2000). Similarly, during exposure to more severe hypoxia (barometric pressure of 290 mmHg), cerebral cortex HIF-1 expression remained elevated for 14 days – which facilitated 45 increases in 1) VEGF, 2) EPO in the brain, 3) cerebral microvascular density [from 411±38 microvessels/mm2 at baseline to 628±45 microvessels/mm2 at 3 weeks (Benderro & LaManna, 2014)]. In the brain of mice, a recent study has highlighted the importance of PHDs [i.e. PHD1-3; serves as a crucial step in iron’s influence on HIF stabilization (Knowles et al., 2003)] in regulating neurovascular angiogenesis and pericyte proliferation – with the greatest induction with PHD1,2,3-/- (Urrutia et al., 2020). While PHD2 knockout in mice is lethal, conditional somatic inactivation of murine PHD2 (Minamishima et al., 2008) and mice rendered PHD2 deficient (Takeda et al., 2008) show profound polycythemia. Moreover, HIF-1 expression was tied to astrocyte activation post-ischemia, that aims to alleviate neuronal damage (Amin et al., 2021). Cerebral astrocytes (but not neurons) exposed to extreme hypoxia and DFO (iron chelator) showed an increase in EPO expression via HIF-2 up-regulation (Chavez et al., 2006). In healthy humans, however, there exists limited data with respect to iron manipulation on the cerebral vasculature. The only human study to date is via the assessment of intracranial blood velocity (i.e. middle cerebral artery) following iron chelation [via DFO – an up-regulator of HIF expression (Ren et al., 2000)] by Sorond and colleagues (Sorond et al., 2009; Sorond et al., 2015). These extremely small changes in middle cerebral artery velocity are unlikely to be of physiological significance – especially if there is constriction/dilation of the middle cerebral artery (Ainslie & Hoiland, 2014). 2.6. Iron metabolism in high altitude adaptation Using census and geographic information system data, it has been estimated that approximately 80 million people live above 2500 m (Tremblay & Ainslie, 2021). Among these residents of high altitude, there are three classically-defined highlander populations: Tibetan Sherpa, Andeans, and Ethiopian. Each group have developed distinct adaptive (and even maladaptive) strategies over hundreds of generations [~1100, ~550, and ranging between 500-1000+, respectively (Aldenderfer, 2003; Beall, 2014)], to combat the reductions in PO2 levels [reviewed in: (Beall, 2007; Simonson, 2015)]. Tibetan Sherpa have been reported to present with unique phenotypic adaptations to high altitude, characterized by: a blunted ventilatory response to hypoxia (Beall et al., 1997), a more efficient 46 plasma volume-hemoglobin ratio to aid exercise capacity (Stembridge et al., 2019), a reduced prevalence of excessive erythrocytosis (Simonson et al., 2010; Gassmann et al., 2019), less pronounced pulmonary hypertension (Figure 2.13), and higher lung diffusing capacity [reviewed in:(Gilbert-Kawai et al., 2014)]. Cerebral O2 delivery is also lower in Sherpa compared to lowlanders – and thus, potentially suggestive that Sherpa experience less deleterious cerebral consequences to hypoxia compared to lowlanders at high altitude (Garrido et al., 1996; Hoiland et al., 2019). Healthy high-altitude adapted Andeans also demonstrate numerous attributes that enhance their hypoxic tolerance compared to lowlanders, including: elevated birth weights, increased exhaled NO concentrations, larger lungs, improved aerobic capacity and genotypic adaptations (Lenfant & Sullivan, 1971; Beall et al., 2001; Moore et al., 2004; Frisancho, 2013; Storz, 2021), and display preserved endothelial function at high altitude (Tymko et al., 2019). Data in Ethiopian highlanders are scarce; however, these highlanders have historically demonstrated unique and enhanced physiological adaptations to high altitude – most notably elevated Hb O2 affinity (Beall et al., 2002; Cheong et al., 2017). This unique characteristic is notable, in that it is evident in high-altitude bar-headed geese, who’s migratory flight pattern enables them, at times, to fly above Mt. Everest (Hawkes et al., 2011). Conversely, other data indicates that Amhara Ethiopians have similar adaptive signaling regions related to HIF system as Sherpa [e.g. PPARA and EPAS1; (Simonson, 2015)], and Amhara Ethiopians at 3700 m exhibit elevated pulmonary artery pressures (without concurrent increase in pulmonary vascular resistance) compared to lower altitude Amhara (Hoit et al., 2011). Ethiopians also seem to have more efficient plasma volume to red cell mass ratio (Claydon et al., 2005). 47 Figure 2.13. Relationship between pulmonary pressure and cardiac output in lowlanders and high-altitude natives. Pulmonary pressure displayed as either direct pulmonary artery pressure measures (via catheter, Ppa) or estimated from pulmonary artery systolic pressure measurements (via ultrasound, PASP). Solid lines represent mean data at altitudes from 2380-8848 m (mean 4732  1903 m). Dash line illustrates young lowlanders (15.3±1.4 years; Hb 15.6±1.0 g dl-1) whom have lived at 3100 m for 12.6±4.9 years (Vogel et al., 1962). Summary of mean data from nine studies – in studies where cardiac index was not provided, mean BSA was used. Lowlander data: (Vogel et al., 1962; Kronenberg et al., 1971; Sime et al., 1974; Groves et al., 1987; Groepenhoff et al., 2012). Sherpa data: (Groves et al., 1993). Andean data: (Banchero et al., 1966; Lockhart et al., 1976; Groepenhoff et al., 2012; Pratali et al., 2013). While Andean and Sherpa highlanders display adaptive characteristics to hypoxia, both also display positive selection for HIF pathway candidate genes (Bigham et al., 2010; Foll et al., 2014). It seems plausible, therefore, that iron status or iron metabolic mechanisms may be differentially regulated in healthy highlanders given their many naturally selective traits to high altitude. However, limited data exists on iron regulation in highlander populations. Interestingly, in 1998, using a radioisotope technique (Warner, 1973), Pizzaro and colleagues demonstrated that Peruvian Andeans at 3450 m did not exhibit an elevated iron absorption, compared to Chilean sea level controls [iron absorption from reference dose 32% and 31% respectively; (Pizarro et al., 1998)]. More recently, Pizarro’s hypothesis has been (re)highlighted in a review, which discusses the plausibility that iron metabolism in high altitude highlanders may have acquired some evolutionary advantage, compared to sea level residents (Muckenthaler et al., 2020). The rationale for such an 48 adaptational trait pertaining to iron metabolism certainly seems reasonable on a number of accounts, including: 1) sea level residents characteristically demonstrate iron depletion over prolonged stay at altitude, but iron deficiency is relatively uncommon amongst high altitude highlanders (0.6% and 8.2% in Bolivian males and females at 3800-3900 m [n=266 (Beall et al., 1990)]; 2) Ethiopian highlanders (Amhara and Oromo) at high altitude (3700-4000m) show comparable body iron levels to their low altitude counterparts (1200-1500 m), and hepcidin levels were comparable or elevated at high altitude compared to sea level, indicating a low demand for iron [n=279 (Lundgrin et al., 2013)]; and 3) military recruits resident of moderate altitude (≥1500 m for the preceding ≥2 years) had higher serum ferritin levels, than recruits resident of sea level (~300 m), for the entirety of the 46-week stationing at 2210 m [n=55 (Brothers et al., 2010)]. However, there exists a paucity of experimental data explicitly supporting this notion and, as such, future studies are needed to address these important areas. 2.7. Literature review synopsis This literature review briefly encompassed the role iron plays in the palaeontological progression of life on Earth; its necessity in human health; how it is interwoven in high altitude physiology; and why ancestral adaptation to high altitude may give rise to iron regulatory enhancements. The experimental chapters herein contain four studies designed to explore the physiological regulation of iron during hypoxia, in the pulmonary (Chapters 3 and 4), peripheral (Chapter 5) and cerebral (Chapter 6) vasculature. 49 Chapter 3. Influence of iron manipulation on hypoxic pulmonary vasoconstriction and pulmonary reactivity during ascent and acclimatization to 5050 m 3.1. Background Oxygen and iron are both essential for life and, in particular, have unique influences on the pulmonary vasculature (Frise & Robbins, 2015; Gassmann & Muckenthaler, 2015). The human pulmonary vasculature vasoconstricts in response to a reduction in alveolar O2 tension, a phenomenon termed HPV (Swenson, 2013; Lumb & Slinger, 2015). This HPV response is influenced by a multitude of factors (Sylvester et al., 2012), including the notably influence of iron status via its role as an obligate cofactor in the stabilization of HIF system (Prabhakar & Semenza, 2015), and its capacity to modulate the responsiveness of the HIF system to hypoxia (Jiang et al., 1996). There appears to be robust inverse relationship between iron status and HPV (Frise & Robbins, 2015). For example: infusion of iron [iron(III)-hydroxide sucrose] or iron-chelators (via DFO) respectively blunts or aggravates HPV (Smith et al., 2008a); iron chelation in the absence of hypoxia increases pulmonary pressure (Balanos et al., 2002); and iron-deficient individuals demonstrate exaggerated hypoxic pulmonary hypertension compared to iron-replete individuals (Frise et al., 2016). Although it does not appear that iron deficiency directly mediates the disproportionate increases in HPV in individuals who are susceptible to HAPE (Altamura et al., 2015), animal models reveal an associated link between iron deficiency (Cotroneo et al., 2015) and the development of pulmonary hypertension – via mechanisms likely attributable to a deficit in hepcidin-functionality (Lakhal-Littleton et al., 2019). Recently, Holdsworth et al. (2020) demonstrated how iron status can alter cardiopulmonary function during ascent. However, it is unclear if during prolonged exposure to high altitude, there is further attenuation of iron stores that might regulate ‘normal’ changes in HPV, since several studies have reported reductions in serum iron levels at high altitude (Mairbäurl et al., 1990; Smith et al., 2009; Piperno et al., 2011; Goetze et al., 2013; Holdsworth et al., 2020). In one of the only studies that has manipulated iron levels at altitude, individuals were taken rapidly (via automobile) from sea level to 4340 m in Peru and. after three days, were randomized to receive either intravenous iron (200 mg iron-sucrose) or saline (Smith et al., 2009). Those administered iron, when compared to saline, showed a significant fall in PASP of ∼16%, 4 hrs following iron infusion (Smith et al., 2009). 50 The Tibetan Sherpa have been reported to present with a unique phenotypic adaptation to high altitude characterized by a blunted ventilatory response to hypoxia (Beall et al., 1997), a more efficient plasma volume-hemoglobin ratio to aid exercise capacity (Stembridge et al., 2019), a reduced prevalence of excessive erythrocytosis (Simonson et al., 2010; Gassmann et al., 2019), and a higher lung diffusing capacity [reviewed in: (Gilbert-Kawai et al., 2014)]. Although not a universal finding (Foster et al., 2014), Tibetans have also been suggested to exhibit lower pulmonary arterial pressures [via cardiac catherization (Groves et al., 1993) and echocardiographic imaging (Faoro et al., 2014)] compared to lowlanders at altitude or other high-altitude populations. Tibetan highlanders residing at sea level, exhibit a blunted HPV during an acute (10-min) hypoxic challenge, as well as a diminished reactivity of the pulmonary vasculature during a sustained (8 hr) hypoxic exposure; when compared to Han Chinese (Tibetans closest ethic group) (Petousi et al., 2014). Although it is now well-established that acute alterations in iron availability affect the pulmonary vascular response to hypoxia (Frise & Robbins, 2015), apart from the aforementioned study at 4340 m (Smith et al., 2009), these data are almost exclusively based at sea level during conditions at rest, and no related studies have been performed in Himalayan natives. Whether acute alterations in iron bioavailability also influence the extent of HPV following acclimatization (e.g. lowlanders) or adaption (e.g. Tibetan natives) to high altitude and/or during exercise, remains unknown. The aim of the current series of studies was to examine the adaptational role of iron bioavailability on the pulmonary vascular responses to acute and chronic hypobaric hypoxia during rest, exercise and acute changes in inspired PO2 (PIO2). To address this aim, lowlanders and partially de-acclimatized Sherpa were studied during progressive ascent to 5050 m (Study 1), and following partial acclimatization to 5050 m, where each group received either an infusion of iron or an iron-chelator (Study 2). Our two primary hypotheses were: 1) during progressive ascent to high altitude (5050 m), the increase in HPV will be attenuated in Sherpa compared to lowlanders due, in part, to Sherpa demonstrating an improved iron status (and a theoretically hypo-responsive HIF system), and; 2) following ~2 weeks of partial acclimatization to 5050 m, iron infusion will attenuate, and iron chelation will amplify the HPV both at rest and during acute changes in PIO2, and submaximal exercise; however, these responses would again be blunted in Sherpa compared to lowlanders. 51 Given the emerging evidence on the relationship between iron regulation, inflammation and hypoxia (Ross, 2017; Simmen et al., 2019; Duarte et al., 2020), complimentary measures of inflammation were also collected in order to explore their temporal relationship with iron availability and HPV. 3.2. Materials and methods 3.2.1. Ethical approval This study was approved by the Clinical Research Ethics Board of the University of British Columbia (H16-01028) and the Nepal Health Research Council, and conformed to the Declaration of Helsinki, except for registration in a database. All lowlander participants gave written informed consent in English prior to participating. All Sherpa participants read an in-depth study information form, spoke with a Nepalese physician and gave written informed consent in Nepalese prior to participating. 3.2.2. Experimental design In line with our two hypotheses, this study was performed in two arms – each inclusive of both lowlanders and high-altitude Sherpa during ascent to (Study 1) and following (11±4 days) partial acclimatization (Study 2) at 5050 m. 3.2.2.1. Study 1 – Ascent to 5050 m A cohort of 20 healthy lowlanders (2 females; 28±6 years; 177±8 cm; 73±9 kg; 23±2 kg/m2) who were recruited from the research team. All lowlander participants were free from cardiovascular, respiratory and neurological diseases and were non-smokers. Twelve high altitude Sherpa (35±11 years; 167±7 cm; 67±14 kg; 24±5 kg/m2; Table 3.1) were recruited and are henceforth referred to as the “Ascent Sherpa”. These Sherpa were free of cardiovascular, respiratory and neurological diseases – with 4 current smokers (1.31.1 [range: 0.3-2.5] pack years). Notably, three of the Ascent Sherpa had summited Mount Everest (8848 m) in the previous year, while the remaining Sherpa had reached a maximum altitude of 4800 m to 7800 m (median: 5545 m) in the last year. All but one ascent Sherpa (44 years of age) had a comparably age-matched lowlander (when n=12 in each group the ages were 3310 years and 3511 years for lowlanders and Sherpa respectively). 52 The logistics of the UBC-Nepal Expedition have been detailed elsewhere (Willie et al., 2018); therefore, only features of the expedition pertinent to the current study will be outlined. All lowlander participants spent 3-9 days in Kathmandu (1400 m) prior to flying to Lukla (2860 m) to begin the ascent to the EV-K2-CNR Pyramid Research laboratory (5050 m), while the Ascent Sherpa group had descended to Kathmandu and remained there for 5-15 days (median: 7 days) prior to flying to Lukla with the lowlanders. Ascent to the Pyramid Laboratory took place over a slow and safe 9-day trekking protocol without the use of any acute mountain sickness prophylactics (e.g., acetazolamide or phosphodiesterase inhibitors). Participants spent one night in Monjo (2800 m), three nights in Namche Bazaar (3400 m), one night in Deboche (3820 m), and then three nights in Pheriche (4370 m) followed by the final trekking day to 5050 m. In Kathmandu prior to the ascent and on the day following arrival to Namche, Pheriche, and the Pyramid laboratory, all participants underwent experimental measurement of arterial blood gases, MAP, heart rate (HR), and echocardiography. At each of these timepoints, venous blood samples were obtained, processed and stored in liquid nitrogen for latter biochemical determination of iron metrics and related hormones. These measurements are detailed below. 3.2.2.2. Study 2 – Iron or DFO infusion following partial acclimatization to 5050 m Following 13 ± 3 days at 5050 m, 18 lowlanders (1 female; 30±9 years; 177±8 cm; 73±8 kg; 23 ± 2 kg/m2) from Study 1, were recruited to participate in the infusion arm of the study. A total cohort of 20 Sherpa (25±7 years; 169 ± 6 cm; 64±11 kg; 22±3 kg/m2; Table 3.1) were recruited for Study 2 – consisting of 8 Ascent Sherpa (following 7±3 days at 5050 m) and 12 additional Sherpa of Tibetan lineage (who were recruited from local villages in the Solukhumbu and thus, did not descend to Kathmandu prior to testing). These “Altitude Sherpa” typically ascended from 3800-4200 m in 1-2 days, and were tested 1-2 days following arrival to 5050 m. These Sherpa were free of cardiovascular, respiratory and neurological diseases – with 7 current smokers (0.40.2 [range: 0.1-0.75] pack years). Four of these Sherpa had summited Mount Everest in the previous year, while the remaining had reached a maximum altitude of 3800 m to 5545 m (median: 5545 m) in the last year. 53 3.2.2.2.1. Changes in PIO2 Echocardiographic images, blood pressure, and ventilation (via wright spirometer) were collected during resting supine breathing of 1) ambient air (PIO2 of 87 mmHg, 2) hyperoxia (PIO2 of 416 mmHg), 3) hypoxia (PIO2 of 67 mmHg; simulating an additional elevation gain of ~2000 m), both before and after the infusion. At PIO2 of 416 and 67 mmHg, echocardiographic measurements were collected between 15-30 min into each respective exposure – with 30 minutes of rest given between each stage. 3.2.2.2.2. Changes in cardiac output Both lowlanders and Sherpa performed a maximal oxygen uptake (VO2max) exercise test on a recumbent cycle ergometer (Lode, Netherlands), 1-2 days prior to infusion, to enable standardized exercise outputs relative to 25% and 50% of peak wattage (Wpeak). Echocardiographic images, blood pressure and ventilation (via Wright spirometer) were collected during steady-state supine sub-maximal exercise at 25% and 50% Wpeak. The submaximal exercise was performed following the altered PIO2 stages. Due to challenges in collecting echocardiographic measurements at 50% Wpeak, this exercise stage is only included, when pooling of lowlanders and Sherpa could be performed (i.e. prior to infusion; results of Study 2a). For Study 2b (i.e. pre- to post-infusion analysis), only the 25% Wpeak stage is presented, since the 50% Wpeak stage lacked an adequate sample size and was therefore excluded from any further inferences. 3.2.2.2.3. Infusion Each lowlander and Sherpa were randomized in a double-blinded fashion to receive either an i.v. infusion of either DFO (7 mg/kg/hour over 4 hr) or iron (iron (III)-hydroxide sucrose; 200 mg over 30 min followed by 3.5 hours of slow-drip saline; the total infusion time for iron was 4 hr to mirror the time-course of the DFO infusion). The infusions were performed by one of the expedition physicians (whom was not involved in the collection or analysis of data). Randomization was conducted in stages to avoid disrupting coinciding studies, given the >40-day longevity of downstream effects of iron (Bart et al., 2016). In addition, an opaque wrapping [i.e. due to the dark color of the iron infusion] was used to blind subjects and investigators to infusion group allocation. Before and after the infusion, arterial and venous blood samples were collected. 54 3.2.3. Experimental Measures 3.2.3.1. Echocardiography Stroke volume was estimated using the following equation: Equation 3.1. Stoke volume (SV). SV=(π[aortic diameter 2⁄ ]2) LVOT VTI where the left ventricular outflow tract (LVOT) velocity time integral (VTI) was measured at the point of aortic leaflet insertion during systole and using a pulsed wave Doppler signal. Cardiac output (CO) was calculated as the product of stroke volume and heart rate (via electrocardiogram). PASP was measured by Doppler echocardiography based upon the measurement of the maximum velocity of the tricuspid regurgitation jet (TR velocity) (Bertini et al., 2009). The peak systolic pressure gradient of the right ventricle (∆Pmax) to the right atrium was calculated according to the simplified Bernoulli equation (4V2), where V is the peak systolic velocity of the tricuspid regurgitate. PASP was then determined by adding the right atrial pressure. Right atrial pressure (RAP) was estimated by evaluating inferior vena cava diameter and response (i.e., collapse) to a sniff inspiration. All participants were assigned a RAP of 3 mmHg, based off vena cava diameter (i.e. <2.1 cm) and collapse (i.e. by at least 50% during a deep inspiration) – aligning with standard American Society of Echocardiography guidelines (Lang et al., 2015). Equation 3.2. Pulmonary artery systolic pressure (PASP). PASP=(4V2)+RAP Mean pulmonary artery pressure (mPAP) was calculated from right ventricular outflow tract (RVOT) acceleration time (AT) and ejection time (ET), using Doppler tissue velocities and time intervals obtained at the Lateral Tricuspid Valve Annulus: Equation 3.3. Mean pulmonary artery pressure (mPAP). mPAP=10-2.8(RVOT AT RVOT ET)+2.4⁄ PVR was calculated from the ratio of the TR velocity to the VTI obtained in the RVOT in all participants using the following equation (Rudski et al., 2010): Equation 3.4. Pulmonary vascular resistance (PVR). PVR = 10 ∙ (TR velocity RVOT VTI)⁄ + 0.16 Once PVR was determined for both lowlanders and Sherpa, we calculated PVR at an Hct of 45% in all individuals using the following equation (Vanderpool & Naeije, 2018): Equation 3.5. Pulmonary vascular resistance proportional to hematocrit (PVRHct) PVR(Hct) = PVR exp[2(φ − 0.45)]⁄ 55 Where  represents the measured hematocrit level. Given pressure is proportional to resistance, we also used the equation to calculate PASP(Hct). 3.2.3.2. Blood sampling Following 10 min supine rest at each time point, arterial blood samples were taken from the radial artery. A 23-G self-filling catheter (SafePico, Radiometer) was advanced into the radial artery under local anesthesia (Lidocaine, 1.0%) and ultrasound guidance (Terason, uSmart 3300). Approximately 1 ml of blood was withdrawn anaerobically and immediately assessed using an arterial blood gas analyzer for the partial pressure of arterial oxygen (PaO2), partial pressure of arterial carbon dioxide (PaCO2), arterial oxygen saturation, SaO2; pH, bicarbonate (HCO3-), standard base excess [BE(ecf)], Hb concentration ([Hb]), and hematocrit (Hct) (i-STAT 1, Abbott Point of Care, USA; ABL90 FLEX, Radiometer, Denmark). 3.2.3.3. Biochemical analysis Venous blood samples [via venipuncture of the median ante-cubital vein (21G needle, BD Vacutainer eclipse); 50 ml total] were separated by microcentrifugation, with plasma and serum then frozen in liquid nitrogen at -196 C for subsequent analysis. Serum iron, ferritin and total iron binding capacity (TIBC) were analysed according to clinical standards (Samyak Diagnostic Pvt. Ltd., Kathmandu, Nepal). For logistical reasons, baseline iron metrics in lowlanders were collected [and analyzed according to clinical standards] at sea level, 2-3 weeks prior to departure to Nepal (Valley Medical Laboratories, Kelowna, Canada). This difference in the collection of baseline iron metrics between lowlanders and Sherpa (at 330 and 1400 m, respectively) was not ideal, but was meant to reflect baseline-equivalent levels. When iron metric concentrations were below the lower limit of detection, the minimum value of detection was assigned. Transferrin saturation (TrF Sat) was calculated as: Equation 3.6. Transferrin saturation (TrF Sat). Transferrin saturation = (serum ironTIBC5.586592⁄ ) 100 Where 5.586592 is for the unit conversion for TIBC. 56 Plasma hepcidin, soluble transferrin receptor (sTfR) and VEGF were measured by human enzyme-linked immunosorbent assay (ELISA; Stemcell Technologies, Vancouver, BC, Canada; Biomart Canada, Toronto, ON, Canada; Thermofisher Scientific, Calgary, AB, Canada). To explore the potential interplay of inflammation on the iron regulation and the pulmonary vasculature at 5050 m, inflammatory markers (IL-1α, IL-1ß, IL-6, IL-8, IL-10, CRP, TNFα, PDGF-AB/BB, GDF-15) were measured in serum blood samples using a commercially available Human Multiplex immunoassay (Millipore, Billerica, MA, USA) according to manufacturer’s instructions. 3.2.4. Indexes of iron stores To assess total iron stores (i.e. the degree of iron deficiency), two complimentary measures (i.e. body iron and sTfR index) have been included – both of which rely on sTfR and ferritin, and are consistently used to identify iron stores and/or iron deficiency (Skikne et al., 1990; Cook et al., 2003; Skikne et al., 2011), including at high altitude (Cook et al., 2005; Lundgrin et al., 2013). 3.2.4.1. Body store index 1 Body iron stores (mg kg-1) were calculated as previously described (Cook et al., 2003): Equation 3.7. Body iron. Body iron = − [log (sTfRserum ferritin) − 2.8229] 0.1207⁄ Where sTfR conversion is based on 1 nmol l-1 = 85 ug l-1. 3.2.4.2. Body store index 2 sTfR Index was calculated as previously described (Skikne et al., 2011). Equation 3.8. Soluble transferrin receptor (sTfR) index. sTfR Index = sTfRlog(serum ferritin) Where sTfR is inputted as nmol l-1. 57 3.2.4.3. Iron recruitment index Based on the recent review by Muckenthaler and colleagues (Muckenthaler et al., 2020) and the delineated hepcidin-ferroportin axis on iron regulation (Lakhal-Littleton et al., 2019), the iron recruitment index was calculated as: Equation 3.9. Iron recruitment index Iron recruitment index= ∆ hepcidin∆ iron store index Where the change in hepcidin and iron store index (i.e. either body iron or sTfR index) is calculated across ascent – in this case, the change () between first day at 3400 m and 5050 m. This recruitment index is based on the following premises: if, 1) hepcidin limits the release of iron into the circulation and availability to peripheral tissues by binding to and inducing internalization of ferroportin (Lakhal-Littleton et al., 2019), and 2) the increase in hepcidin is attenuated during states of iron deficiency (Frise et al., 2016) – then quantifying the reduction in hepcidin based on the total available iron stores would provide a valuable estimate of the ability to utilize/mobilize available iron. 3.2.5. Statistical Analyses Data from Studies 1 & 2 were analyzed using a linear mixed effects model with a compound symmetry repeated measure co-variance structure (IBM SPSS Statistics). For Study 1, the fix factors for the model were altitude and ethnicity – altitude was a repeated factor with a compound symmetry repeated covariance structure. For Study 2, the fix factors for the model were ethnicity with either PIO2, intensity, or time – for each respective model. PIO2, intensity, or time was the repeated factor with a compound symmetry repeated covariance structure. Subjects were included as a random effect. When a significant interaction effect (e.g., altitudeethnicity) was detected, Bonferroni adjusted post-hoc tests were utilized to test pairwise comparisons. In an attempt to better understand the role of iron regulation on the pulmonary vasculature, a multiple regression analysis was conducted to determine whether metrics of iron markers (serum iron, sTfR, ferritin, and hepcidin), cardiovascular parameters (SV, CI, MAP) or CaO2, predicted PASP and PVR during ascent in lowlanders and Sherpa. Durbin-Watson values were between 1.7 and 2.0, which is acceptable that there was no autocorrelation between observations. Multicollinearity between predictive variables was negligible – for example, multicollinearity statistics of variance inflation 58 factor was <1.6 and tolerance was 0.6. Indexes of the functional recruitment of iron stores, absolute exercise intensities, comparison between Ascent and Altitude Sherpa, and inflammatory markers (pre-infusion) were assessed with independent samples t-tests, and noted where applicable within the text. All results are reported as mean±SD and significance was assumed at p<0.05. There was no difference in the primary outcome variables between current and non-smokers in either Sherpa group. 3.3. Results 3.3.1. Study 1: Progressive reductions in serum iron and comparable pulmonary pressures in both lowlanders and Sherpa upon ascent to high altitude 3.3.1.1. Iron homeostasis during ascent As illustrated in Figure 3.1 and Table 3.2, all markers of iron bioavailability (i.e. serum iron, TIBC, Ferritin, TrF Sat) were decreased in both groups (main effect of ascent p0.001 for all). Likewise, markers of erythropoietic demand (i.e. a decrease in hepcidin and increase in sTfR) were also evident in both groups (main effect of ascent p<0.001 for both). Aligning with Sherpa having descended from high altitude within 5-15 days, at each groups respective baseline (i.e. 330 m for lowlanders and 1400 m for Sherpa), serum iron, TrF sat, Hb and Hct were all elevated in Sherpa compared to lowlanders at baseline (p<0.001 for all). 59 Figure 3.1. Iron metric during ascent to 5050 m in lowlanders (black) and Sherpa (red). Baseline values in lowlanders and Sherpa we collected at 330 and 1400 m respectively. Bold and faint lines denote mean and individual data, respectively. *Significant difference from baseline, P<0.05. †Significant difference between Sherpa and Lowlander at an altitude, P<0.05. Main effect of ethnicity denoted with vertical line and asterisk (linear mixed model with Bonferroni correction). Ascent to high altitude coincided with reductions in arterial oxygen content (CaO2), PaO2, PaCO2 and HCO3- in both groups with ascent (main effect p<0.001 for all; Table 3.3). 3.3.1.2. Cardiopulmonary status during ascent There were comparable increases in PASP, PVR and mPAP between lowlanders and Sherpa upon ascent from 1400 m to 5050 m (main effect of ethnicity p=0.353, p=0.203 and p=0.177, 60 respectively; Figure 3.2; Table 3.3). However, the rise of PASP in Sherpa was not evident until 4200 m (+28%; p<0.001), compared to 3400 m in lowlanders (+44%; p<0.001). There was a main effect for both cardiac output and cardiac index (uncorrected and corrected, respectively, to body surface area) to increase with ascent in both groups (p=0.001 for both); these changes were attributable to an increased heart rate (main effect p<0.001), rather than a change in stroke volume (main effect p=0.813). However, SV was lower in Sherpa across all time points compared to lowlanders (main effect p<0.001). Figure 3.2. Serial cardiopulmonary measurements in 20 lowlanders (black) and 12 Sherpa (red) during ascent from Kathmandu (1400 m) to 5050 m at the Pyramid laboratory. Bold and faint lines denote mean and individual data, respectively. *Significant difference from baseline, P<0.05. †Significant difference between Sherpa and Lowlander within an altitude, P<0.05. Main effect of ethnicity denoted with vertical line and asterisk (linear mixed model with Bonferroni correction). PASP, pulmonary artery systolic pressure; PVR, pulmonary vascular resistance; CI, cardiac index; mPAP, mean pulmonary artery pressure. Given the influence of Hct on the pulmonary vasculature (Vanderpool & Naeije, 2018), and diverging responses of Hct during ascent in lowlanders (i.e. increased) and Sherpa (i.e. remained steady), PVR and PASP were corrected to [Hct]. Both PVR(Hct) and PASP(Hct) remained 61 significantly increased with ascent in both groups (main effect p<0.001 for both; Table 3.3). While there was no difference in PVR(Hct) between lowlanders (1.66±0.24 wood units) and Sherpa (1.67±0.38 wood units; main effect p=0.885), there was a tendency for lowlanders across all time points to have higher PASP(Hct) (28.2±5.6 mmHg) when compared to Sherpa (25.2±4.6 mmHg; main effect p=0.099). Multiple regression analysis (adjusted r2=0.40, p<0.001) revealed that sTfR (=0.34, t=3.5, p=0.001), SV (=0.26, t=2.7, p=0.009) and MAP (=0.33, t=3.5, p=0.001) had significant impacts on PASP – explaining 46 % of the variance in PASP. The iron requirement index is illustrated in Figure 3.3, using the two different indexes of total iron store status (i.e. body iron and sTfR index) across ascent. In both circumstances, the change in hepcidin (i.e. the restrainment of iron release; from 3400 m to 5050 m) per given change in total iron stores (indicated by either body iron or sTfR index) were significantly improved in Sherpa, compared to lowlanders (p=0.016 and p=0.029 respectively). 62 Figure 3.3. Indices of total iron stores in 20 lowlanders (black) and 11 Sherpa (red) during ascent to high altitude. Bold and faint lines denote mean and individual data, respectively. For each total iron store index, is presented a corresponding iron recruitment (IR) index, which describes the efficiency to mobilize stored iron based on the change in hepcidin (i.e., the iron regulatory protein that limits release of iron into the circulation and availability to peripheral tissues by binding to and inducing internalization of ferroportin) between 3400 and 5050 m, for a given change in 1) body iron (i.e., total iron status determined by the logarithmic ratio of sTfR and ferritin) and 2) sTfR Index [total iron status determined by the ratio of sTfR to the log(ferritin)]. For both of these two indexes, a more positive value reflects a more responsive reduction in hepcidin per unit change in total iron stores). *Significant difference between groups, P<0.05 (independent samples t-test). Main effect of ethnicity denoted with vertical line and asterisk, P<0.05 (linear mixed model with Bonferroni correction). 63 3.3.2. Study 2a: Comparable pulmonary vascular reactivities to changes in PIO2 – but not during submaximal exercise at 50% Wpeak – in lowlanders and Sherpa following partial acclimatization. Prior to the infusion portion of Study 2 (when within-group pooling could be performed in lowlanders and Sherpa), there were no between-group differences in pulmonary O2 reactivity (i.e. PASP or PVR; depicted in relation to SpO2 at each PIO2 stage; Figure 3.4 and Table 3.4). Sherpa demonstrated a blunted HVR (i.e. VE/SPO2 during PIO2 67 mmHg), compared to lowlanders (p=0.009) – however, despite lower SpO2 (p<0.001) and presumably lower PAO2, PASP was not disproportionately elevated between lowlanders and Sherpa. During changing exercise intensities (depicted in relation to CI at each exercise intensity at 25% and 50% Wpeak), the only between-group difference was noted at 50% Wpeak, where Sherpa demonstrated an elevated PASP at compared to lowlanders (p=0.045). Interestingly, SpO2, VE, and reactivity (VE/SpO2 and PASP/SpO2) were similar between lowlanders and Sherpa at 50% Wpeak. As indicated in Table 3.1, the majority of the Sherpa from Study 1, also participated in Study 2 (n=8/12) – it is relevant to note that there were no differences in age (p=0.492), PASP (p=0.729), mPAP (p=0.616), PVR (p=0.670), serum iron (p=0.134), ferritin (p=0.219), TIBC (p=0.570) between the ascent Sherpa and altitude Sherpa recruited for Study 2. Figure 3.4. Pooled pulmonary vascular reactivities to changing inspired O2 and cardiac output in both lowlanders and Sherpa prior to infusion. Bold and faint lines denote mean and individual data, respectively. Pulmonary vascular reactivity in lowlanders (n=17; black) and Sherpa (n=20; red) to varying PIO2 (416, 87, and 67, mmHg from left to right, respectively; left panel) and to increasing cardiac output (right panel), coinciding to 64 baseline, and 25 % & 50 % peak wattage during recumbent cycling (lowlander, n=18, n=14, and n=12; Sherpa, n=19, n=12, n=9, at each timepoint respectively). *Significant difference from baseline, P<0.05. †Significant difference between Sherpa and Lowlander within an altitude, P<0.05. Main effect of intensity denoted with line and colour-coded asterisk, P<0.05. 3.3.3. Study 2b: Manipulating iron bioavailability led to similar pulmonary vascular responses in lowlanders and Sherpa to changing PIO2 at rest, but partially divergent reactivities at 25% Wpeak. In both groups, compared to baseline, iron infusion increased mean serum iron and TrF Sat (by 432% and 365%, respectively), whereas DFO infusion decreased mean serum iron and TrF Sat (by 78% and 82%, respectively; main effect of time p<0.001 for all; Figure 3.5; Table 3.5). Figure 3.5. Impact of iron infusion [via iron (III)-hydroxide sucrose] and iron chelation (via desferrioxamine) in lowlanders and Sherpa at 5050 m. Iron infusion decreased resting PASP and PVR in both partial acclimatized lowlanders and Sherpa (main effect p=0.005 and p=0.004 respectively), with no between-group differences. The overall reduction in resting PASP across both groups was 11%, which was likely predominantly driven by lowlanders, who exhibited a 18% reduction, whereas Sherpa only displayed a 6% reduction. These changes also mirror those occurring in PVR. Administration of DFO did not affect resting PASP and PVR in either lowlanders or Sherpa (main effect p>0.05 for both). Correcting PVR or PASP to Hct, did not alter the influence of iron or DFO-infusion at rest. As summarized in Table 3.6, manipulating iron status, produced similar pulmonary vascular responses to altered PIO2 between partially acclimatized lowlanders and Sherpa at 5050 m. 65 3.3.3.1. Sub-maximal recumbent exercise at 25% Wpeak Peak wattages during the recumbent VO2max test were 20115 W and 15429 W for lowlanders and Sherpa respectively, with a tendency for peak heart rates to be lower in lowlanders (15314 bpm) compared to Sherpa (16417 bpm; unpaired t-test p=0.076). Submaximal exercise (at 25% Wpeak) was therefore performed at 504 W in lowlanders and 387 W in Sherpa. There was a mostly consistent, main effects of PASP increasing from baseline to 25% Wpeak (main effect of intensity, p<0.001 in all groups, except lowlander DFO group; Table 3.7). However, only Sherpa demonstrated higher PASP at 25% Wpeak at post-infusion, compared to pre-infusion – an increase by 6% following iron (p=0.016), and increase by 30% following DFO (p=0.003; Figure 3.6, Table 3.7). Figure 3.6. Individual and mean pulmonary reactivity to increasing cardiac output – coinciding to baseline, and 25 % peak wattage during recumbent cycling – in lowlanders and Sherpa. Bold and faint lines denote mean and individual data, respectively. Iron – Lowlanders (n=11 and 8) and Sherpa (n=12-11 and 6) at each CI respectively, pre- and post-iron. DFO – Lowlanders (n=7 and 6-5) and Sherpa (n=7 and 6-5) at each CI respectively, pre- and post-DFO. * Significant effect from rest, P<0.05. 66 3.3.4. Study 2c: Inflammatory status is not different between lowlanders and Sherpa and is unaffected by iron manipulation. Prior to any infusion at 5050 m, we found subtle differences in certain pro-inflammatory markers, with lowlanders demonstrating a tendency for higher IL-6, IL-8 and TNF compared to Sherpa – although both groups exceeded North American reference ranges, except for IL-8 in Sherpa (Table 3.8). Following iron infusion, IFN (pro-inflammatory) and GDF-15 (anti-inflammatory) were significantly altered (i.e. decreased and increased respectively) in both groups. There were no differential responses in inflammatory markers between lowlanders and Sherpa (Table 3.9). In lowlanders, serum ferritin was correlated with IL-1 (r=0.779, p<0.001), IL-1 (r=0.848, p<0.001), IL-6 (r=0.789, p=0.001) and CRP (r=0.516, p=0.041); body iron was correlated with IL-1 (r=0.568, p=0.017), IL-1 (r=0.503, p=0.047) and IL-6 (r=0.595, p=0.032); and sTfR was only correlated with PDGF-AB/BB (r=0.583, p=0.011). In Sherpa, IFN and IL-1 concentration were positively correlated to PVR (r=0.493, p=0.045 and r=0.642, p=0.033, respectively). 67 Table 3.1. Sherpa demographics and time at altitude. Ascent study Infusion study Subject ID Sex Age (years) Childhood elevation (m) Childhood residence Total years at altitude yes yes 101 m 22 3800 Thame 17 yes yes 102 m 32 3440 Thamo 31 yes yes 103 m 31 3400 Namche 12 yes yes 104 m 25 3440 Thamo 25 yes no 105 m 38 3440 Salleri 24 yes no 106 m 44 3790 Khumjung 39 yes no 107 m 44 3790 Khumjung 44 yes yes 108 m 26 3440 Thamo 21 yes yes 109 m 44 3985 Pangbouche 41 yes yes 110 m 22 3440 Thamo 20 yes no 111 m 59 3840 Kunde 59 yes yes 112 m 31 4217 Pheriche 16 no yes 113 m 42 3985 Pangbouche 43 no yes 114 m 19 3840 Kunde 14 no yes 115 m 24 2042 Darjeeling 6 no yes 116 m 26 4217 Pheriche 26 no yes 117 m 18 3800 Thame 18 no yes 118 m 20 3800 Thame 16 no yes 119 m 18 3800 Thame 18 no yes 120 m 18 3800 Thame 18 no yes 121 m 20 3800 Thame 20 no yes 122 m 23 3800 Thame 23 no yes 123 m 22 3440 Thamo 25 no yes 124 m 25 3440 Thamo 10 Modified from (Hoiland et al., 2019). 68 Table 3.2. Iron homeostasis during ascent to altitude in lowlanders and Sherpa. Variable Group 330 / 1400 m 3400 m 4370 m 5050 m Altitude Ethnicity Inter. Iron (mol l-1) Lowlander 16.9±6.6† 8.6±6.2†* 8.4±7.2* 7.1±6.0* <0.001 0.010 0.004 Sherpa 26.3±6.2 15.6±8.7* 7.5±3.9* 7.7±4.3* TIBC (g dl-1) Lowlander 313.8±47.4 216.1±48.6 204.6±68.8 195±72.1 <0.001 0.065 0.103 Sherpa 245.5±32.6 223.5±42.6 186.0±69.4 174.9±88.5 Ferritin (ng ml-1) Lowlander 53.0 ± 40.5 85.1 ± 83.3 72.9 ± 98.4 34.5 ± 28.5 <0.001 0.002 0.198 Sherpa 158.8±115.6 218.8±136.7 152.9±111.3 113.8±95.6 Hepcidin (ng ml-1) Lowlander 5.6±1.6 7.0±3.9† 4.5±2.0§ 3.6±2.3§ <0.001 0.003 0.020 Sherpa 11.0±0.2 5.6±1.7§ 5.1±1.9§ TrF Sat (%) Lowlander 30.6±12.2† 22.5±14.1† 23.5±18.0 19.1±11.8* <0.001 0.001 <0.001 Sherpa 60.6±16.2 38.6±21.4* 22.2±6.9* 25.3±6.0* sTfR (nmoll-1) Lowlander 16.2 ± 2.2 21.1 ± 3.3 22.9 ± 6.4 24.9 ± 5.6 <0.001 0.672 0.956 Sherpa 20.6±4.6 22.4±6.4 24.0±6.2 Body iron (mgkg-1) Lowlander 10.2 ± 3.2 11.0 ± 3.4 9.4 ± 4.3 7.7 ± 2.8 <0.001 0.001 0.990 Sherpa 15.2 ± 3.3 13.2 ± 3.2 11.8 ± 4.0 sTfR Index Lowlander 11.4 ± 3.9 12.7 ± 3.6 17.5 ± 11.3 18.5 ± 6.6 <0.001 0.011 0.536 Sherpa 9.5 ± 3.2 11.5 ± 4.5 13.7 ± 5.8 VEGF (pgml-1) Lowlander 277.3 ± 200.1 296.6 ± 352.0 380.6 ± 748.9 395.2 ± 615.3 0.209 0.575 0.726 Sherpa 250.6 ± 135.4 223.8 ± 114.6 344.4 ± 266.8 Mean ± SD for n=20 for lowlanders, and n=11-12 for Sherpa. Baseline measures in lowlanders and Sherpa coincide with 330 and 1400 m respectively. * Significant difference from baseline (or § from Namche when Sherpa have no KTM data), P<0.05. † Significant difference between Sherpa and Lowlander within an altitude, P<0.05. Linear mixed model with Bonferroni correction. Inter, interaction; TIBC, total iron binding capacity; TrF sat, transferrin saturation; sTfR, soluble transferrin receptor; VEGF, vascular endothelial growth factor. 69 Table 3.3. Cardiopulmonary variables during ascent to altitude in lowlanders and Sherpa. Variable Group 1400 m 3400 m 4370 m 5050 m Altitude Ethnicity Inter. PASP (mmHg) Lowlander 19.7±2.8 28.2±6.3†* 28.9±5.7* 29.6±6.1* <0.001 0.353 0.005 Sherpa 21.5±4.0 23.8±4.5 27.3±6.3* 27.6±4.3* PVR (wood units) Lowlander 1.32±0.19 1.58±0.17 1.67±0.25 1.68±0.25 <0.001 0.203 0.053 Sherpa 1.59±0.36 1.65±0.38 1.66±0.35 1.79±0.41 mPAP (mmHg) Lowlander 18.0 ± 7.1 26.9 ± 10.0 27.6 ± 11.6 25.3 ± 9.9 <0.001 0.177 0.275 Sherpa 16.6 ± 8.4 25.6 ± 9.4 22.6 ± 8.6 17.9 ± 7.3 MAP (mmHg) Lowlander 84.9±6.7† 94.8±9.9†* 96.0±9.0* 101.4±9.8* <0.001 0.079 0.001 Sherpa 95.2±9.2 101.5±10.7 100.7±9.6 96.5±5.3 SV (ml) Lowlander 63.7 ± 12.3 64.4 ± 10.5 67.0 ± 11.9 66.2 ± 13.1 0.813 <0.001 0.484 Sherpa 47.7 ± 10.0 50.2 ± 8.3 47.6 ± 5.7 47.4 ± 8.5 HR (bpm) Lowlander 58 ± 11 62 ± 14 61 ± 15 64 ± 16 <0.001 0.149 0.163 Sherpa 61 ± 10 66 ± 10 69 ± 10 74 ± 8 CO (l min-1) Lowlander 3.6 ± 0.8 4.0 ± 1.2 4.0 ± 0.9 4.2 ± 1.3 0.001 0.024 0.976 Sherpa 2.9 ± 0.7 3.3 ± 0.6 3.3 ± 0.6 3.5 ± 0.8 CI (l min-1 m2) Lowlander 2.0 ± 0.5 2.2 ± 0.6 2.2 ± 0.5 2.3 ± 0.7 0.001 0.024 0.977 Sherpa 1.6 ± 0.4 1.8 ± 0.3 1.8 ± 0.3 2.0 ± 0.5 SaO2 (%) Lowlander 95.6±1.9 92.2±2.1 86.9±2.7 81.7±2.8 <0.001 0.744 0.309 Sherpa 95.6±1.7 93.5±2.5 86.8±2.6 81.3±1.7 PaO2 (mmHg) Lowlander 76.1 ± 5.2 51.8 ± 4.2 47.2 ± 3.4 40.5 ± 4.0 <0.001 0.808 0.877 Sherpa 74.8 ± 7.6 52.2 ± 4.6 46.7 ± 4.5 40.6 ± 4.3 PaCO2 (mmHg) Lowlander 40.7 ± 2.0 34.5 ± 1.3* 32.4 ± 1.3†* 30.3 ± 1.7†* <0.001 0.083 0.003 Sherpa 39.8 ± 1.6 35.4 ± 2.3* 34.3 ± 2.9* 32.1 ± 2.7* CaO2 (ml dl-1) lowlander 17.9 ± 1.1† 17.3 ± 1.0 16.7 ± 0.9* 15.5 ± 1.3* <0.001 0.176 0.009 Sherpa 19.3 ± 1.1 17.3 ± 1.4* 16.8 ± 1.0* 15.7 ± 1.4* pH Lowlander 7.42 ± 0.02 7.43 ± 0.02 7.43 ± 0.02 7.46 ± 0.02 <0.001 <0.001 0.902 Sherpa 7.40 ± 0.01 7.41 ± 0.02 7.40 ± 0.02 7.44 ± 0.02 BE(ecf) (mmol l-1) Lowlander 2.0 ± 1.6 -0.8 ± 1.6 -2.7 ± 1.5 -2.2 ± 1.8 <0.001 0.030 0.068 Sherpa -0.1 ± 1.4 -2.2 ± 1.8 -3.3 ± 2.1 -2.7 ± 2.2 HCO3- (mmol l-1) Lowlander 26.4 ± 1.4† 23.4 ± 1.3†* 21.7 ± 1.2* 21.6 ± 1.4* <0.001 0.084 0.003 Sherpa 24.6 ± 1.2 22.3 ± 1.6* 21.5 ± 1.8* 21.7 ± 2.0* PASP[Hct] (mmHg) Lowlanders 21.8 ± 3.3 29.6 ± 6.5 30.5 ± 6.2 31.1 ± 6.2 <0.001 0.099 0.081 Sherpa 21.4 ± 3.7 24.3 ± 4.1 27.5 ± 6.8 27.8 ± 3.9 PVR[Hct] (wood units) Lowlanders 1.5 ± 0.2 1.7 ± 0.2 1.8 ± 0.3 1.8 ± 0.3 <0.001 0.885 0.272 Sherpa 1.6 ± 0.4 1.6 ± 0.3 1.7 ± 0.4 1.8 ± 0.4 Hct (%) Lowlander 40.1 ± 2.2† 42.5 ± 2.1* 42.5 ± 1.5* 42.6 ± 2.2* 0.016 0.021 0.002 Sherpa 43.6 ± 2.7 42.6 ± 2.2 43.6 ± 2.6 43.7 ± 2.2 Hb (g dl-1) Lowlander 13.6 ± 0.7† 14.4 ± 0.7* 14.5 ± 0.5* 14.4 ± 0.7* 0.018 0.019 0.001 Sherpa 14.8 ± 0.9 14.5 ± 0.7 14.8 ± 0.9 14.8 ± 0.7 Mean ± SD for n=20 for lowlanders, and n=11-12 for Sherpa. * Significant difference from baseline, P<0.05; † Significant difference between Sherpa and Lowlander within an altitude, P<0.05. Linear mixed model with Bonferroni correction. 70 Table 3.4. Pulmonary vascular reactivities following partial acclimatization at 5050 m. Variable Group 67 mmHg 87 mmHg 416 mmHg Ethnicity PIO2 Inter PASP (mmHg) Lowlander 24.1 ± 9.0 23.7 ± 5.8 18.7 ± 4.7 0.863 <0.001 0.321 Sherpa 26.2 ± 7.9 22.0 ± 6.9 20.0 ± 5.1 PVR (wood units) Lowlander 1.6 ± 0.5 1.6 ± 0.3 1.5 ± 0.3 0.838 0.624 0.592 Sherpa 1.6 ± 0.3 1.5 ± 0.4 1.6 ± 0.4 SpO2 (%) Lowlander 74.8 ± 6.6*† 87.3 ± 2.6 98.5 ± 0.8* 0.015 <0.001 0.005 Sherpa 67.7 ± 10* 85.9 ± 3.3 98.2 ± 0.8* VE (l min-1) Lowlander 18.5 ± 6.9 10.7 ± 2.2 11.1 ± 2.7 0.499 <0.001 0.063 Sherpa 17.0 ± 3.4 12.8 ± 3.8 12.7 ± 4.0 VE/SPO2 (l min-1 %-1) Lowlander 0.26 ± 0.11 0.12 ± 0.02 0.11 ± 0.03 0.374 <0.001 0.360 Sherpa 0.25 ± 0.06 0.15 ± 0.04 0.13 ± 0.04 PASP/SPO2 (mmHg %-1) Lowlander 0.33 ± 0.13 0.27 ± 0.07 0.19 ± 0.05 0.431 <0.001 0.088 Sherpa 0.39 ± 0.14 0.26 ± 0.08 0.20 ± 0.05 ΔVE/ΔSPO2 (Δl min-1 Δ%-1) Lowlander 0.94 ± 1.49*† ⋅ -0.03 ± 0.20 0.087 0.005 0.047 Sherpa 0.22 ± 0.71 ⋅ 0.04 ± 0.19 ΔPASP/ΔSPO2 (ΔmmHg Δ%-1) Lowlander -0.12 ± 0.76 ⋅ 0.48 ± 0.55 0.759 0.021 0.204 Sherpa 0.05 ± 0.67 ⋅ 0.24 ± 0.38 71 Variable Group Rest 25% Wpeak 50% Wpeak Ethnicity Intensity Inter PASP (mmHg) Lowlander 23.7 ± 5.8 27.9 ± 6.0* 31.8 ± 6.7*† 0.794 <0.001 0.002 Sherpa 22.0 ± 6.9 28.1 ± 5.3 39.9 ± 8.4* PVR (wood units) Lowlander 1.6 ± 0.3 1.5 ± 0.2 1.4 ± 0.2 0.576 0.415 0.051 Sherpa 1.5 ± 0.4 1.6 ± 0.2 1.8 ± 0.3 CI (l⋅min-1 m-2) Lowlander 2.5 ± 0.5 4.1 ± 0.7 5.5 ± 0.8 0.567 <0.001 0.373 Sherpa 2.6 ± 0.5 3.9 ± 0.7 5.1 ± 1.9 MAP (mmHg) Lowlander 95.1 ± 8.9 82.5 ± 4.1 79.0 ± 4.3 0.024 <0.001 0.137 Sherpa 88.4 ± 9.1 80.1 ± 3.7 78.7 ± 4.9 SpO2 (%) Lowlander 87.4 ± 2.7 80.5 ± 2.8* 77.9 ± 4.3* 0.676 <0.001 0.025 Sherpa 85.8 ± 3.4 79.4 ± 4.6* 79.8 ± 5.1* VE (l min-1) Lowlander 10.8 ± 2.2 86.6 ± 11.1 105.9 ± 29.0 0.104 <0.001 0.577 Sherpa 12.7 ± 3.9 93.5 ± 10.0 115.5 ± 13.4 VE/SPO2 (l min-1 %-1) lowlander 0.12 ± 0.02 1.08 ± 0.15 1.37 ± 0.4 0.153 <0.001 0.356 Sherpa 0.15 ± 0.04 1.19 ± 0.17 1.44 ± 0.2 PASP/SPO2 (mmHg %-1) lowlander 0.27 ± 0.06 0.34 ± 0.08 0.42 ± 0.1 0.503 <0.001 0.123 Sherpa 0.26 ± 0.08 0.37 ± 0.08 0.49 ± 0.13 ΔVE/ΔSPO2 (Δl min-1 Δ%-1) lowlander ⋅ 11.73 ± 4.79 15.92 ± 22.6 0.609 0.944 0.389 Sherpa ⋅ 18.5 ± 20.41 13.97 ± 17.52 ΔPASP/ΔSPO2 (ΔmmHg Δ%-1) lowlander ⋅ 0.50 ± 0.70 0.71 ± 0.46 0.852 0.701 0.877 Sherpa ⋅ 0.64 ± 0.87 0.71 ± 2.45 Mean ± SD. N=18 and 19 in lowlanders and Sherpa respectively. At 50% Wpeak, the sample size was reduced to n=12 and 9, in lowlanders and Sherpa respectively, due to measurement challenges. Linear mixed model with Bonferroni correction. 72 Table 3.5. Iron homeostasis and hematological measures prior to and following iron or DFO infusion. Variable Group Pre-IRON Post-IRON Ethnicity Time Inter. Pre-DFO Post-DFO Ethnicity Time Inter. Iron (mol l-1) Lowlander 10.8±7.5 53.8±22.6 0.606 <0.001 0.936 11.7±7.5 2.4±0.9 0.206 <0.001 0.243 Sherpa 8.9±7.1 51.2±15.7 7.6±4.7 2.0±0.0 TIBC (g dl-1) Lowlander 196.5±83.3 213.7±76.4 0.260 0.230 0.614 244.1±31.4† 271.3±91.4 0.087 0.001 0.016 Sherpa 161.9±72.9 203.4±55.4 145.0±73.4 268.1±36.8* Ferritin (ng ml-1) Lowlander 60.3±90.8 67.7±111.5 0.525 0.195 0.541 33.0±14.5 28.7±16.6 0.269 0.176 0.070 Sherpa 73.9±44.8 93.1±47.2 30.0±19.4 56.9±41.7 Tr Sat (%) Lowlander 29.8±14.6 135.5±17.5 0.911 <0.001 0.738 26.2±16.5 6.2±5.0 0.731 <0.001 0.438 Sherpa 28.9±15.5 137.8±13.3 30.7±12.0 4.3±0.7 sTfR (nmol l-1) Lowlander 35.9±8.1 32.3±5.2 0.520 <0.001 0.831 28.7±4.5 27.0±3.6 0.286 0.017 0.500 Sherpa 33.3±13.6 29.3±12.4 24.7±11.4 21.9±9.4 VEGF (pg ml-1) Lowlander 691.8±987.8 311.3±406.3* 0.243 0.028 0.048 417.7±460.6 287.5±248.0 0.776 0.189 0.251 Sherpa 264.6±179.0 242.4±156.8 317.6±171.8 308.3±168.0 SaO2 (%) Lowlander 81.8±3.2 80.7±5.6 0.022 0.622 0.681 84.3±4.7 80.0±2.6 0.022 0.672 0.069 Sherpa 77.1±7.0 77.0±3.0 74.9±6.2 77.9±5.6 PaO2 (mmHg) Lowlander 42.7 ± 3.7 42.5 ± 5.6 0.291 0.562 0.670 45.6 ± 9.3 41.4 ± 2.0 0.449 0.285 0.069 Sherpa 41.6 ± 6.4 40.3 ± 2.6 40 ± 4.9 41.5 ± 5.3 PaCO2 (mmHg) Lowlander 30.2 ± 2.0 29.6 ± 2.2 0.151 0.480 0.500 28.5 ± 2.4 29.1 ± 1.9 0.001 0.798 0.333 Sherpa 31.5 ± 3.4 31.5 ± 2.8 33.2 ± 2.1 32.3 ± 2.0 pH Lowlander 7.44 ± 0.02 7.45 ± 0.02 0.002 0.390 0.054 7.46 ± 0.02 7.50 ± 0.12 0.010 0.201 0.540 Sherpa 7.43 ± 0.02 7.42 ± 0.02 7.41 ± 0.02 7.42 ± 0.02 BE(ecf) (mmol l-1) Lowlander -3.6 ± 1.6 -3.2 ± 1.7 0.590 0.877 0.240 -3.7 ± 1.1 -3.8 ± 1.2 0.871 0.845 0.626 Sherpa -3.6 ± 1.9 -3.9 ± 1.5 -3.9 ± 2.4 -3.5 ± 1.2 HCO3- (mmol l-1) Lowlander 20.5 ± 1.4 20.8 ± 1.4 0.864 0.959 0.248 20.1 ± 1.1 20.1 ± 1.0 0.152 0.941 0.880 Sherpa 20.7 ± 1.8 20.4 ± 1.5 20.9 ± 2.1 21.0 ± 0.9 Hct (%) Lowlander 45.5±3.1 45.2±3.0 0.355 0.053 0.197 46.6±3.1 45.0±2.8 0.678 0.184 0.820 Sherpa 47.4±3.7 45.7±2.9 45.9±2.9 45.3±2.8 Hb (g dl-1) Lowlander 15.5 ± 1.0 15.4 ± 1.0 0.405 0.040 0.256 15.8 ± 0.9 15.3 ± 1.0 0.870 0.071 0.895 Sherpa 16.0 ± 1.1 15.5 ± 1.0 15.7 ± 0.9 15.4 ± 1.0 Mean ± SD for lowlanders and Sherpa (Iron: n=11 and n=12, respectively; DFO: n=7 and n=8 respectively) at 5050 m. PVR, PASP and mPAP during rest. * Significant difference from Pre-drug, P<0.05. † Significant difference between Sherpa and lowlander within an altitude, P<0.05. Linear mixed model with Bonferroni correction. 73 Table 3.6. Cardiopulmonary variables prior to and following iron or DFO infusion. Variable PIO2 Group Pre-Iron Post-Iron Ethnicity Time Inter. Pre-DFO Post-DFO Ethnicity Time Inter. PASP (mmHg) Baseline Lowlander 24.2 ± 5.3 19.9 ± 2.3 0.193 0.005 0.197 23.0 ± 6.9 26.0 ± 8.6 0.991 0.470 0.518 Sherpa 20.6 ± 5.8 19.3 ± 4.3 24.3 ± 8.4 24.5 ± 4.5 416 mmHg Lowlander 18.5 ± 5.1 17.6 ± 2.7 0.982 0.413 0.997 19.0 ± 4.4 19.9 ± 5.6 0.320 0.926 0.664 Sherpa 18.5 ± 4.0 17.6 ± 4.5 23.2 ± 6.3 21.6 ± 7.6 67 mmHg Lowlander 24.4 ± 9.3 24.3 ± 5.1 0.702 0.750 0.788 23.7 ± 9.1 21.9 ± 10.5 0.243 0.497 0.229 Sherpa 26.2 ± 7.1 24.9 ± 7.8 26.1 ± 9.9 31.7 ± 10.4 PVR (wood units) Baseline Lowlander 1.6 ± 0.4 1.4 ± 0.2 0.679 0.004 0.465 1.5 ± 0.1 1.6 ± 0.2 0.957 0.515 0.947 Sherpa 1.5 ± 0.4 1.4 ± 0.3 1.5 ± 0.5 1.6 ± 0.3 416 mmHg Lowlander 1.5 ± 0.3 1.5 ± 0.3 0.916 0.162 0.263 1.5 ± 0.3 1.5 ± 0.2 0.800 0.978 0.951 Sherpa 1.6 ± 0.4 1.4 ± 0.3 1.5 ± 0.4 1.5 ± 0.4 67 mmHg Lowlander 1.6 ± 0.4 1.6 ± 0.3 0.648 0.307 0.826 1.6 ± 0.5 1.4 ± 0.4 0.305 0.406 0.308 Sherpa 1.6 ± 0.3 1.7 ± 0.3 1.7 ± 0.4 1.9 ± 0.5 mPAP (mmHg) Baseline Lowlander 13.3 ± 5.0 15.3 ± 6.3 0.123 0.679 0.227 12.0 ± 3.8 13.6 ± 5.2 0.901 0.775 0.558 Sherpa 12.3 ± 3.7 11.3 ± 3.6 13.4 ± 5.5 12.8 ± 7.7 416 mmHg Lowlander 14.4 ± 4.3 10.1 ± 2.9* 0.866 0.108 0.003 9.7 ± 3.4 11.3 ± 4.1 0.188 0.316 0.908 Sherpa 11.8 ± 3.2 13.2 ± 4.5 11.6 ± 2.1 12.9 ± 3.4 67 mmHg Lowlander 14.3 ± 4.0 15.3 ± 6.6 0.169 0.382 0.098 15.7 ± 7.4 12.0 ± 8.8 0.722 0.755 0.317 Sherpa 14.4 ± 3.4 11.3 ± 2.5 13.9 ± 1.6 15.8 ± 7.0 MAP (mmHg) Baseline Lowlander 98.2 ± 8.4 99.0 ± 6.3 0.005 0.779 0.891 88.9 ± 6.6 90.1 ± 6.6 0.644 0.277 0.078 Sherpa 87.8 ± 9.4 88.1 ± 12.4 89.8 ± 8.7 85.1 ± 12.5 416 mmHg Lowlander 93.6 ± 6.8 95.5 ± 7.3 0.175 0.295 0.763 86.8 ± 8.5 86.4 ± 5.0 0.543 0.614 0.447 Sherpa 89.5 ± 8.1 90.5 ± 10.7 82.4 ± 10.7 84.7 ± 13.1 67 mmHg Lowlander 98.9 ± 9 101.0 ± 6.2 0.007 0.009 0.108 97.2 ± 3.7 86.8 ± 7.5* 0.331 0.037 0.034 Sherpa 85.8 ± 10.5 94.0 ± 10.2 87.8 ± 8.3 87.9 ± 11.5 VE (l min-1) Baseline Lowlander 11.1 ± 2.5 11.9 ± 3.1 0.885 0.293 0.850 10.1 ± 1.4 12.1 ± 2.6 0.006 0.091 0.705 Sherpa 11.4 ± 3.5 11.9 ± 4.9 14.8 ± 3.5 16.0 ± 3.8 416 mmHg Lowlander 10.9 ± 3.2 11.0 ± 2.8 0.646 0.423 0.345 11.5 ± 2.1 11.2 ± 2.0 0.113 0.296 0.175 Sherpa 12.3 ± 3.4 10.9 ± 4.2 13.1 ± 4.8 15.0 ± 3.8 67 mmHg Lowlander 19.2 ± 8.5 16.6 ± 3.9 0.332 0.052 0.667 17.4 ± 3.7 17.5 ± 5.1 0.523 0.228 0.250 Sherpa 16.9 ± 3.8 15.2 ± 3.9 17.0 ± 3.0 20.7 ± 6.3 74 Variable PIO2 Group Pre-Iron Post-Iron Ethnicity Time Inter. Pre-DFO Post-DFO Ethnicity Time Inter. SpO2 (%) Baseline Lowlander 87.3 ± 3.0 85.3 ± 2.6* 0.745 0.201 0.025 87.4 ± 2.0 86.9 ± 2.0 0.482 0.542 0.838 Sherpa 85.6 ± 3.6 86.2 ± 3.3 86.4 ± 3.0 86.1 ± 2.9 416 mmHg Lowlander 98.5 ± 0.9 98.5 ± 1.0 0.307 0.273 0.347 98.4 ± 0.7 99.6 ± 0.8*† 0.040 0.434 0.034 Sherpa 98.0 ± 0.7 98.5 ± 1.1 98.6 ± 0.7 98.1 ± 1.2 67 mmHg Lowlander 74.1 ± 8.0 72.7 ± 6.9 0.085 0.636 0.839 76.1 ± 3.1 71.2 ± 11.1 0.068 0.541 0.406 Sherpa 67.7 ± 11.8 67.1 ± 9.7 67.6 ± 7.0 68.3 ± 8.8 HR (bpm) Baseline Lowlander 67.5 ± 10.9 62.4 ± 9.2 0.035 0.164 0.103 59.7 ± 6.7 65.3 ± 10.5 0.030 0.275 0.265 Sherpa 73.7 ± 8.9 74.1 ± 11.8 72.0 ± 9.1 71.9 ± 8.6 416 mmHg Lowlander 59.9 ± 6.4 59.8 ± 8.9 0.188 0.662 0.704 56.8 ± 7.6 62.7 ± 13.7 0.720 0.167 0.532 Sherpa 64.5 ± 8.9 63.3 ± 7.5 60.2 ± 8.6 62.5 ± 9.3 67 mmHg Lowlander 73 ± 10.4 68.5 ± 9.7 0.007 0.025 0.600 65.9 ± 7.0 78.7 ± 6.3 0.034 0.029 0.253 Sherpa 85.4 ± 11.5 77.7 ± 9.5 77.6 ± 9.7 82.1 ± 10.2 SV (ml) Baseline Lowlander 76.2 ± 13.2 71.5 ± 15.4 0.037 0.622 0.326 72.0 ± 17.2 85.3 ± 17.9†* 0.245 0.090 0.006 Sherpa 62.7 ± 14.6 64.3 ± 9.7 71.4 ± 13.5 67.5 ± 8.6 416 mmHg Lowlander 65.5 ± 10.9 71.1 ± 16.9 0.011 0.306 0.641 64.9 ± 13.1 64.6 ± 11.5 0.424 0.838 0.777 Sherpa 57.8 ± 10.5 59.9 ± 8.0 69.7 ± 16.2 70.9 ± 14.3 67 mmHg Lowlander 77.8 ± 13.7 76.3 ± 10.3 0.017 0.733 0.789 85.4 ± 15.2 85.8 ± 20.8 0.042 0.793 0.812 Sherpa 65.7 ± 13.5 65.6 ± 10.5 68.7 ± 11.7 69.3 ± 12.8 CO (l min-1) Baseline Lowlander 4.8 ± 0.8 4.4 ± 0.8 0.856 0.697 0.174 4.3 ± 1.0 5.3 ± 1.1* 0.849 0.223 0.049 Sherpa 4.5 ± 1.0 4.7 ± 1.2 4.8 ± 1.4 4.5 ± 0.9 416 mmHg Lowlander 4.0 ± 0.7 3.9 ± 0.9 0.385 0.638 0.208 3.7 ± 0.6 3.8 ± 0.7 0.489 0.686 0.440 Sherpa 3.5 ± 0.7 3.9 ± 1.2 4.3 ± 1.6 3.8 ± 0.7 67 mmHg Lowlander 5.3 ± 1.2 5.3 ± 0.8 0.951 0.810 0.510 5.1 ± 0.8 6.0 ± 1.4 0.859 0.291 0.358 Sherpa 5.4 ± 1.1 5.3 ± 1.3 5.4 ± 1.4 5.5 ± 0.9 Mean ± SD for lowlanders and Sherpa (Iron: n=11 and n=12, respectively; DFO: n=7 and n=8 respectively) at 5050 m. Baseline PIO2=87 mmHg. * Significant difference from Pre-drug, P<0.05. † Significant difference between Sherpa and Lowlander within an altitude, P<0.05. Linear mixed model with Bonferroni correction. MAP, mean arterial pressure; VE, ventilation; SpO2; peripheral oxygen saturation; HR, heart rate; SV, stroke volume; CO, cardiac output; PVR, pulmonary vascular resistance; PASP, pulmonary artery systolic pressure; mPAP, mean pulmonary pressure. 75 Table 3.7. Cardiopulmonary responses to submaximal exercise in lowlanders and Sherpa, before and after either infusion of iron or DFO. Lowlander Sherpa Variable IRON Baseline 25% Wpeak Time Intensity Inter. Baseline 25% Wpeak Time Intensity Inter. SpO2 (%) Pre 87.4 ± 3.0 80.6 ± 3.0 <0.001 <0.001 0.087 85.3 ± 2.5 80.4 ± 3.6 0.345 <0.001 0.165 Post 85.2 ± 2.9 75.2 ± 4.8 85.8 ± 2.3 77.9 ± 3.3 MAP (mmHg) Pre 98.9 ± 9.3 97.1 ± 9.0 0.426 0.687 0.517 85.1 ± 9.8 94.0 ± 14.0 0.773 0.018 0.696 Post 99.2 ± 6.2 100.1 ± 8.2 87.3 ± 9.5 93.7 ± 12.9 SYS (mmHg) Pre 129.8 ± 7.7 137.6 ± 14.4 0.072 <0.001 0.058 113.9 ± 11.2 125.3 ± 17.9 0.890 0.009 0.990 Post 129.4 ± 8.4 148.9 ± 16.9 114.4 ± 13.3 125.9 ± 17.1 DIA (mmHg) Pre 83.5 ± 11.0 76.9 ± 9.2 0.930 0.014 0.764 70.7 ± 10.0 78.4 ± 14.5 0.754 0.076 0.596 Post 84.1 ± 7.2 75.7 ± 11.7 73.8 ± 8.0 77.6 ± 12.2 HR (bpm) Pre 62 ± 10 87 ± 9 0.396 <0.001 0.924 76 ± 5 102 ± 10 0.410 <0.001 0.498 Post 65 ± 13 90 ± 12 76 ± 8 99 ± 11 SV (ml) Pre 79.3 ± 13.3 84.0 ± 10.1 0.837 0.016 0.208 58.9 ± 16.4 65.0 ± 21.2 0.688 0.985 0.143 Post 74.0 ± 17.6 87.9 ± 16.6 66.5 ± 11.4 60.6 ± 18.4 CO (l min-1) Pre 4.9 ± 0.9 7.3 ± 1.1 0.629 <0.001 0.232 4.4 ± 1.1 6.6 ± 1.8 0.959 0.002 0.138 Post 4.7 ± 0.8 7.8 ± 1.2 5.1 ± 1.2 6.0 ± 1.7 PVR (wood units) Pre 1.6 ± 0.2 1.6 ± 0.2 0.424 0.030 0.009 1.8 ± 0.3 1.4 ± 0.2 0.301 0.539 0.045 Post 1.4 ± 0.2† 1.7 ± 0.3* 1.7 ± 0.3 1.8 ± 0.3† PASP (mmHg) Pre 24.6 ± 3.9 29.1 ± 6.4* 0.294 <0.001 0.016 24.9 ± 4.2 26.2 ± 5.0 0.806 <0.001 0.003 Post 19.9 ± 2.7† 31.1 ± 5.3* 20.8 ± 5.1† 30.8 ± 3.7*† 76 Lowlander Sherpa Variable DFO Baseline 25% Wpeak Time Intensity Inter. Baseline 25% Wpeak Time Intensity Inter. SpO2 (%) Pre 87.4 ± 2.3 82.1 ± 2.3 0.448 <0.001 0.636 85.1 ± 2.9 78.1 ± 4.9 0.680 <0.001 0.863 Post 87.2 ± 1.9 81.1 ± 3.4 85.7 ± 2.8 77.8 ± 5.4 MAP (mmHg) Pre 87.6 ± 6.1 94 ± 4.3 0.127 0.003 0.471 87.7 ± 9.6 90.3 ± 13.1 0.558 0.023 0.131 Post 89.5 ± 6.7 99.8 ± 8.4 81.8 ± 15.3 93.9 ± 8.2 SYS (mmHg) Pre 118.3 ± 3.7 145.9 ± 9.4 0.826 <0.001 0.785 123.0 ± 14.7 131.3 ± 21.8 0.736 <0.001 0.025 Post 120.0 ± 8.6 144.5 ± 14.3 115.9 ± 14.2 140.4 ± 19.7* DIA (mmHg) Pre 72.3 ± 9.4 68.1 ± 5.9 0.050 0.489 0.233 70.0 ± 8.4 69.8 ± 14.4 0.477 0.587 0.553 Post 74.3 ± 7.7 77.4 ± 6.1 64.8 ± 16.4 70.6 ± 12.8 HR (bpm) Pre 58 ± 6 80 ± 4 0.644 <0.001 0.806 70 ± 9 92 ± 11 0.917 <0.001 0.469 Post 60 ± 9 80 ± 10 68 ± 9 94 ± 9 SV (ml) Pre 75.9 ± 10.8 97.5 ± 16.8 0.035 0.001 0.178 75.3 ± 14.3 82.3 ± 16.0 0.065 0.147 0.773 Post 90.2 ± 13.6 101.0 ± 12.4 68.7 ± 9.2 73.5 ± 17.2 CO (l min-1) Pre 4.4 ± 0.5 7.8 ± 1.4 0.059 <0.001 0.249 5.3 ± 1.3 7.5 ± 1.1 0.157 <0.001 0.967 Post 5.4 ± 0.8 8.0 ± 0.6 4.7 ± 0.8 6.9 ± 1.5 PVR (wood units) Pre 1.5 ± 0.1 1.3 ± 0.01 0.017 0.001 0.942 1.8 ± 0.3 1.7 ± 0.2 0.686 0.331 0.142 Post 1.7 ± 0.1 1.4 ± 0.2 1.7 ± 0.3 1.9 ± 0.2 PASP (mmHg) Pre 22.8 ± 4.0 24.7 ± 4.8 0.198 0.804 0.353 27.9 ± 6.3 31.8 ± 2.7 0.096 <0.001 0.006 Post 29.1 ± 8.3 25.8 ± 6.8 25.8 ± 2.5 39.0 ± 6.7*† Mean ± SD. Supine submaximal exercise at 25% of peak wattage (Wpeak) in lowlanders (n=8 Iron; n=5 DFO) and Sherpa (n=6 Iron; n=5 DFO) at 5050 m. * Significant difference from baseline, † Significant difference from pre- to post-drug within a condition, P<0.05. Linear mixed model with Bonferroni correction. 77 Table 3.8. Inflammatory markers in lowlander and Sherpa at 5050 m Variable Lowlander Sherpa p-value IFN (pg/mL) 72.4 ± 74.9 38.3 ± 46 0.119 IL-1 (pg/mL) 332.6 ± 361.7 202 ± 283.7 0.309 IL-1 (pg/mL) 38.7 ± 55.8 33.6 ± 63.7 0.828 IL-6 (pg/mL) 29.7 ± 25.3 14.2 ± 13.9 0.073 IL-8 (pg/mL) 63.5 ± 76.2 25.6 ± 24.7 0.058 IL-10 (pg/mL) 18.6 ± 21.1 13.1 ± 28.5 0.594 TNF (pg/mL) 46.6 ± 38.2 27.5 ± 13.7 0.059 CRP (ng/L) 12.5 ± 14.8 13.8 ± 15.5 0.804 GDF-15 (ng/mL) 0.44 ± 0.16 0.46 ± 0.11 0.601 PDGF-AB/BB (pg/L) 54.5 ± 20.2 53.4 ± 17.8 0.862 VEGF (pg/mL) 585.2 ± 817.2 285.8 ± 173.6 0.145 Mean ± SD. Pooled mean values in lowlanders (n=13-18) and Sherpa (n=10-20). Independent samples t-test with unequal variances, based on Levene’s test for equality of variances. Interferon, IFN; interleukin, IL; tumor necrosis factor, TNF; c-reactive protein, CRP; growth differentiating factor, GDF; platelet derived growth factor, PDGF; vascular endothelial growth factor, VEGF. 78 Table 3.9. Impact of iron manipulation on inflammatory markers at 5050 m Variable Iron Lowlander Sherpa Ethnicity Time Inter. DFO Lowlander Sherpa Ethnicity Time Inter. IFN (pg/mL) Pre 67.9 ± 68.3 25.0 ± 21.5 0.059 0.032 0.106 Pre 78.9 ± 88.7 55.0 ± 63.0 0.755 0.045 0.101 Post 45.3 ± 41.1 20.1 ± 17.4 Post 49.7 ± 55.4 53.2 ± 65.5 IL-1 (pg/mL) Pre 361.7 ± 444.4 127.9 ± 254.8 0.212 0.109 0.316 Pre 291.1 ± 221.9 276.0 ± 320.1 0.557 0.743 0.924 Post 232.4 ± 230.7 75.2 ± 159.6 Post 376.1 ± 171.2 192.1 ± 337.8 IL-1 (pg/mL) Pre 42.1 ± 66.2 11.4 ± 9.9 0.160 0.379 0.464 Pre 31.3 ± 25.1 49.5 ± 81.6 0.554 0.514 0.620 Post 27.6 ± 24.7 7.7 ± 8.4 Post 26.5 ± 27.3 46.7 ± 82.3 IL-6 (pg/mL) Pre 33.6 ± 31.5 10.9 ± 6.0 0.024 0.317 0.186 Pre 23.5 ± 10.1 20.0 ± 22.4 0.278 0.194 0.447 Post 29.9 ± 20.9 14.6 ± 9.5 Post 24 ± 11.8 16.0 ± 20.7 IL-8 (pg/mL) Pre 59.5 ± 76.0 21.6 ± 21.9 0.130 0.222 0.049 Pre 69.7 ± 82.2 31.6 ± 29.0- 0.235 0.118 0.688 Post 53.6 ± 69.2* 23.1 ± 17.5 Post 64.9 ± 74.9 28.7 ± 31.2 IL-10 (pg/mL) Pre 15.2 ± 18.3 5.7 ± 6.2 0.127 0.108 0.191 Pre 23.9 ± 25.8 26.1 ± 47.8 0.822 0.822 0.412 Post 11.8 ± 16.8 4.1 ± 4.5 Post 22.1 ± 19.3 20.0 ± 44.1 TNF (pg/mL) Pre 46.4 ± 45.1 27.9 ± 11.1 0.255 0.518 0.083 Pre 46.9 ± 27.4 26.9 ± 17.8 0.133 0.225 0.543 Post 39.3 ± 31.1 31.3 ± 11.2 Post 42.8 ± 29.9 25.5 ± 14.6 CRP (ng/L) Pre 12.9 ± 14.7 15.7 ± 16.9 0.838 0.746 0.123 Pre 11.8 ± 16.5 11.7 ± 14.5 0.999 0.186 0.753 Post 10.1 ± 11.9 13 ± 18.7 Post 9.2 ± 11.7 7.1 ± 10.8 GDF-15 (ng/mL) Pre 0.41 ± 0.14 0.49 ± 0.11 0.309 <0.001 0.451 Pre 0.48 ± 0.19 0.43 ± 0.11 0.287 0.001 0.393 Post 0.54 ± 0.14 0.57 ± 0.16 Post 0.62 ± 0.16 0.52 ± 0.13 PDGF-AB/BB (pg/L) Pre 63.7 ± 18.9 51.6 ± 14.3 0.007 0.412 0.203 Pre 40.0 ± 12.6 56.0 ± 23.0 0.270 0.063 0.477 Post 74.5 ± 28.6 49.3 ± 11.2 Post 56.7 ± 22.9 63.9 ± 28.4 Mean ± SD [n]. Lowlanders (n=8-11 Iron; n=5-7 DFO) and Sherpa (n=5-12 Iron; n=4-8 DFO). Linear mixed model with Bonferroni correction. Inter, Interaction; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; CRP, c-reactive protein; GDF, growth differentiating factor; PDGF, platelet derived growth factor. 79 3.4. Discussion This is the first study to examine in detail: a) the potential relationship(s) between iron status and pulmonary vascular responsiveness to progressive trekking to high altitude in lowlanders and Sherpa; and b) following ~2 weeks of acclimatization to high altitude, how iron infusion (i.e. HIF down-regulation) or iron chelation (i.e. HIF up-regulation) alters PASP and PVR (i.e. HPV) at rest, during acute changes in PIO2, and exercise in lowlanders and Sherpa highlanders. Aligning with our first hypothesis, the rise in PASP during ascent was delayed in Sherpa, compared to lowlanders. The mechanism(s) underpinning these findings are unclear but could be related to Sherpa demonstrating a more efficient capacity to mobilize stored iron. Despite these subtle differences in PASP between lowlanders and Sherpa, there were no major differences in HPV upon arrival at 5050 m. In relation to our second hypothesis, iron-infusion induced a reduction PASP during rest in both lowlanders (-18%) and Sherpa (-6%). Interestingly, the PASP during exercise, both before and following infusions, were consistently elevated in Sherpa, compared to lowlanders. Additionally, iron-infusion led to similar HIF-downregulation in lowlanders and Sherpa, as indexed via reductions in select markers of downstream gene expression (specifically sTfR and VEGF). Aside from a few subtle differences, the infusion of DFO, did not noticeably induce exaggerated HPV during rest, manipulation of PIO2 or exercise in either group. The following discussion considers the evidence, experimental limitations and the relevance underlying the findings of this study. 3.4.1. Dissociation of iron status and PASP during ascent to high altitude? Since only about 1-2 mg of daily iron requirements come from intestinal absorption, most iron is endogenously obtained via recycling of damaged/senescent erythrocytes and release of stored iron from the liver, muscle, and other tissues (Neves et al., 2019) . As shown in the current study, iron demand – as reflected in reductions in serum iron, ferritin, TrF Sat, hepcidin, body iron and increases in sTfR – is increased upon exposure to high altitude, primarily to meet the erythropoietic demand (Beguin, 2003; Cook et al., 2005; Robach et al., 2007; Gassmann & Muckenthaler, 2015). Indeed, iron is a critical cofactor for the activation of prolyl-hydroxylases (and factors inhibiting HIF; FIH), thus playing an important role in the O2 sensing pathway (Kaelin & Ratcliffe, 2008; Hirota, 2019). Therefore, diminishing iron levels would further contribute to the stabilization of HIF via prolyl hydroxylases inactivation, resulting in sustained or further elevation in the 80 transcription of erythropoietin and other HIF-dependent genes (Altamura et al., 2015). Surprisingly, in the current study, only sTfR levels – and not progressive reductions in serum iron (or other iron metrics) – were related to (or predicted) changes in PASP. Our findings are consistent with those of Altamura and coworkers who reported that, at least within the first two days of arrival at 4559 m neither the physiological increase in PASP nor the exaggerated pressure response in HAPE are related to differences in serum iron and that changes in serum iron within the physiological range seem not to affect PASP (Altamura et al., 2015). Likewise, even though serum iron was not reported in the recently study by Holdsworth et al. (2020), indices of iron demand (i.e. increased sTfR, erythropoietin and decreased hepcidin levels) in saline-receiving versus iron-receiving subjects, coincided with elevated right ventricular afterload during ascent to 5050. Together, implicating that exaggerated iron-regulatory signalling (e.g. sTfR), rather than circulating serum iron, may be the more potent stimulus for the pulmonary vasculature and contribute to the ergogenic role of iron supplementing during ascent to altitude. 3.4.2. Divergent HPV between lowlanders and Sherpa at altitude? Previous reports have shown that Sherpa/Tibetans present a lower HPV compared to other high-altitude populations and lowlanders (Groves et al., 1993; Faoro et al., 2014; Petousi et al., 2014). The mechanism to explain this finding, has been suggested to be consequent to a HIF transcriptional system that is hypo-responsive to hypoxia (Beall et al., 2010; Lorenzo et al., 2014; Petousi et al., 2014). Our findings of Sherpa demonstrating a delayed rise in PASP, compared to lowlanders, broadly supports this notion; however, only a weak tendency existed for Sherpa to have a lower PASP (when corrected to Hct) than lowlanders across ascent. It seems reasonable to speculate that the interaction(s) between a hypo-responsive HIF transcriptional system and pulmonary vascular manifestations differ when iron levels are reduced alongside progressive and sustained hypoxemia that is also accompanied with exercise. A complimentary hypothesis, however, is that Sherpa may exhibit an improved efficiency for recruiting iron during ascent. This notion was evident by their more responsive reduction in hepcidin per unit change in total iron stores (via two indices of total iron stores, or rather, the likelihood of iron deficiency – body iron and sTfR index) between 3400 and 5050 m (Figure 3.3). The physiological implications of this interesting observation are difficult to discern, especially given that Sherpa demonstrated elevated serum iron levels in Kathmandu (1400 m) compared to lowlanders. Elevated iron stores are likely 81 attributable to Sherpa having descended to Kathmandu, where the removal of the hypoxic stimulus could lead to increases in ferritin and red blood cell destruction/re-allocation of iron stores away from [Hb]. These observations have been reported in lowlanders descending from high altitude (Ryan et al., 2014) and consistent with recent evidence that hemoglobin mass levels (and plasma volume) is substantially higher in Sherpa compared to lowlanders at sea level (Stembridge et al., 2019). Ultimately, the evaluation of sea level resident Sherpa, during re-exposure and ascent to high altitude, will be pivotal in fully elucidating these mechanisms. 3.4.3. Iron and the pulmonary vascular reactivity to changing inspired PO2 and submaximal exercise The inclusion of hyperoxic and hypoxic stages in Study 2, served two main purposes: 1) to investigate the reversibility of the pulmonary vasculature (i.e. HPV; thus the degree of vascular remodelling); 2) and as an additional method to assess any vasoactive reserve within the pulmonary vasculature (Swenson, 2013). We found that after partially acclimatizing to 5050 m, HPV did not abate with hyperoxia in either group, which were also unaltered by either iron infusion or chelation. Although the initial changes in PASP would have unfolded, we acknowledge that 15 min of allotted wash-in of low and high PIO2 (67 and 416 mmHg, respectively) was potentially not long enough to determine the steady-state state changes in HPV. Interestingly, however, from the small cohort of lowlanders tested in both Studies 1 and 2 (n=10), we can provide some insight into the role of acclimatization on efficacy of iron supplementation. For example, following iron infusion, the degree of advanced acclimatization (i.e., greater elevations in SpO2 and reductions in arterial HCO3- from arrival at 5050 m to time of Study 2) was related to an attenuated rise in PASP during hypoxia (PIO2 67 mmHg; r=0.656, p=0.039 [SpO2 vs. PASP] and r=0.611, p=0.061 [HCO3- vs. PASP]). This finding remains equivocal (given PVR and mPAP were unrelated), but it indicates that ventilatory and related acid-base acclimatization could attenuated/confounded the potency of iron supplementation on the pulmonary vasculature and, therefore, could play a determining factor as to why iron supplementation did not proffer major benefits to our lowlanders at high altitude. To the best of our knowledge, this is the first study to explore the influence of acute iron manipulation on PASP during exercise at high altitude. Following both iron or DFO, Sherpa demonstrated a significant rise in PASP during recumbent 25% Wpeak – with PASP rising more 82 (by 24%) with DFO than following iron. Likewise, the rise in PASP during exercise was not attenuated by iron in Sherpa, or lowlanders. These findings differ from a recent study that reported, following 8-weeks of iron supplementation at sea level, there was a reduction in the rise in PASP in healthy older adults during normoxic mild intensity exercise (Cheng et al., 2019). Perhaps not surprising, the influence of iron status on acute exercise at high altitude is influenced by a myriad of other factors (e.g. iron deficiency, exercise-induced hypoxemia, changes in venous PO2, etc.) that could likely mask any potential influence of iron per se on the PASP response to exercise. Future studies are needed to explore these mechanism(s) and potential implications further. 3.4.4. Chemoreflex, pulmonary responsiveness and iron Our observations of a blunted hypoxic ventilatory response in the Sherpa (0.22 vs. 0.94 lmin-1SpO2-1 in lowlanders; p=0.009) is consistent with early reports (Lahiri & Milledge, 1967; Milledge & Lahiri, 1967). The implications of a lower HVR on the pulmonary vascular should equate to a stronger HPV via reductions in alveolar PO2 (i.e., one of the main stimuli for HPV). However, in view of the comparable PASP responses between lowlanders and Sherpa during hypoxia (i.e., PIO2=67 mmHg), it would suggest that factors other than alveolar PO2, are influencing the degree of HPV. Additionally, although there are multiple drivers to respiration during exercise, it is also noteworthy that PASP was generally higher during exercise in Sherpa, despite Sherpa demonstrating similar changes in VE/SpO2 sensitivity to lowlanders. Despite the removal of one statistical outlier, iron did not significantly reduce VE in hypoxia (main effect p=0.052), HVR (main effect p=0.095) or the change in VE/SpO2 sensitivity during 25% Wpeak (main effect p=0.069) in both lowlanders and Sherpa. These findings were surprising, since a recent study in mice, demonstrated that HIF-2 antagonism reduced the ventilatory response to hypoxia (Cheng et al., 2020). However, previous laboratory studies in humans at sea level, have reported no influence of DFO infusion on the chemoreflex control of breathing (Ren et al., 2000). Likewise, similar laboratory-based studies have shown SpO2 to be unaltered during acute hypoxic exposure (Talbot et al., 2014) and unaltered at high altitude, when iron was administered prior to ascent (Talbot et al., 2011). However, a more recently study utilizing a higher dosing of iron (administration pre-ascent), appeared to be improve SpO2 at high altitude (Holdsworth et al., 2020). Ultimately, future work is needed to reconcile some of these discrepant findings, but the 83 available studies to date seem to indicate a dissociation between the influence of iron on the chemoreflex and pulmonary circulation. 3.4.5. Inflammation and iron homeostasis Sojourn to high altitude is associated with an elevated pro-inflammatory status (Goetze et al., 2013; Boos et al., 2016; Song et al., 2016) that seems to be particularly marked in the presence of various high altitude illness [e.g., AMS (Boos et al., 2016) and high altitude cerebral edema (Song et al., 2016)]. Interestingly, there is substantial overlap and confounding influence between inflammatory responses and iron regulation (Ross, 2017), especially when compounded by hypoxia (Simmen et al., 2019). For example, ferritin, hepcidin and ceruloplasmin are all acute phase reactants of inflammation; however, they are also directly involved with iron regulation and integrated within the activity derived from HIF family (Hirota, 2019). Surprisingly, in the absence of altitude illness, the evaluation of inflammatory markers in lowlander and Sherpa, and their potential relationships with iron hemostasis and HPV at altitudes >5000m seems to be poorly explored. At least at 5050 m (i.e. pre-infusion baseline of Study 2), we found no clear differences in inflammatory status at rest. Moreover, during iron manipulation (Table 3.8), only IFN and GDF-15 were significantly altered (i.e. decreased and increased respectively), acting similarly with both iron and DFO, but with no differential responses between lowlanders and Sherpa. The physiological significance of these observations remains to be established. 3.4.6. Is iron supplementation useful at high altitude? If progressive reductions in iron status lead to iron-deficiency, as previously demonstrated to occur at high altitude (Robach et al., 2007; Gassmann & Muckenthaler, 2015), impairments to iron-dependent regulatory processes (e.g., erythropoiesis, angiogenesis, etc.) will inevitably occur. The mechanisms underlying these changes are complex, and influenced by multiple factors related to the high altitude environment – including diet (Okazaki et al., 2019), hypoxic inducible factor (Liu et al., 2012); erythropoietin levels (Gammella et al., 2015); erythroid proliferation signals, such as GDF-15 (Tantawy et al., 2014) and erythroferrone (Kautz et al., 2014; Buratti et al., 2015; Renassia & Peyssonnaux, 2019); hypoxia-induced inflammatory cytokines, such as interleukin (IL)-1 and IL-6 (Liu et al., 2018); together, all affecting hepcidin and iron balance (Hintze & McClung, 2011; Talbot et al., 2012; Gassmann & Muckenthaler, 2015; Lee, 2019). However, precisely identifying 84 iron-deficient status in individuals at high altitude, is not completely straightforward, given the multitude of thresholds, and their varying reliancy/dependency upon iterations of different iron metrics. For example, in the current study, using threshold TrF Sat values of either <16% (Bainton & Finch, 1964) or <20%, (Wish, 2006), 60-65% of lowlanders and only 9% of Sherpa were categorized as iron deficiency at 5050 m. Alternatively, sTfR levels of 21 mol l-1, (70% of lowlanders and 73% of Sherpa) or ferritin levels of <15 ng ml-1, (65% of lowlanders and 9% of Sherpa) or sTfR index cut off of >14 (80% of lowlanders and 45% of Sherpa) categorized individuals as iron deficient at 5050 m (Skikne et al., 2011). Ultimately, a consistent pattern of greater iron deficiency prevalence in lowlanders versus Sherpa at 5050 m, was consistently demonstrated, irrespective of index/mean of quantifying iron deficiency. Further study is required, but it seems reasonable to speculate that the enhanced protection against iron deficiency in Sherpa at high altitude is derived from evolutionary pressure and adaptation. In terms of lowlanders sojourning to high altitude, replete iron status prior to (Holdsworth et al., 2020) and during ascent (Talbot et al., 2011) and shortly upon arrival (Smith et al., 2009) have shown to proffer benefits for the trekker/mountaineer. For example, in the recent study by Holdworth (2020), iron supplementation prior to ascent to 5050 m improved SpO2 and enhanced SV via reduced pulmonary vascular resistance and improved right heart function. Ultimately, ensuring iron surplus (and not a deficit) would seem advantageous upon further ascent to extreme altitude. 3.4.7. Experimental considerations It is not possible to directly quantify HIF in the human lung, and not all peripheral concentrations of HIF-regulated gene products (e.g., erythropoietin, VEGF), necessarily reflect HIF activity in the pulmonary vasculature. It is noteworthy, however, that at sea level iron chelation and iron supplementation emulated the pulmonary vascular manifestations of up- and down-regulation of HIF that are seen in Chuvash polycythemia (Smith et al., 2006) and HIF+/− mice (Brusselmans et al., 2003), respectively. It seems plausible that such changes would also occur at high altitude. Likewise, our inflammatory markers reflect circulating concentrations rather than cellular protein expression and the mechanism(s) mediating these changes remain to be explored. A larger dose could have been administered and may have augmented some of our findings. However, our 85 current doses of iron-sucrose and DFO significantly increased and decreased serum iron levels in the blood (main effect p<0.001, for both), and aligned to other studies which showed a PASP-reducing effect (Smith et al., 2008a; Smith et al., 2009; Talbot et al., 2011; Talbot et al., 2014). Additionally, we fully acknowledge the temporal responsiveness of the pulmonary vasculature to hypoxia (Dorrington et al., 1997) and appreciate that our shortened hypoxic and hyperoxic conditions may not have been sufficient for complete stabilization of the pulmonary vasculature. Nevertheless, although this was a field study, and diet was permitted ad libitum, it forms one of the most comprehensive and mechanistic studies to date to examine the influence of iron hemostasis, acclimatization and adaption on the pulmonary vascular responses to high altitude. 3.5. Conclusion The current study aimed to establish whether the influence of iron on the pulmonary vasculature is differentially regulated between in lowlanders compared to high altitude adapted Sherpa. Our findings provide new evidence on the complex interplay of iron regulation on pulmonary vascular regulation during acclimatization and adaptation to high altitude. Future studies should evaluate the longer-term implications of iron manipulation in these groups, especially during acclimatization and/or further ascent to extreme altitude, to allow for any differential propagation – or lack thereof – of downstream gene expression to physiologically manifest in the pulmonary vasculature. 86 Chapter 4. GLOBAL REACH 2018: Iron infusion at high altitude reduces hypoxic pulmonary vasoconstriction equally in both lowlanders and healthy Andean highlanders 4.1. Background With ascent to high altitude, there is a progressive reduction in the PO2 and a progressive decline in iron bioavailability (Robach et al., 2007; Gassmann & Muckenthaler, 2015; Willie et al., 2021). As such, there is a robust inverse relationship between iron status and the pulmonary vasculature in hypoxia (reviewed in (Frise & Robbins, 2015)). Part of this relationship stems from the integral role of iron on propyl hydroxylase activity (Knowles et al., 2003; Kaelin & Ratcliffe, 2008) and contribution to the stabilization of HIF. The HIF pathway acts as a cellular oxygen sensor (Semenza et al., 1998; Prabhakar & Semenza, 2015), and controls a broad range of transcriptional responses to hypoxic exposure, including a variety of down-stream gene products that can influence HPV (e.g. endothelin, VEGF and transient receptor potential channels) (Sylvester et al., 2012; Frise & Robbins, 2015). Experimentally, iron manipulation has been used as: 1) a tool to attenuate pulmonary vascular responsiveness of lowlanders exposed to acute normobaric hypoxia (Bart et al., 2016), and upon ascent and stay at high altitude (Smith et al., 2008a; Smith et al., 2009; Talbot et al., 2011; Holdsworth et al., 2020; Willie et al., 2021); and 2) a method to interrogate HIF-dependent differences associated with clinical pulmonary hypertension (Balanos et al., 2002) and adaptation to high altitude – such as Sherpa of Tibetan descent (Willie et al., 2021), and Andeans suffering with CMS (Smith et al., 2009). In both lowlanders and Sherpa, iron infusion attenuates HPV at high altitude; however, this does not occur in Andeans with CMS. Andeans with CMS characteristically display marked pulmonary remodeling and hypertension (reviewed in (Penaloza & Arias-Stella, 2007)). Therefore, in the study by Smith and colleagues (Smith et al., 2009), it is perhaps not surprising that 400 mg of iron(III)-sucrose [200 mgday-1 over 2 days preceded by 4 days of venesection (at 500 mlday-1)], was ineffective at reducing PASP, even after 5 days (Smith et al., 2009). While Andeans have had a putatively shorter high-altitude lineage, especially compared to Sherpa/Tibetans (Aldenderfer, 2003), healthy Andeans still demonstrate numerous attributes that enhance their hypoxic tolerance compared to lowlanders including: elevated birth weights, increased exhaled nitric oxide concentrations, larger lungs, improved aerobic capacity and genotypic adaptations (Lenfant & Sullivan, 1971; Beall et al., 2001; Moore et al., 2004; Frisancho, 87 2013; Storz, 2021). With the recently proposed notion that high altitude adaptation may proffer an enhanced state of iron metabolism (Muckenthaler et al., 2020; Willie et al., 2021), exploring whether healthy Andeans are receptive to iron would provide insight into the pathological divergence of CMS on the pulmonary vasculature. Therefore, the aim of this study was to examine the role of iron bioavailability on the pulmonary vascular regulation to hypoxia in high-altitude adapted Andeans, with and without, acute reductions in PIO2. To address this aim, partially acclimatized lowlanders and healthy Andeans received either an infusion of iron or saline at 4300 m, in a double-blinded and block-randomized design. Our primary hypothesis was that healthy Andeans and lowlanders would demonstrate comparable pulmonary vascular responses to hypoxia, and both groups would be sensitive to iron infusion. 4.2. Methods 4.2.1. Ethical Approval All experimental procedures were approved by the University of British Columbia Research Ethics Board (H17-02687 and H18-01404) and the Universidad Peruana Cayetano Heredia Comité de Ética (no. 101686), and conformed to the Declaration of Helsinki, except for registration in a database. All participants received both written and oral information about the study and provided written informed consent. Forms were translated into Spanish, and Andeans participants spoke with a Peruvian research assistant and provided written informed consent in Spanish before participating. 4.2.2. Participants A cohort of 24 healthy Andean highlanders and 22 age-matched lowlanders completed the study (Table 4.1). All were normotensive, and did not report a history of cardiovascular, cerebrovascular, or respiratory diseases during completion of a medical history questionnaire. All Andeans were born at high altitude (n=2 at 1880-2900 m, n=22 at 4300m; as were their parents) and resided at 4300 m, were non-smokers, did not exhibit excessive erythrocytosis (Hb 19 g/dl for females and 21 g/dl for males) and had a Qinghai CMS questionnaire score of 0.5±0.8 – and thus were not considered to suffer from CMS (León-Velarde et al., 2005). Of the few lowlander participants who opted for high altitude prophylaxis (i.e., acetazolamide) upon immediate arrival to 4300 m, all 88 lowlander participants had discontinued use for at least a week, prior to being tested in the current study. This study was part of the Global Research Expedition on Altitude Related Chronic Health (REACH) to Cerro de Pasco, Peru [4300m; barometric pressure of 457 mmHg; PIO2 of ~96 mmHg] (Tymko et al., 2020a). Upon arrival in Lima and finalizing logistics, lowlander participants (of which were members of the expedition and sea-level residents of European descent) were driven up to Cerro de Pasco in 8 hours. Lowlanders were tested after 84 days at 4300 m – lowlander infusion groups were equally weighted, for number of days at 4300 m. 4.2.3. Experimental Design Participants arrived at the laboratory having only eaten a small meal ~2 hours prior, and abstained from caffeine and exercise for >6 and >12 hours, respectively. Following 10 min of supine rest an indwelling venous catheter (21G needle, BD Vacutainer eclipse) was inserted into the median antecubital vein for repeated blood sampling and the infusion. Echocardiography was performed at baseline, and at 2 and 4 hours after receiving an infusion of either iron or saline. Between time-points, participants were allowed to sit up, but all participants resumed supine rest for at least 10 minutes before any measurement. At baseline and at 4 hours, echocardiography was also performed during a hypoxic stage (15 min of FIO2=0.16; PIO2=73 mmHg; simulating ~1700 m gain in elevation). At 2.5 minutes into the hypoxic stage, automated blood pressure was collected in duplicate using brachial oscillometry. After 10 min of reduced PIO2, echocardiographic imaging was repeated, and at the end of the hypoxic stage, SPO2 was recorded. 4.2.3.1. Infusion In a double-blinded, block-randomized design, Andean and lowlander participants received an i.v. infusion of either iron (iron (III)-hydroxide sucrose; 200 mg in 250 ml 0.9% NaCl saline) or saline (250 ml of 0.9% NaCl saline) over 30 min. Both participants and investigators were blinded during collection (via opaque bags to cover the infusion and lines), and maintained throughout analysis. Due to the >40-day longevity of iron infusion (Bart et al., 2016) and the logistic constraints of an expedition (Tymko et al., 2020a), a cross-over design was not feasible. Therefore, the purpose of block-randomizing participants was two-fold: 1) enabled us to optimize logistics and coordination 89 with other ongoing studies, and 2) ensured that lowlanders allocated to iron or saline conditions, were appropriately weighted, in terms of the number of days at 4300 m, to limit any potentially confounding influence of hypobaric exposure on iron stores [lowlanders receiving saline and iron were tested after 95 and 63 days, respectively (unpaired t-test P=0.158)]. 4.2.4. Experimental Measures 4.2.4.1. Echocardiography SV was estimated using the following Equation 3.1: SV = (π[aortic diameter 2⁄ ]2)LVOT VTI where the LVOT VTI was measured at the point of aortic leaflet insertion during systole using a pulsed wave Doppler signal. CO was calculated as the product of stroke volume and heart rate (via electrocardiography). CI is the quotient of CO and body surface area, calculated as (Mosteller, 1987): Equation 4.1. Mosteller body surface area Body surface area=√(height×weight) 3600⁄ PASP was measured by Doppler echocardiography based upon the measurement of the maximum velocity of the TR velocity (Bertini et al., 2009). The ∆Pmax to the right atrium was calculated according to the simplified Bernoulli equation (4V2), where V is the peak systolic velocity of the tricuspid regurgitate. PASP (Equation 3.2) was then determined by adding the RAP, which was estimated by evaluating inferior vena cava diameter and collapsibility index – in line with the standard American Society of Echocardiography guidelines (Lang et al., 2015). PASP = (4V2) + RAP PASP was normalized to a hematocrit (Hct) of 45% in all individuals using the following Equation 3.5 (Vanderpool & Naeije, 2018): PASP[Hct] = PASP exp[2(φ − 0.45)]⁄ Where  represents the measured hematocrit level. Total pulmonary resistance (TPR) was calculated as an index of PASP against CO (i.e., PASP/CO), as previously described (Foster et al., 2014). 90 4.2.4.2. Biochemical analysis Venous blood samples were separated by microcentrifugation, with serum samples frozen in liquid nitrogen at -196 C for subsequent analysis. Serum iron, ferritin, transferrin, and EPO were analysed according to clinical laboratory standards (Medlab clinical laboratories, Lima, Peru). TIBC was calculated as (2016): Equation 4.2. Total iron binding capacity (TIBC). TIBC = transferrin ∙ 1.389 TrF Sat was calculated as (Equation 3.6): Transferrin saturation=(serum iron TIBC⁄ )∙100 Hb and Hct were obtained from whole venous blood sample using microcentrifugation, and analyzed immediately. The Hct at 2 hours, was estimated as the median value between the baseline and 4-hour timepoints. 4.2.5. Statistical analysis Data were analyzed using a linear mixed effects model with a compound symmetry repeated measure co-variance structure (SPSS v24, IBM Statistics). The fixed factors for the model were ancestry and PIO2 (i.e., 96 to 73 mmHg) or time (i.e. pre to post infusion) – with the latter, PIO2 or time, being a repeated factor with a compound symmetry repeated covariance structure. Subjects were included as a random effect. When a significant interaction effect (e.g., altitudeancestry) was detected, Bonferroni adjusted post-hoc tests were utilized to test pairwise comparisons. Comparison of hematological measures at baseline, of pooled lowlanders and Andeans, were assessed with independent samples t-test. The absolute difference between normoxia and hypoxia (Δ), is also presented. A linear regression was used to evaluate the prevalence of decreasing serum iron, across days at altitude. All results are reported as mean ± SD and significance was set at P<0.05. 4.3. Results 4.3.1. Comparison of lowlanders and healthy Andeans at baseline 4.3.1.1. Iron metrics At baseline, when all participants were pooled, ferritin was 82% higher in Andeans at baseline, compared to lowlanders (97.354.6 ng/ml vs. 53.537.1 ng/ml, respectively; P=0.003; Table 4.1). 91 There were no other differences in markers of iron status (e.g., serum iron, transferrin, TIBC, TrF Sat, or EPO) at baseline between Andeans and lowlanders. 4.3.1.2. Hypoxic pulmonary vasoconstriction At baseline (prior to infusion), there was no difference in pulmonary vascular reactivity to hypoxia (PIO2=73 mmHg) between lowlanders and Andeans (for both uncorrected and corrected to 45% Hct; 92 Table 4.2). LV SV slightly decreased during hypoxia in Andeans (7517 ml at baseline to 7218 ml during hypoxia; P=0.047); however, both lowlanders and Andeans demonstrated comparable increases in CO, CI, TPR, and MAP during hypoxia. 4.3.2. Effects of intravenous iron 4.3.2.1. Iron metrics The iron infusion increased serum iron (by 382193% and 288100% in lowlanders and Andeans, respectively; main effect P<0.001), increased serum ferritin (by 1816% and 1410% in lowlanders and Andeans, respectively; main effect P<0.001) and decreased transferrin (by 47% and 56% in lowlanders and Andeans, respectively; main effect P=0.010; Figure 4.1). Consistent with previous reports in CMS (Smith et al., 2009), EPO remained unaltered following iron or placebo in both groups. There were no significant changes in iron metrics during the saline condition. 93 Figure 4.1. Impact of saline (grey bars) or iron infusion (red bars) on iron metrics in lowlanders and healthy Andeans. *P<0.05 main effect of time. 94 4.3.2.2. Time course over 4 hours at high altitude In lowlanders and Andeans receiving saline, PASP did not significantly change across the 4 hours (main effect of time P=0.262; Table 4.3). Following iron infusion, there was a decrease in PASP by 7% and 4% at 2 hours and 11% and 7% at 4 hours in lowlanders and Andeans, respectively (main effect of time P<0.001; Figure 4.2 and Table 4.4). Following iron, both lowlanders and Andeans demonstrated a tendency for an increased MAP (main effect of time P=0.052; Table 4.4). Even though there were no change in TPR across the 4 hours in lowlanders and Andeans with iron (Table 4.4), TPR was higher in Andeans compared to lowlanders at 4 hours (P=0.034) – this difference is likely driven by the tendency for TPR to decrease across the 4 hours in lowlanders following iron (P=0.051). Figure 4.2. Impact of saline and iron infusion on the PASP in lowlanders and healthy Andeans. Horizonal bar with asterisk denotes a main effect significance of time (*P<0.05). 4.3.2.3. Hypoxic pulmonary vasoconstriction The rise in PASP during hypoxia was unaltered following the placebo condition in both groups (main effect P=0.326; Table 4.5). During acute hypoxia (i.e., PIO2=73 mmHg), iron infusion attenuated PASP by 4 mmHg, in both Andeans and lowlanders (main effect P=0.01; Table 4.6 and Figure 4.3), which coincided with a decrease in TPR (main effect P=0.001) and an increase in LV SV (main effect P=0.031). While the expected decrease in SpO2 was apparent in the exaggerated hypoxia trial, this reduction was not altered by iron infusion in lowlanders and Andeans; however, 95 the rise in PASP for a given drop in SpO2 was attenuated by iron infusion (main effect P=0.011; Table 4.6). Figure 4.3. Pulmonary vascular reactivity to hypoxia in lowlanders and Andeans receiving either saline or iron infusion. * main effect P<0.05. 96 Table 4.1. Baseline summary of pooled lowlanders and pooled Andeans. Variable Lowlander Andean p-value Sex (males/females) 16 / 6 21 / 3 Age (years) 29 ± 7 29 ± 11 0.898 Height (cm) 175 ± 8 162 ± 5 <0.001 Weight (kg) 71 ± 10 63 ± 7 <0.001 BMI (kg/m2) 23.3 ± 2.3 23.8 ± 2.8 0.530 Hb (g/dl) 16.2 ± 1.4 [17] 16.9 ± 1.9 [14] 0.235 Hct (%) 48.4 ± 3.9. [20] 53.2 ± 4.1 [18] 0.001 EPO (mIU/ml) 17.3 ± 9.5 14.1 ± 8.9 0.257 Serum Ferritin (ng/ml) 53.5 ± 37.1 97.3 ± 54.6 [23] 0.003 Serum Iron (ug/dL) 81.8 ± 40.9 94.2 ± 29.7 [23] 0.250 Serum Transferrin (mg/dl) 275.4 ± 24.4 268 ± 28.3 [23] 0.353 TIBC (ug/dl) 382.5 ± 33.9 372.2 ± 39.3 [23] 0.353 TrF Sat (%) 21.9 ± 11.5 25.3 ± 7.6 [23] 0.240 SpO2 (%) 89 ± 3 88 ± 3 0.528 PASP (mmHg) 31.0 ± 6.4 [21] 29.5 ± 5.9 [23] 0.403 PASP[Hct] (mmHg) 29.5 ± 7.3 [19] 25.4 ± 5.8 [17] 0.072 Mean ± SD in 22 Lowlanders and 24 Andeans (unless otherwise noted [n]). Independent samples t-test. 97 Table 4.2. Cardiopulmonary measures in pooled lowlanders and Andeans (prior to infusion) during room air breathing and reduced PIO2. Variable Group Room air Hypoxia Ancestry PIO2 Inter PASP (mmHg) Lowlander 31.0 ± 6.4 [21] 40.2 ± 8.4 [21] 0.866 <0.001 0.343 Andean 29.5 ± 5.9 [23] 40.9 ± 12.9 [23] PASP[hct] (mmHg) Lowlander 29.5 ± 7.3 [19] 38.4 ± 10.7 [19] 0.242 <0.001 0.586 Andean 25.4 ± 5.8 [17] 35.7 ± 12.3 [17] TPR (mmHg/l) Lowlander 6.4 ± 1.5 [21] 7.0 ± 1.8 [21] 0.401 <0.001 0.120 Andean 6.5 ± 1.6 [23] 7.7 ± 2.3 [23] LV SV (ml) Lowlander 83 ± 18 86 ± 19 0.041 0.931 0.009 Andean 75 ± 17 72 ± 18*† HR (beats/min) Lowlander 62 ± 12 71 ± 15 0.320 <0.001 0.061 Andean 63 ± 9 77 ± 12 CO (l/min) Lowlander 5.0 ± 1.0 6.0 ± 1.3 0.173 <0.001 0.167 Andean 4.7 ± 0.9 5.4 ± 1.2 CI (ml/min/m2) Lowlander 2.7 ± 0.5 3.2 ± 0.7 0.751 <0.001 0.349 Andean 2.8 ± 0.5 3.2 ± 0.7 MAP (mmHg) Lowlander 90 ± 8 94 ± 10 <0.001 0.001 0.813 Andean 78 ± 8 82 ± 10 SpO2 (%) Lowlander 89 ± 3 72 ± 7 0.916 <0.001 0.506 Andean 88 ± 3 73 ± 10 [23] Mean ± SD in 22 Lowlanders and 24 Andeans (unless otherwise noted [n]). Linear mixed model. * Significant difference from baseline, P<0.05; † Significant difference between Andeans and Lowlanders, P<0.05. 98 Table 4.3. Resting cardiopulmonary measures at baseline and following the infusion of saline at 4300 m. Variable Group Baseline 2 hours 4 hours Ancestry Time Inter PASP (mmHg) Lowlander 32.1 ± 7.5 31.5 ± 6.2 31.1 ± 5.8 0.366 0.262 0.751 Andean 29.7 ± 6.4 [11] 29.6 ± 7.8 [11] 27.8 ± 8.1 [11] PASP[hct] (mmHg) Lowlander 30.5 ± 9.0 29.7 ± 7.7 29.3 ± 8.4 0.094 0.174 0.549 Andean 24.8 ± 6.3 [11] 25.5 ± 6.1 [9] 23.1 ± 7 [9] TPR (mmHg/l) Lowlander 6.1 ± 1.4 6 ± 1.2 5.8 ± 1.1 0.739 0.307 0.594 Andean 6.1 ± 1.5 [11] 6.5 ± 1.7 [11] 5.5 ± 2.4 HR (beats/min) Lowlander 60 ± 10 59 ± 13 57 ± 13 0.592 0.067 0.648 Andean 63 ± 10 59 ± 9 60 ± 9 LV SV (ml) Lowlander 90 ± 17 92 ± 11 98 ± 22 0.021 0.129 0.099 Andean 80 ± 16 78 ± 9 79 ± 11 CO (l/min) Lowlander 5.3 ± 1.0 5.3 ± 1 5.4 ± 1.3 0.079 0.430 0.278 Andean 5.0 ± 0.8 4.6 ± 0.7 4.7 ± 0.5 CI (l/min/m2) Lowlander 2.8 ± 0.6 2.8 ± 0.6 2.9 ± 0.7 0.855 0.398 0.227 Andean 2.9 ± 0.6 2.7 ± 0.5 2.7 ± 0.4 MAP (mmHg) Lowlander 92 ± 8 90 ± 7 91 ± 10 0.027 0.914 0.305 Andean 81 ± 8 84 ± 11 83 ± 14 SpO2 (%) Lowlander 89 ± 3 89 ± 3 88 ± 4 0.607 0.557 0.806 Andean 88 ± 2 88 ± 3 88 ± 2 Mean ± SD in 11 lowlanders and 12 Andeans (unless otherwise noted [n]). PIO2=96mmHg. Significant p-values are denoted in bold. Linear mixed model. 99 Table 4.4. Resting cardiopulmonary measures at baseline and following the infusion of iron at 4300 m. Variable Group Baseline 2 hours 4 hours Ancestry Time Inter PASP (mmHg) Lowlander 29.8 ± 5.1 [10] 27.7 ± 5.6 [10] 26.6 ± 6.3 [10] 0.967 <0.001 0.587 Andean 29.3 ± 5.6 28.1 ± 7.2 27.2 ± 6.9 PASP[hct] (mmHg) Lowlander 28.2 ± 4.3 [8] 26.5 ± 4.8 [8] 25.1 ± 5.5 [10] 0.183 0.001 0.485 Andean 26.5 ± 5.2 [6] 26.6 ± 6.9 [4] 21.3 ± 4.3 [8] TPR (mmHg/l) Lowlander 6.7 ± 1.6 [10] 6.4 ± 1.8 [10] 5.3 ± 2.6† 0.190 0.867 0.008 Andean 6.8 ± 1.7 7 ± 2 7.5 ± 2.9 HR (beats/min) Lowlander 63 ± 14 63 ± 20 64 ± 16 0.483 0.098 0.125 Andean 63 ± 7 58 ± 8 58 ± 8 LV SV (ml) Lowlander 76 ± 17 75 ± 15 77 ± 19 0.439 0.764 0.181 Andean 71 ± 18 73 ± 18 68 ± 19 CO (l/min) Lowlander 4.7 ± 0.9 4.5 ± 1 4.7 ± 0.9 0.262 0.277 0.135 Andean 4.4 ± 0.9 4.2 ± 1.2 3.9 ± 1.3 CI (l/min/m2) Lowlander 2.6 ± 0.4 2.5 ± 0.5 2.6 ± 0.5 0.854 0.256 0.096 Andean 2.7 ± 0.5 2.5 ± 0.6 2.4 ± 0.7 MAP (mmHg) Lowlander 89 ± 8 89 ± 10 91 ± 7 0.001 0.052 0.647 Andean 75 ± 6 76 ± 8 79 ± 11 SpO2 (%) Lowlander 88 ± 3 88 ± 2 88 ± 3 [10] 0.444 0.160 0.315 Andean 88 ± 4 87 ± 3 87 ± 3 Mean ± SD in 11 lowlanders and 12 Andeans (unless otherwise noted [n]). PIO2=96mmHg. Significant p-values are denoted in bold. Linear mixed model. 100 Table 4.5. Cardiopulmonary reactivities to hypoxia, prior-to and following the infusion of saline. Variable Group Pre-saline Post-saline Ancestry Time Inter ΔSpO2 (%) Lowlander -14.9 ± 6.9 -15.5 ± 6.7 0.949 0.899 0.610 Andean -15.8 ± 9.1 -14.9 ± 8 ΔPASP (mmHg) Lowlander 9 ± 5.3 10.6 ± 5.6 0.995 0.326 0.989 Andean 9.1 ± 5.7 10.6 ± 8.5 ΔPASP/ΔSpO2 (mmHg/%) Lowlander -0.6 ± 0.4 -0.9 ± 0.9 0.565 0.070 0.691 Andean -0.5 ± 0.7 [10] -0.9 ± 0.5 ΔPASP[hct] (mmHg) Lowlander 8.7 ± 5.8 10.1 ± 5.9 0.654 0.258 0.854 Andean 7.5 ± 4.9 9.8 ± 6.4 [9] ΔPASP[hct]/ΔSpO2 (mmHg/%) Lowlander -0.6 ± 0.4 -0.9 ± 0.8 0.427 0.045 0.477 Andean -0.3 ± 0.6 [9] -0.8 ± 0.3 [9] ΔTPR (mmHg/l) Lowlander 0.4 ± 0.8 1.0 ± 1.9 0.420 0.137 0.922 Andean 0.9 ± 1.3 1.4 ± 2 ΔHR (beats/min) Lowlander 10.0 ± 9.5 9.6 ± 7.2 0.091 0.702 0.849 Andean 15.0 ± 4.8 13.9 ± 8.6 ΔSV (ml) Lowlander 3.6 ± 8.1 1.5 ± 7.5 0.004 0.433 0.651 Andean -6.2 ± 8.8 -6.8 ± 6.9 ΔCO (ml/min) Lowlander 1.1 ± 0.6 1.1 ± 0.9 0.055 0.665 0.591 Andean 0.7 ± 0.6 0.6 ± 0.4 ΔCI (ml/min/m2) Lowlander 0.6 ± 0.4 0.6 ± 0.5 0.123 0.624 0.657 Andean 0.4 ± 0.3 0.3 ± 0.3 ΔMAP (mmHg) Lowlander 5.1 ± 6.6 5.9 ± 5.5 0.495 0.760 0.888 Andean 3.9 ± 8.1 4.2 ± 5.1 Mean ± SD. N=10-11 and n=11-12 for lowlanders and Andeans respectively, unless otherwise noted [n]. Δ, the absolute change between normoxia and hypoxia. 101 Table 4.6. Cardiopulmonary reactivities to hypoxia, prior-to and following the infusion of iron. Variable Group Pre-iron Post-iron Ancestry Time Inter. ΔSpO2 (%) Lowlander -18.4 ± 3.3 -18 ± 4.2 0.238 0.966 0.749 Andean -14.8 ± 7.7 -15.2 ± 9.8 ΔPASP (mmHg) Lowlander 9.2 ± 3.9 6.8 ± 3.1 0.451 0.010 0.219 Andean 13.6 ± 12.9 7.4 ± 8.4 ΔPASP/ΔSpO2 (mmHg/%) Lowlander -0.5 ± 0.2 -0.4 ± 0.2 [9] 0.979 0.011 0.051 Andean -0.9 ± 0.7 -0.2 ± 0.9 ΔPASP[Hct] (mmHg) Lowlander 9 ± 4.4 [8] 6.6 ± 3.1 [10] 0.556 0.075 0.300 Andean 15.5 ± 16.6 [5] 7.7 ± 10 [6] ΔPASP[Hct]/ΔSpO2 (mmHg/%) Lowlander -0.51 ± 0.25 [8] -0.4 ± 0.23 [9] 0.509 0.057 0.176 Andean -0.85 ± 0.7 [5] -0.001 ± 1.04 [6] ΔTPR (mmHg/l) Lowlander 0.7 ± 1.3 0.02 ± 1.4 0.620 0.001 0.160 Andean 1.5 ± 1.9 -0.1 ± 2.1 ΔHR (beats/min) Lowlander 9.1 ± 5.9 6.9 ± 10.9 0.256 0.303 0.671 Andean 11.9 ± 6.8 11 ± 7.2 ΔSV (ml) Lowlander 2.8 ± 5.6 7.3 ± 8.3 0.234 0.031 0.729 Andean -0.6 ± 9.3 2.7 ± 11 ΔCO (l/min) Lowlander 0.9 ± 0.5 1.1 ± 0.6 0.505 0.264 0.929 Andean 0.8 ± 0.7 0.9 ± 0.7 ΔCI (ml/min/m2) Lowlander 0.5 ± 0.3 0.6 ± 0.3 0.809 0.244 0.997 Andean 0.5 ± 0.4 0.6 ± 0.4 ΔMAP (mmHg) Lowlander 2.6 ± 9.3 1.1 ± 5.9 0.718 0.145 0.468 Andean 4.9 ± 6.9 0.5 ± 6.1 Mean ± SD. N=10-11 and n=11-12 for lowlanders and Andeans respectively, unless otherwise noted [n]. Δ, the absolute change between normoxia and hypoxia. 102 4.4. Discussion The pulmonary vasculature of adapted Andeans remained responsive to iron infusion – a feature consistent among lowlanders and high-altitude Sherpa (Willie et al., 2021). Because the pulmonary vasculature of Andeans suffering with CMS does not exhibit this responsiveness to iron (Smith et al., 2009), this may provide valuable insight into the pathological divergence of CMS. The following discussion outlines the implications of these findings, including the physiological significance of elevated ferritin levels in the Andeans. 4.4.1. Pulmonary vascular responsiveness to hypoxia and iron Aligning with our hypothesis, this is the first study to confirm that the pulmonary vasculature of adapted and otherwise healthy Andeans is responsive to iron infusion in hypoxia – a feature akin to the well adapted Sherpa and lowlanders (Willie et al., 2021). Such congruent responses across different populations (i.e. acclimatizing lowlanders and high altitude adapted Andeans and Sherpa), highlight a conserved phenotype reflected in an otherwise healthy pulmonary vasculature. Healthy Andeans have demonstrated either slightly higher PASP (at rest and during exercise) (Groepenhoff et al., 2012), or similar PASP (at rest) (Schwab et al., 2008; de Bisschop et al., 2010), compared to partially acclimatized lowlanders at the same altitude. Therefore, it is perhaps not surprising in the current study that PASP at rest and during further hypoxia was again similar between partially acclimatized lowlanders and healthy Andeans. The unremarkable influence of a comparable iron infusion on PASP in Andeans with CMS (Smith et al., 2009) is intriguing. Certainly, CMS has long been established to coincide with pulmonary vascular remodeling and persistence of pulmonary hypertension (during rest and exercise) (Rotta et al., 1956; Arias-Stella & Saldana, 1963; Peñaloza et al., 1963; Lockhart et al., 1976; Grover, 1990; Groepenhoff et al., 2012). However, CMS has also been linked to the dysfunction of a myriad of HIF-related factors [including sentrin-specific protease 1 (Villafuerte, 2015), prevailing circulatory status of vasoactive peptides (i.e. vascular endothelial growth factor and endothelin-1 (Ge et al., 2011)), methylation of propyl hydroxylase activity (Julian, 2017), and/or mutations to the von Hippel-Lindau protein (Ang et al., 2002)] – these factors could blunt or counteract the efficacy of iron infusion on pulmonary vascular tone. Ultimately, the notable divergence of healthy 103 adapted Andeans from the CMS pulmonary phenotype, hints at some facet of HIF-dysregulation, that may share mechanistic basis with other clinical pathologies. 4.4.2. Iron metabolism – higher ferritin levels in Andeans Ascent to high altitude is associated with a progressive decline in iron bioavailability (Robach et al., 2007; Gassmann & Muckenthaler, 2015; Willie et al., 2021), which can exaggerate hypoxia-induced HIF stabilization and contribute to pulmonary hypertension (Frise et al., 2016). Therefore, in the context of iron regulation, the observed higher ferritin (a stimulus for iron storage) in Andeans could imply a greater state of iron sufficiency and a lower iron/erythropoietic demand, compared to lowlanders. While this feature might have resonance in the interesting dialogue of enhanced iron metabolism in adapted highlander (Muckenthaler et al., 2020; Willie et al., 2021), using ferritin as an index of iron sufficiency is limited, as elevated ferritin levels (e.g., >200-300 ng/ml) are indicative of a myriad of medical concerns [e.g., liver disease, hepatitis, infection, state of inflammation, and hemochromatosis] (Wallace & Subramaniam, 2009; Wessling-Resnick, 2010). As such, Sherpa do not present with elevated ferritin levels. For example, in the study by Willie and colleagues, serum ferritin of Sherpa was 46.737.8 ng/ml (Willie et al., 2021) – values which more closely resemble our lowlander data at 4300 m, rather than healthy Andeans (Table 4.1). Coincidingly, similar to lowlanders, Sherpa also did not exhibit any obvious inflammatory profile (Willie et al., 2021). Conversely, previous reports of Andeans with CMS (and/or excessive erythrocytosis), show ferritin levels of 169±90 ng/ml (n=11) (Smith et al., 2009), 184±28 ng/ml (n=42) (Villafuerte et al., 2014) and 156±132 ng/ml (n=41) (De Ferrari et al., 2014). However, in the latter two studies, ferritin in healthy Andeans were comparable to Andeans with CMS (De Ferrari et al., 2014; Villafuerte et al., 2014). While it is unclear why the ferritin in our “healthy” Andeans appear substantially lower than the ferritin values in other “healthy” Andeans (De Ferrari et al., 2014; Villafuerte et al., 2014), one possibility may be related to CMS score severity [0.5±0.8 in this study; 2.7±0.38 in (Villafuerte et al., 2014); no score reported in (De Ferrari et al., 2014)]. Future studies are needed to consider how and why CMS severity might influence ferritin levels, or why ferritin level can be so variable between otherwise healthy individuals. The factors potentially mediating these elevations in ferritin, in both healthy and CMS Andeans compared to lowlanders, may have some basis when considering the city of Cerro de Pasco itself. 104 First, it has been demonstrated that more than half of the soil sample sites evaluated in Cerro de Pasco, had lead (Pb) levels exceeding 1200mg/kg (Van Geen et al., 2012), which is ~10-fold higher the health recommendations in Canada [140 mg/kg(Health Canada, 2013)]. Recently, and not surprisingly, whole blood Pb concentrations in residents of Cerro de Pasco (with Hb 10-25 g/dl) were >2-fold greater than Pb concentrations in sea-level residents of Lima [4.751.53 ug/dl, range: 1.5-9.8 ug/dl versus 2.030.62 ug/dl, range: 1.0-4.6 ug/dl, respectively (Childebayeva et al., 2021)]. While Pb toxicity may not be prevalent in our cohort [due to the lack of iron deficiency and anemia (reviewed in: (Kwong et al., 2004))], consequence(s) of Pb exposure on upstream inflammatory factors (Hosseinirad et al., 2021) and ferroportin activity (Harada et al., 2011), have been demonstrated in animal and cell-preparation models. Whether Pb exposure could extend beyond iron deficiency/anemia and potentially contribute to the elevated ferritin levels in Andeans, compared lowlanders, remains to be determined. Second, the city of Cerro de Pasco starkly contrasts those of small towns/villages in the Himalayan highlands, in terms of lifestyle (e.g. diet, physical activity) and level of industrialization (e.g. drive-in and mining versus hike-in and agriculture/tourism, respectively). As such, comparisons between populations are certainly multifaceted and future studies are needed to consider the potential detrimental influence of even mildly elevated Pb levels on inflammation (Brewster et al., 2021; Hosseinirad et al., 2021) and explore the mechanistic links to contributing to the elevated ferritin levels in Andeans. Importantly, our findings that iron attenuated the rise in PASP during hypoxia, with a coinciding increase in left ventricular SV and decrease in TPR (Table 4.6), is broadly consistent with the recent study by Holdsworth and colleagues (Holdsworth et al., 2020). Here, the authors reported that increasing iron bioavailability (via infusion of 1g of ferric carboxymaltose) in lowlanders, prior to ascent to high altitude, translated into enhanced right ventricular SV and lower pulmonary vascular resistance at 5050 m. 4.4.3. Considerations of iron status during prolonged stay at high altitude In our lowlander cohort, a pattern of decreasing serum iron levels appeared to develop over the 17 days at 4300 m (r2=0.204, P=0.035; Figure 4.4) – a reproducible feature of high-altitude sojourn (Robach et al., 2007; Gassmann & Muckenthaler, 2015; Willie et al., 2021). Even though our cohort of Andeans did not travel and resided at 4300 m, our groups were also appropriately 105 weighted for serum iron (unpaired t-test P=0.522; Figure 4.4). Our study appropriately weighted lowlanders to saline and iron conditions, so the confounding influence of time at altitude would be less. However, the progressive reduction in iron with stay at altitude, highlights the need to be cognisant of such changes in iron status, if the outcome variable of interest hinges or relies upon downstream iron-regulatory processes. Figure 4.4. Baseline serum iron measurements in lowlanders and Andeans. Lowlander data is presented as 1) to correspond to the number of days at 4300 m, before being tested (with linear regression; left panel) and 2) highlight the equal weighting of iron status between the saline and iron groups (with unpaired t-test; middle panel). Andean data demonstrates iron levels in the saline and iron groups at baseline were comparable (with unpaired t-test; right panel). 4.5. Interpretation Unlike Andeans with CMS (Smith et al., 2009), yet aligning with Sherpa (Willie et al., 2021), the pulmonary vasculature of adapted Andeans remained responsive to iron infusion. Therefore, these findings highlight a feature of healthy pulmonary vascular regulation, and a trait of high-altitude adaptation that remains divergent from the typical pathological characteristics of high-altitude maladaptation (i.e., CMS). 106 Chapter 5. Global REACH 2018: Iron infusion improves peripheral blood flow and microvascular function in lowlanders and healthy Andeans at high altitude 5.1. Background With ascent to high altitude, there is a progressive reduction in the PO2 and stabilization of the HIF family – the key cellular oxygen sensor (Semenza et al., 1998; Kaelin & Ratcliffe, 2008). The HIF- complex binds to hypoxia-responsive elements in gene promoters to up-regulate expression of >100 genes to coordinate increased oxygen supply to hypoxic tissue. Iron – due to its constituent role in HIF- stabilization, via PHD activity (Knowles et al., 2003) – influences the coordination of HIF-mediated responses that are designed to maintain oxygen delivery (Gassmann & Muckenthaler, 2015). Surprisingly, although reductions in iron status or deficiency commonly occur with sojourn to high altitude (Robach et al., 2007; Gassmann & Muckenthaler, 2015; Patrician et al., 2021b; Willie et al., 2021), which influences pulmonary vascular tone (Smith et al., 2008a; Smith et al., 2009; Smith et al., 2011b; Frise et al., 2016; Cheng et al., 2019; Lakhal-Littleton et al., 2019; Holdsworth et al., 2020; Patrician et al., 2021b; Willie et al., 2021), the potential link between iron status and changes in peripheral blood flow regulation, vascular function, and ultimately tissue O2 delivery at high altitude have yet to be explored. Intravenous iron is widely performed clinically and there exists a myriad of studies exploring the negative (and lack there-of) influence of iron on a variety of pre-clinical and human (patient and healthy populations) models of varying vascular dysfunction states [reviewed in: Koskenkorva-Frank et al. (2013)]. At high altitude, there exists an extensive breadth of studies exploring vascular function in sea-level and high-altitude residents [reviewed in: Tymko et al. (2019)]. However, the link between HIF- and the systemic and peripheral vasculature originates from an elegant study where skin HIF was demonstrated to regulate blood pressure in an isoform-dependent action (i.e. blood pressure was increased with HIF-1𝛼 and decreased with HIF-2𝛼 expression) in both mouse and human models (Cowburn et al., 2013). Therefore, utilizing an integrative approach of a HIF-up regulated state (i.e. high-altitude exposure, where the combined effects of hypoxia and iron store depletion are expected to be prevalent) is a novel next step to evaluate the iron-mediating role on HIF- and the peripheral vasculature in humans. 107 Healthy high-altitude adapted Andeans demonstrate numerous attributes that enhance their hypoxic tolerance compared to lowlanders including: elevated birth weights, increased exhaled NO concentrations, larger lungs, improved aerobic capacity and genotypic adaptations (Lenfant & Sullivan, 1971; Beall et al., 2001; Moore et al., 2004; Frisancho, 2013; Storz, 2021). However, healthy Andeans also display 1) preserved endothelial function at high altitude (Tymko et al., 2019) and 2) positive selection for HIF pathway candidate genes (Bigham et al., 2010). It seems plausible, therefore, that iron infusion (and thus HIF-down regulation) may also differentially impact vascular function in healthy high-altitude Andeans. To explore these possibilities, we examined the hypotheses that iron infusion would lead to: 1) an increase in peripheral blood flow and 2) an improvement in endothelial function, which would be more apparent in partially acclimatized lowlanders, compared to the high-altitude adapted Andeans. Given the well-established role of NO on blood flow, blood pressure and vascular function (Dietz et al., 1997; Markwald et al., 2011; Green et al., 2014), complimentary measures of NO were also collected in order to explore the temporal relationship with iron bioavailability (Li et al., 2014) and the control of HIF on NO synthase [NOS (Takeda et al., 2010)]. 5.2. Material and Methods 5.2.1. Ethical Approval All experimental procedures were approved by the University of British Columbia Research Ethics Board (H17-02687 and H18-01404) and the Universidad Peruana Cayetano Heredia Comité de Ética (no. 101686), and conformed to the Declaration of Helsinki, except for registration in a database. All participants received both written and oral information about the study and provided written informed consent. Forms were translated into Spanish, and Andean participants spoke with a Peruvian research assistant and provided written informed consent in Spanish before participating. 5.2.2. Participants A cohort of 24 healthy Andean highlanders and 22 age-matched lowlanders completed the study (Table 5.1). All were normotensive, and did not report a history of cardiovascular, cerebrovascular, or respiratory diseases during completion of a medical history questionnaire. All Andeans were born at high altitude (n=2 at 1880-2900 m, n=22 at 4300 m; as were their parents) and resided at 108 4300 m, were non-smokers, did not exhibit excessive erythrocytosis [Hb 19 g/dl for females and 21 g/dl for males] and had a Qinghai CMS questionnaire score of 0.5±0.8, and thus, were not considered to suffer from CMS (León-Velarde et al., 2005). Of the few lowlander participants who opted for high-altitude prophylaxis (i.e., acetazolamide) upon immediate arrival to 4300 m, all had discontinued use for at least a week prior to baseline testing – an important point given the postulated capacity to impact redox-regulation of vascular endothelial function (Bailey et al., 2012). This study was part of the Global REACH to Cerro de Pasco, Peru [4300 m; barometric pressure of 457 mmHg; PIO2 of ~96 mmHg (Tymko et al., 2020a)] Upon arrival in Lima and finalizing logistics, participants were driven up to Cerro de Pasco in ~8 hours. Lowlanders were tested after 84 days at 4300 m – lowlander infusion groups were equally weighted for number of days at 4300 m. 5.2.3. Experimental Design Data collected during this study have previously been published as part of an investigation that focused exclusively on the pulmonary vascular responses to iron infusion (Patrician et al., 2021b). Thus, although the present study adopted an identical experimental design, it constitutes an entirely separate question complemented by de novo experimental measures constrained to the systemic vasculature. Participants arrived at the laboratory having only eaten a small meal ~2 hours prior and abstained from caffeine and exercise for >6 and >12 hours, respectively. Following 10 min of supine rest an indwelling venous catheter (21G needle, BD Vacutainer eclipse) was inserted into the median antecubital vein for repeated blood sampling and the infusion. Flow-mediated dilation (FMD) was performed at baseline (i.e. pre-infusion), and at 2 and 4 hours after receiving an infusion of either iron or saline. Between time-points, participants were allowed to sit up, but resumed supine rest for at least 10 minutes before any measurement. 5.2.3.1. Infusion In a double-blinded, block-randomized design, Andean and lowlander participants received an i.v. infusion of either iron (iron (III)-hydroxide sucrose; 200 mg in 250 ml 0.9% NaCl saline) or saline (250 ml of 0.9% NaCl saline) over 30 min. Both participants and investigators were blinded during 109 collection (via opaque bags to cover the infusion and lines) and maintained throughout analysis. Due to the >40-day longevity of iron infusion (Bart et al., 2016) and the logistic constraints of an expedition (Tymko et al., 2020a), a cross-over design was not feasible. Therefore, the purpose of block-randomizing participants was two-fold: 1) enabled the optimization of logistics and coordination with other ongoing studies, and 2) ensured that lowlanders allocated to iron or saline conditions were appropriately weighted, in terms of the number of days at 4300 m, to limit any potentially confounding influence of hypobaric exposure on iron stores [lowlanders receiving saline and iron were tested after 95 and 63 days, respectively (unpaired t-test P=0.158)]. 5.2.4. Experimental measures 5.2.4.1. Flow-mediated dilation and reactive hyperemia Brachial artery FMD was performed according to standardized guidelines (Thijssen et al., 2011). In brief: following the acquisition of 1 minute of baseline artery diameter and blood velocity (via duplex ultrasound), a pneumatic cuff (placed distal to the epicondyles) was inflated to 250 mmHg for 5 minutes. Upon rapid cuff deflation, ultrasound imaging continued for 3 minutes post-deflation. Screen capture of the recording was saved using an audio-video interface software (Camtasia Studio) for future analysis using edge-detection software (Woodman et al., 2001). Peak and total reactive hyperemia (RH) were acquired as indices of resistance vessel function (Limberg et al., 2020; Rosenberry & Nelson, 2020). 5.2.4.2. Blood pressure, heart rate, and pulse oximetry At each stage prior to FMD, automated blood pressure (duplicate using brachial oscillometry) and pulse oximetry were collected. 5.2.4.3. Nitric oxide sampling and analysis Blood samples were drawn without stasis from the antecubital vein (of the contralateral arm) directly into Vacutainers (Becton, Dickinson and Company, Oxford, UK) before centrifugation at 600g (4°C) for 10 min. Plasma and serum supernatant was decanted into cryogenic vials (Nalgene Labware, Thermo Fisher Scientific Inc, Waltham, MA) and immediately snap-frozen in liquid nitrogen before transport under nitrogen gas (Cryopak, Taylor-Wharton, Theodore, AL). Samples were left to defrost at 37°C in the dark for 5 minutes prior to batch analysis. Total (bioactive) NO 110 represents the combined concentration of plasma nitrite and S-nitrosothiols measured using tri-iodide reductive ozone based chemiluminescence (Bailey et al., 2017). The intra- and inter-assay coefficient of variation for the NO metabolites were <10 %. 5.2.4.4. Iron status Venous blood samples were separated by microcentrifugation, with serum samples frozen in liquid nitrogen at -196 C for subsequent analysis. Serum iron, ferritin and transferrin were analysed according to clinical laboratory standards (Medlab clinical laboratories, Lima, Peru). 5.2.4.5. Viscosity, Hb and Hct Whole venous blood viscosity was obtained using lithium heparin vacutainers and measured within 1 hour of acquisition in duplicate at 37°C at a shear rate of 225 s-1 using a cone and plate viscometer (DV2T Viscometer, Brookfield Amtek) and heated circulating water bath (TC-150, Brookfield Amtek). When repeated measures exceeded the viscometer error, a third viscosity measure was obtained and an average of the two closest measures was reported. Hb was measured using a HemoCue (HemoCue AB, Ängelholm, Sweden). Hct was analyzed immediately using microcentrifugation (4 min at 13,500 rpm). 5.2.5. Data analyses Volumetric blood flow (Q) of brachial artery was calculated as Equation 5.1, using edge-detection software (FMD/Blood Flow Software version 5.1, Reed C, Australia) that continuously tracks vessel diameter and blood velocity (Woodman et al., 2001). Equation 5.1. Blood flow. Q={(peak envelope blood velocity 2⁄ )×[(π(vessel diameter 2⁄ )2]}×60 FMD (used as an index of endothelial function) was measured according to guidelines (Thijssen et al., 2011). Vascular conductance was calculated as brachial artery blood flow / MAP. Peak RH (used as index of microvascular function) was calculated as the greatest 3-sec post-occlusion blood flow. Peak vascular conductance was calculated as peak RH / MAP. Total RH was calculated as the blood flow area under the curve 180 sec post-cuff deflation. To account for changes in baseline diameter, allometric scaling of FMD was performed using a linear mixed model with the difference 111 between the natural log of peak and baseline diameters as the dependent variable, with ancestry and time as factors and natural log of baseline diameter as the covariate (Atkinson & Batterham, 2013). Linear mixed model estimated means were back-transformed [eestimated means–1)×100] to obtain allometrically scaled FMD and standard deviations [((estandard error–1)×100)×√n] (Shenouda et al., 2017). Shear rate was calculated as (4 × peak blood velocity / diameter). FMD was also corrected using its shear stress stimulus 60s post cuff deflation (SSAUC60) as a covariate. Shear stress was calculated from shear rate × viscosity. Hct and viscosity values at 2 hours were estimated as the median value between the baseline and 4-hour timepoints. 5.2.6. Statistical analyses Data were analyzed using a linear mixed effects model with a compound symmetry repeated measure co-variance structure (SPSS v24, IBM Statistics). The fixed factors for the model were ancestry and time (i.e. pre to post infusion) – with the latter being a repeated factor with a compound symmetry repeated covariance structure. When a significant interaction effect (e.g., ancestrytime) was detected, Bonferroni adjusted post-hoc tests were utilized to test pairwise comparisons. Comparison of measures at baseline of pooled lowlanders and Andeans, and between-group baseline total NO were assessed with independent samples t-test. Winsorizing was performed on one outlier NO value in an Andean in the iron group at baseline [(1.95×SD)+mean]. Effect size was estimated by partial eta square (η2) from general linear model with time as a repeated measure factor. Linear regression analysis was performed to determine relationship between blood flow and MAP across time in the iron infusion group. All results are reported as mean ± SD and significance was set at P<0.05. 5.3. Results 5.3.1. Pre-infusion (baseline) Baseline brachial conductance in Andeans was almost double that of lowlanders (0.45±0.38 ml/min/mmHg) vs. 0.25±0.09 ml/min/mmHg; p=0.023) due to reciprocal differences in flow and MAP. Absolute/relative FMD and RH were similar between lowlanders and Andeans (Table 5.1). 112 Apart from elevated serum ferritin levels in the Andeans, all remaining iron metrics were comparable (Patrician et al., 2021b). Andeans exhibited higher total NO at baseline (62.1±39.5 nM) than lowlanders (41.1±24.9 nM; p=0.038). The inter-individual variability in baseline iron or NO were not related to resting peripheral blood flow, conductance, FMD or RH. 5.3.2. Post-infusion (saline versus iron) Following the infusion of saline, there was no change in MAP, brachial blood flow, FMD (absolute or relative), peak RH or total bioactive NO in lowlanders and Andeans (all main effects of time p>0.05; Table 5.2; Figure 5.1). 113 Figure 5.1. Impact of saline infusion. Blood flow (panels A & B), peripheral conductance (panels C & D) and microvascular function (panels E & F) in lowlanders (black) and Andeans (blue) at baseline and 2- and 4-hours post-infusion. Mean and individual data are presented in left and right panels, respectively. Statistical significance was assessed using linear mixed model. Following the infusion of iron, there was a rise in brachial blood flow by 22-25% in both lowlanders and Andeans (main effect of time, p=0.033, η2=0.145; driven by an increase in blood velocity rather than diameter; Figure 5.2). While likely physiologically relevant, the change in the 114 magnitude of blood flow at 4hr, from both BL and 2 hr timepoints did not reach significance (p=0.085 and p=0.060, respectively). There was an almost significant rise in MAP by 3-4 mmHg across the 4 hours in both groups (main effect of time p=0.052, η2=0.097), with Andeans having lower MAP than lowlanders (main effect of ancestry p=0.001). Coincidingly, Andeans had a higher conductance compared to lowlanders (main effect of ancestry p=0.025, η2=0.133; Table 5.3). Diastolic blood pressure at 4 hours was elevated in both lowlanders and Andeans, compared to baseline (p=0.01). While FMD (absolute and relative) was unaltered, microvascular function (indexed via peak RH) in lowlanders and Andeans was increased by 10% at 4 hr following iron infusion [main effect of time p=0.005, η2=0.214; peak RH at 2hr was not different from BL (p=0.161) Table 5.4]. Moreover, peak RH at 4hr was elevated compared to 2hr (p=0.004)]. Total RH was also increased with iron infusion (main effect p=0.004, η2=0.196; total RH at 4hr was elevated compared to BL and 2hr, p=0.028 and p=0.005, respectively). Similar findings were observed for elevations in RHAUC (at 30, 60 and 120 s; all main effects of time p<0.01). Iron infusion did not alter total NO bioavailability in lowlanders, however there was a 48% reduction evident in Andeans (p=0.001; Table 5.3). Total NO between Andean groups at baseline (i.e. Andean groups who were allocated to receive saline or iron) were physiologically comparable (p=0.060). There was no change in SpO2, blood viscosity, Hb or Hct across the 4 hours in both lowlanders and Andeans (Table 5.2). 115 Figure 5.2. Impact of iron infusion. Blood flow (panels A & B), peripheral conductance (panels C & D) and microvascular function (panels E & F) in lowlanders (black) and Andeans (blue) at baseline and 2- and 4-hours post-infusion. Mean and individual data are presented in left and right panels, respectively. Statistical significance was assessed using linear mixed model. Significant pairwise comparisons for main effect of time in both lowlanders and Andeans are denoted using § from baseline, and ‡ from 2hrs (p<0.05). 116 Table 5.1. Measurement summary of pooled lowlanders and healthy Andeans at baseline. Variable Lowlander Andean p-value N (males/females) 16/6 21/3 Age (years) 29±7 29±11 0.898 Height (cm) 175±8 162±5 <0.001 Weight (kg) 71±10 63±7 <0.001 BMI (kg/m2) 23.3±2.3 23.8±2.8 0.530 Hb (g/dl) 16.2±1.4 [17] 16.9±1.9 [14] 0.235 Hct (%) 48.4±3.9 [20] 53.2±4.1 [18] 0.001 Whole blood viscosity (mPa · s) 4.75±0.55 [20] 5.25±0.79 [22] 0.021 Total NO (nM) 41.1±24.9 62.2±39.5 0.038 Systolic (mmHg) 122±11 106±8 <0.001 Diastolic (mmHg) 75±7 64±9 <0.001 MAP (mmHg) 90±8 78±8 <0.001 Baseline diameter (mm) 3.67±0.50 [21] 3.83±059 0.359 Peak diameter (mm) 3.91±0.48 [21] 4.13±0.63 0.201 FMD (mm) 0.24±0.12 [21] 0.30±0.13 0.077 FMD (%) 6.7±3.3 [21] 8.0±3.5 0.187 SSAUC60-adjusted FMD (%) 6.5±3.5 8.1±3.6 0.158 SSAUC60 (au) 1230±398 1251±308 0.846 Time to Peak (sec) 48.8±25.7 [21] 53.5±23.6 0.524 Mean shear stress (dyn/cm2) 1.8±0.6 [19] 2.2±1.4 [22] 0.211 Antegrade shear stress (dyn/cm2) 5.1±1.7 [19] 5.9±2.1 [22] 0.191 Retrograde shear stress (dyn/cm2) -1.5±0.9 [19] -1.4±1.8 [22] 0.884 OSI (AU) 0.21±0.07 [21] 0.18±0.16 0.448 Baseline flow (ml/min) 22.4±8.2 [21] 34.1±28.5 [23] 0.075 Baseline vascular conductance (ml/min/mmHg) 0.25±0.09 [21] 0.45±0.38 [23] 0.023 Peak Reactive Hyperemia (ml/min) 250.7±69.0 [21] 247.1±100.1 0.891 Peak reactive conductance (ml/min/mmHg) 2.77±0.75 [21] 3.21±1.36 0.182 Total reactive hyperemia (ml) 201 ± 72 [20] 251 ± 146 [23] 0.171 Mean ± SD in 22 Lowlanders and 24 Andeans (unless otherwise noted). Without winsorizing, Andean total NO was 65.3 ± 49.7 nM (p=0.046). 117 Table 5.2. Hematological measures by infusion type. Saline infusion group Baseline 2 hours 4 hours Ancestry Time Inter Iron (ug/dL) Lowlander 76.3±35 - 75±33.6 0.238 0.759 0.641 Andean 90.3±31.5 - 96.6±54.5 Ferritin (ng/ml) Lowlander 53.3±25.3 - 51.3±25.3 0.009 0.654 0.926 Andean 110.2±62.7 - 108.9±62.3 Transferrin (mg/dl) Lowlander 278.1±21.3 - 271.6±33.3 0.411 0.304 0.970 Andean 268.3±31.0 - 262.3±35.5 Viscosity (mPa·s) Lowlanders 4.68±0.64 [9] 4.71±0.60 [9] 4.74±0.58 [9] 0.019 0.339 0.987 Andeans 5.53±0.75 [11] 5.57±0.84 [11] 5.61±0.96 [11] Hb (g/dl) Lowlanders 16.1±1.6 [9] 16.1±1.6 [9] 16.1±1.7 [9] 0.048 0.998 0.987 Andeans 17.7±1.9 [6] 17.7±1.9 [6] 18.1±2.0 [7] Hct (%) Lowlanders 48.3±4.3 48.6±4.8 48.8±5.4 0.009 0.866 0.608 Andeans 54.1±3.7 54.3±4.8 [9] 54.2±5.6 [9] SpO2 (%) Lowlanders 89±3 89±3 88±4 0.607 0.557 0.806 Andeans 88±2 88±3 88±2 Total NO (nM) Lowlanders 45.5±29 - 44±39 [10] 0.633 0.307 0.445 Andeans 47.1±30.7 - 32.4±18.4 Iron infusion group Baseline 2 hours 4 hours Ancestry Time Inter Iron (ug/dL) Lowlander 87.4±47.1 - 352.7±43.0 [10] 0.355 <0.001 0.894 Andean 98.5±28.5 [11] - 367.1±55.4 Ferritin (ng/ml) Lowlander 53.8±47.4 - 64.2±50.5 [10] 0.251 <0.001 0.629 Andean 83.3±42.7 [11] - 86.9±50.8 Transferrin (mg/dl) Lowlander 272.6±27.9 - 264.5±34.4 [10] 0.887 0.010 0.991 Andean 267.6±26.5 [11] - 263.7±41.9 Viscosity (mPa·s) Lowlanders 4.80±0.49 4.76±0.42 4.70±0.45 [10] 0.229 0.664 0.062 Andeans 4.98±0.76 [11] 5.12±0.86 5.21±1.01 Hb (g/dl) Lowlanders 16.4±1.3 [8] 16.1±1.3 [7] 16.1±1.3 [9] 0.277 0.900 0.189 Andeans 16.4±1.9 [8] 16.5±2.0 [8] 17.3±2.1 [11] Hct (%) Lowlanders 48±3 [9] 48±3 [8] 48±3 [10] 0.011 0.766 0.906 Andean 51±5 [6] 51±5 [4] 54±5 [8] SpO2 (%) Lowlanders 88±3 88±2 88±3 [10] 0.444 0.160 0.315 Andean 88±4 87±3 87±3 Total NO (nM) Lowlanders 36.8±20.5 - 31.1±23.3 [10] 0.003 0.005 0.028 Andeans 77.2±42.7† - 35.7±22.4* Mean ± SD in 11 lowlanders and 12 Andeans in each infusion group (unless otherwise noted [n]). *denotes significant main effect of time p<0.05. †denotes significant main effect of ancestry p<0.05. Without winsorizing, total NO of Andeans pre-iron was 83.6±59.1 nM (main effect of ancestry, time and interaction, p=0.032, p=0.017, p=0.054). 118 Table 5.3. Peripheral vascular function in lowlander and Andeans receiving saline infusion. Variable Group Baseline 2 hours 4 hours Ances-try Time Inter Baseline (mm) Lowlander 3.96±0.52 [10] 4.03±0.6 [10] 4.0±0.58 0.278 0.642 0.909 Andean 3.81±0.57 3.81±0.37 3.75±0.37 Peak Response (mm) Lowlander 4.22±0.47 [10] 4.32±0.52 [10] 4.31±0.54 0.288 0.937 0.909 Andean 4.09±0.61 4.08±0.37 4.10±0.38 FMD (mm) Lowlander 0.26±0.14 [10] 0.28±0.20 [10] 0.32±0.15 0.805 0.174 0.802 Andean 0.29±0.15 0.28±0.20 0.35±0.15 FMD (%) Lowlander 6.8±3.9 [10] 7.5±6.0 [10] 8.3±4.3 0.725 0.176 0.747 Andean 7.7±4.1 7.5±5.5 9.5±4.4 Allometrically scaled FMD (%) Lowlander 7.9±3.5 8.5±3.5 9.2±3.3 0.599 0.181 0.716 Andean 5.2±3.5 5.2±3.5 5.8±3.5 SSAUC60-adjusted FMD (%) Lowlander 6.4±4.3 [8] 5.3±2.8 [8] 7.3±4 [9] 0.385 0.223 0.854 Andean 7.7±4.3 [11] 7.8±5.7 [11] 9.9±4.4 [11] Time to Peak (sec) Lowlander 51.7±32.1 [10] 58.4±23.8 [10] 47.5±18.9 0.809 0.373 0.290 Andean 47.3±14.3 56.3±22 58.9±16.7 Baseline blood flow (ml/min) Lowlander 24.1±9.0 [10] 23.1±9.1 [10] 24.1±11.7 0.446 0.710 0.635 Andean 33.4±30.6 [11] 26.2±17.3 26.1±15.6 Baseline conductance (ml/min/mmHg) Lowlander 0.26±0.10 [10] 0.26±0.11 [10] 0.27±0.14 [11] 0.249 0.674 0.521 Andean 0.42±0.41 [11] 0.32±0.21 0.31±0.19 Baseline oscillatory shear index Lowlander 0.201±0.075 [10] 0.209±0.116 [10] 0.191±0.124 [11] 0.962 0.486 0.984 Andean 0.222±0.164 0.205±0.123 0.189±0.159 Peak reactive hyperemia (ml/min) Lowlander 291.3±70.3 [10] 281.5±70.1 [10] 271.5±75.9 0.208 0.866 0.601 Andean 238.5±99.2 242.9±80.9 245.7±81.8 Peak vascular conductance (ml/min/mmHg) Lowlander 3.13±0.77 [10] 3.10±0.72 [10] 2.98±0.80 0.688 0.958 0.653 Andean 2.96±1.24 2.89±0.91 3.00±1.05 Total reactive hyperemia (ml) Lowlander 230 ± 85 [9] 244 ± 84 [9] 218 ± 102 [10] 0.953 0.965 0.367 Andean 234 ± 156 214 ± 104 232 ± 114 Mean Shear Stress (dyn/cm2) Lowlander 1.48±0.58 [8] 1.34±0.78 [8] 1.53±0.98 [9] 0.061 0.939 0.970 Andean 2.09±1.48 [11] 2.17±0.93 [11] 2.19±1.14 [11] Antegrade Shear Stress (dyn/cm2) Lowlander 4.04±1.47 [8] 4.01±1.71 [8] 4.13±1.59 [9] 0.007 0.949 0.864 Andean 6.02±2.31 [11] 5.66±1.59 [11] 5.71±1.23 [11] Retrograde Shear Stress (dyn/cm2) Lowlander -1.08±0.62 [8] -1.33±0.82 [8] -1.08±0.77 [9] 0.554 0.451 0.493 Andean -1.84±2.25 [11] -1.32±0.93 [11] -1.35±1.54 [11] SSAUC60 (au) Lowlander 1072±431 [8] 919±389 [8] 930±423 [9] 0.015 0.984 0.424 Andean 1278±293 [11] 1337±306 [11] 1368±264 [11] MAP (mmHg) Lowlander 92±8 90±7 91±10 0.027 0.914 0.305 Andean 81±8 84±11 83±14 Systolic (mmHg) Lowlander 123±12 122±10 124±12 0.012 0.775 0.410 Andean 109±7 112±11 111±16 Diastolic (mmHg) Lowlander 76±7 74±7 75±9 0.063 0.970 0.350 Andean 66±9 69±11 69±13 Heart rate (bpm) Lowlander 68±11 66±15 62±14 § 0.769 0.019 0.974 Andean 66±12 65±11 61±9 § Mean ± SD in 11 lowlanders and 12 Andeans (unless otherwise noted [n]). Significant pairwise comparisons for main effect of time in both lowlanders and Andeans are denoted using § from baseline (p<0.05). 119 Table 5.4. Peripheral vascular function in lowlander and Andeans receiving iron infusion. Variable Group Baseline 2 hours 4 hours Ances-try Time Inter Baseline (mm) Lowlander 3.41±0.32 3.46±0.32 3.4±0.28 0.092 0.586 0.060 Andean 3.84±0.63 3.59±0.51 [11] 3.86±0.74 Peak Response (mm) Lowlander 3.63±0.29 3.68±0.28 3.64±0.24 0.039 0.469 0.086 Andean 4.16±0.68 3.92±0.54 [11] 4.2±0.76 FMD (mm) Lowlander 0.22±0.09 0.21±0.09 0.23±0.13 0.006 0.663 0.949 Andean 0.32±0.12 0.32±0.11 [11] 0.34±0.10 FMD (%) Lowlander 6.5±3.0 6.3±2.9 7.1±4.2 0.042 0.643 0.805 Andean 8.4±2.8 9.0±2.8 [11] 9.1±3.0 Allometrically scaled FMD (%) Lowlander 7.8±3.3 7.7±3.3 9.5±3.3 0.024 0.287 0.858 Andean 8.3±3.5 8.8±3.3 9.4±3.5 SSAUC60-adjusted FMD (%) Lowlander 6.5±3.0 6.3±2.9 6.6±4.1 [10] 0.120 0.250 0.900 Andean 8.4±2.9 [11] 9.0±2.8 [11] 9.1±3.0 Time to Peak (sec) Lowlander 46.1±19.4 54.9±15.2 48.6±18.7 0.161 0.717 0.644 Andean 59.7±29.6 58.2±20.5 [11] 56.8±12.2 Baseline blood flow (ml/min) Lowlander 20.8±7.6 20.2±7.2 25.5±13 0.073 0.033 0.520 Andean 34.8±27.7 29.6±21.8 [11] 46.9±36.8 Baseline conductance (ml/min/mmHg) Lowlander 0.23±0.08 0.22±0.07 0.28±0.15 0.025 0.064 0.561 Andean 0.48±0.38 0.39±0.26 [11] 0.58±0.42 Baseline oscillatory shear index Lowlander 0.22±0.073 0.18±0.101 0.145±0.118 § 0.115 0.007 0.952 Andean 0.144±0.144 0.117±0.125 [11] 0.082±0.123 § Peak reactive hyperemia (ml/min) Lowlander 213.7±43.8 214.8±48.5 232.4±67.5 ‡ 0.327 0.005 0.102 Andean 255.6±104.7 [12] 201.1±83.6 [11] 286.9±114 ‡ Total reactive hyperemia (ml) Lowlander 178 ± 51 191 ± 77 232 ± 101 [10]‡§ 0.087 0.004 0.311 Andean 271 ± 139 [11] 223 ± 120 [11] 343 ± 197 [11]‡§ Peak vascular conductance (ml/min/mmHg) Lowlander 2.43±0.57 2.42±0.54 2.57±0.74 ‡ 0.051 0.008 0.055 Andean 3.46±1.48 2.67±0.99 [11] 3.63±1.32 ‡ Mean Shear Stress (dyn/cm2) Lowlander 2.02±0.49 1.99±0.78 2.5±1.29 [10] 0.282 0.040 0.779 Andean 2.37±1.37 [11] 2.54±1.4 [11] 3.2±2.06 Antegrade Shear Stress (dyn/cm2) Lowlander 5.79±1.54 5.17±1.38 6.15±2.02 [10] 0.688 0.224 0.689 Andean 5.71±2.02 [11] 5.84±2.29 [11] 6.66±4.01 Retrograde Shear Stress (dyn/cm2) Lowlander -1.76±0.99 -1.19±0.84 -1.15±1.1 [10] § 0.053 0.004 0.633 Andean -0.98±1.11 [11] -0.76±1.01 [11] -0.33±0.34 § SSAUC60 (au) Lowlander 1344±346 1343±443 1486±524 [10] ‡§ 0.556 0.013 0.547 Andean 1224±334 [11] 1222±275 [11] 1513±416 ‡§ MAP (mmHg) Lowlander 89±8 89±10 91±7 0.001 0.052 0.647 Andean 75±6 76±8 79±11 Systolic (mmHg) Lowlander 120±10 118±10 120±10 <0.001 0.235 0.743 Andean 103±8 102±11 106±11 Diastolic (mmHg) Lowlander 73±8 75±10 76±6 § 0.002 0.013 0.671 Andean 61±7 63±8 66±11 § Heart rate (bpm) Lowlander 71±14 66±16 66±16 § 0.447 <0.001 0.552 Andean 69±9 62±9 60±8 § Mean ± SD in 11 lowlanders and 12 Andeans (unless otherwise noted [n]). Significant pairwise comparisons for main effect of time in both lowlanders and Andeans are denoted using § from baseline, and ‡ from 2hrs (p<0.05). 120 5.4. Discussion Our primary finding was that iron infusion increased brachial blood flow and microvascular function, in both lowlanders and healthy Andeans at high altitude (Figure 5.3). Since iron infusion has been shown to induce HIF-down regulatory influences on the pulmonary vasculature (Smith et al., 2008a; Talbot et al., 2014; Frise & Robbins, 2015; Frise et al., 2016; Patrician et al., 2021b; Willie et al., 2021), our similarly suggestive role of iron on HIF down-regulation in the peripheral vasculature, is notable. The following discussion outlines the implications of these findings and highlights a functional role for iron status on HIF in the peripheral vasculature of humans at high altitude. Figure 5.3. Summary of the impact of iron infusion on peripheral vascular function in hypoxia. Top panel: Saline infusion did not lead to any significant changes in blood flow, macrovascular function (via FMD), total NO, blood pressure or microvascular function [via peak reactive hyperemia (RH) and total RH]. Bottom panel: Both lowlanders and Andeans demonstrated an increase in resting brachial flow and microvascular function, with subtle increases in MAP and diastolic blood pressure (DIA) – percent change from baseline from significant measures are included. * indicates a main effect of time for both lowlanders and Andeans. † indicates that NO was only reduced in Andeans post-iron. The putative role of iron on systemic and peripheral HIF regulation is conceptually included, with green line denoting activation and purple line denoting attenuation. 121 5.4.1. Iron and peripheral tissue perfusion The HIF- pathway acts as a cellular oxygen sensor (Semenza et al., 1998; Kaelin & Ratcliffe, 2008) and controls a broad range of transcriptional responses to hypoxic exposure, in a dose-dependent (Jiang et al., 1996) and organ-specific (Stroka et al., 2001) manner. While it is logical to postulate that HIF-down regulation could exert influence over the peripheral vasculature, there exists little literature exploring this relationship in humans in vivo [reviewed in: Rey and Semenza (2010)]. The exception is a notable study by Cowburn and colleagues (2013) which – by utilizing an extensive array of techniques in the skin of mice and humans – revealed that 1) blood pressure was 14% higher in mice with HIF-1a knockout (and 18% lower in HIF-2𝛼 knockout), compared to wild-type controls, and 2) in human participants, the severity of arterial hypertension is negatively and positively associated with HIF-1𝛼 and HIF-2𝛼 expression, respectively (Cowburn et al., 2013). Therefore, these findings 1) support our rise in peak vascular conductance and perfusion pressure by 3-8 % across the 4 hours in our cohort following iron infusion, and 2) provide mechanistic insight into the HIF-isoform responsible for the down regulatory influence of iron. While our data did not directly assess O2 delivery via sampling of arterial blood gases, both SpO2 and Hb were unaltered after iron infusion (Table 5.2); therefore, the elevations in brachial blood flow would likely be reflective of increased O2 delivery, and hence, peripheral tissue perfusion. Together, our findings provide preliminary evidence for a functional link between iron infusion and peripheral HIF-down regulation via its influence on vascular conductance and tissue perfusion. Further studies are needed to delineate the influence of iron on vasodilatory function from subtle changes in perfusion pressure. 5.4.2. Iron and NO NO is a potent regulator of blood flow and vascular function (Dietz et al., 1997; Markwald et al., 2011; Green et al., 2014). Links between NO and both NTB (i.e. ‘free iron’) (Li et al., 2014)] and reactive oxygen species (Valko et al., 2016) are emerging, and thus highlighting a complex interplay between iron, NO and vascular function. Although speculative, because plasma NO was selectively decreased in Andeans following iron infusion, it remains plausible that there exists potential for differential HIF regulation between groups. However, in a mouse model (albeit with 122 a different sample preparation protocol), knocking out either HIF-1𝛼 and HIF-2𝛼 had no effect on altering plasma NO (Cowburn et al., 2013). Interestingly, in cell preparation models, HIF activation (and NO production) can be differentially activated in macrophages, depending on stimulus and interplay between HIF-1𝛼 and HIF-2𝛼 isoforms (Takeda et al., 2010). This is a relevant finding given 1) macrophages are one of the primary sites of iron storage (via hepcidin) and iron recycling (via senescent red blood cells) (Hirota, 2019), and 2) during low interferon- (INF) concentration states [such as occurring after an infusion of iron sucrose during a similarly designed study in lowlanders and Sherpa at 5050 m (Willie et al., 2021)], HIF-2α is primarily activated and inducible NOS expression is decreased (Takeda et al., 2010). The potential for an iron- or oxidative/inflammatory-related mechanism(s) that might be accountable for the decline of plasma NO in Andeans in the current study should be explored in future studies. 5.4.3. Clinical basis of iron infusion on endothelial dysfunction Intravenous iron administration is widely performed clinically, however, there is growing evidence that suggests intravenous iron can increase oxidative stress and, therefore, could compromise endothelial function subsequent to NO inactivation [reviewed in: (Bishu & Agarwal, 2006; Koskenkorva-Frank et al., 2013)]. For example, at ~10 min following the infusion of ferric saccharate (100 mg), there was a transient reduction in endothelium-dependent FMD in healthy humans (Rooyakkers et al., 2002). Likewise, in a human experimental model of endothelial dysfunction (via oral methionine to induce homocysteine-induced endothelial dysfunction), infusion of iron-sucrose (100 mg) exaggerated the decline in FMD at 1-hour post-infusion, compared to placebo (Zheng et al., 2006). However, it is important to note that in both studies, the transient endothelial dysfunction was no longer different from the control condition at 4 hours (Rooyakkers et al., 2002; Zheng et al., 2006). Additionally, forearm blood flow in dialysis patients was unaltered at 2 hours after infusion of iron sucrose [300 mg (Schaller et al., 2005)]. It is interesting to note that iron sucrose has a pronounced effect of increasing NTBI levels post-infusion – which peaks at ~4 hours (Garbowski et al., 2020). While this peak in NTBI aligns with the 5-fold increase in NTBI at 4 hours in the study by Zheng and colleagues (Zheng et al., 2006), the lack of significant decline in metrics of vascular function [in ours and others (Rooyakkers et al., 2002; Schaller et al., 2005; Zheng et al., 2006)] at rising/peak NTBI levels is surprising, since NTBI is capable of catalyzing ROS production (Kruszewski, 2003), including superoxide 123 (Rooyakkers et al., 2002). Such elevations in superoxide can act as a NO scavenger to produce peroxynitrite (Radi et al., 2001) and ultimately vasoconstriction. Therefore, our finding of elevations in peripheral blood flow suggest that endogenous anti-oxidative mechanisms act to buffer elevated NTBI levels (and downstream ROS formation) and preserve endothelial-dependent vasodilator function. 5.4.4. Experimental limitations and practical implications It is not possible to quantify HIF in the vascular endothelium of humans, and not all systemically circulating concentrations of HIF-regulated gene products (e.g., erythropoietin) necessarily reflect HIF activity in the peripheral vasculature. Previous studies have quantified HIF expression in skeletal muscle (Robach et al., 2007) and in the duodenum (Goetze et al., 2013). In a study by Cowburn and colleagues, peripheral HIF expression was quantified and inferred upon from epithelial skin biopsies (Cowburn et al., 2013). It is noteworthy that HIF up-regulation during hypoxia appears to be a conserved response across multiple organs and tissues in a dose-dependent manner (Jiang et al., 1996; Stroka et al., 2001). Likewise, acute iron depletion (via chelation) has been shown to facilitate HIF activation in liver cells during normoxic (Bianchi et al., 1999) and hypoxic conditions (Wang & Semenza, 1993b). Additionally, at sea level iron chelation and iron supplementation emulated the pulmonary vascular manifestations of up- and down-regulation of HIF that are seen in Chuvash polycythemia (Smith et al., 2006) and HIF+/− mice (Brusselmans et al., 2003), respectively. Therefore, given the constituent role in HIF- stabilization, via prolyl hydroxylase activity (Maxwell et al., 1999; Knowles et al., 2003)], it seems reasonable to expect that such changes in HIF in peripheral vascular tissues, would also be iron-dependent at high altitude. A larger dose could have been administered and may have augmented some of our findings. However, our current doses of iron-sucrose significantly increased serum iron levels in the blood (main effect p<0.001 for both), and aligned to other studies which showed effects of reducing pulmonary artery systolic pressure (Smith et al., 2008a; Smith et al., 2009; Talbot et al., 2011; Talbot et al., 2014). The implications of elevated brachial blood flow and microvascular function is of particular interest, whether these findings could be extended to other vascular beds, such as the brain or heart, where matching of tissue metabolism to O2 delivery is of crucial importance. Therefore, further studies should explore these important future research topics. 124 5.5. Perspectives and significance Aligning with previous work in mice and humans – whereby HIF-down regulation has been linked to improved tissue perfusion (Cowburn et al., 2013) – our primary finding was that iron infusion increased brachial blood flow and microvascular function, in both lowlanders and healthy Andeans. While further investigation is necessary to fully elucidate the mechanism, our study highlights a functional role for HIF/iron status in the peripheral vasculature of humans at high altitude. 125 Chapter 6. Iron status and manipulation on cerebral blood flow at high altitude in lowlanders and adapted highlanders 6.1. Background The brain is highly O2 dependent and, as such, the related CBF responses to hypobaric hypoxia have been well described (Severinghaus et al., 1966; Huang et al., 1987; Willie et al., 2015; Hoiland et al., 2019). For example, upon exposure to hypoxia, CBF increases in order to compensate for the initial reductions in CaO2 in order to maintain cerebral O2 delivery (Willie et al., 2014a; Hoiland et al., 2016). Eventually, the acute rise in CBF is attenuated, coinciding with an increase in CaO2 via erythropoiesis, ventilatory acclimatization and compensation of the initial respiratory alkalosis (Severinghaus et al., 1966; Ainslie & Subudhi, 2014; Swenson, 2016). In hypoxia, the HIF family – the key cellular O2 sensor (Semenza et al., 1998; Kaelin & Ratcliffe, 2008) – binds to hypoxia-responsive elements in gene promoters to up-regulate expression of >100 genes to coordinate increased O2 supply to hypoxic tissue. While HIF-1 expression within the human brain has not been quantified during hypoxia, data from rodent models shows that cortical HIF-1 expression during hypoxia follows a similar trajectory to the CBF responses – i.e., HIF-1 peak expression occurring with 6-12 h of exposure to hypoxia, which is halved by day 7, and normalized within ~3 weeks (Chávez et al., 2000). The potential for HIF expression to influence cerebrovascular function also stems from murine models, that have shown that within 4-hours of exposure to extreme hypoxia, there is an increase in downstream products of HIF-1 (e.g. VEGF, EPO) in the brain, that increase cerebral microvascular density and Hct within 3 weeks (Benderro & LaManna, 2014). Similarly, inactivation of prolyl-hydroxylase [the site of iron’s influence on HIF stabilization (Knowles et al., 2003)] leads to HIF expression and neurovascular angiogenesis and pericyte proliferation in mice (Urrutia et al., 2020). Finally, cerebral astrocytes (but not cerebral neurons) exposed to extreme hypoxia and DFO (iron chelator) showed an increase in EPO expression via HIF-2 up-regulation (Chavez et al., 2006). In humans, it is reasonable that the increase in CBF that occurs to counteract the reduction in arterial O2 content during hypoxia, also coincides with HIF stabilization. However, it has yet to be determined whether acute HIF down- or upregulation can acutely modulate hypoxic vasodilation of the cerebral vasculature. 126 Iron and iron-chelation are typically utilized to down- and up-regulate HIF expression – due to iron’s constituent role in HIF stabilization, via PHD activity (Knowles et al., 2003) – which have repeatedly demonstrated notable implications in pulmonary (Smith et al., 2008a; Smith et al., 2009; Smith et al., 2011b; Frise et al., 2016; Cheng et al., 2019; Lakhal-Littleton et al., 2019; Holdsworth et al., 2020; Patrician et al., 2021b; Willie et al., 2021), and peripheral vascular regulation (Cowburn et al., 2013; Patrician et al., 2021a). However, limited data exists with respect to the cerebral vasculature. As far as the authors are aware, the only human study to date is via the assessment of intracranial blood velocity (i.e. middle cerebral artery) following iron chelation [via DFO – an up-regulator of HIF expression (Ren et al., 2000)] by Sorond and colleagues (Sorond et al., 2009; Sorond et al., 2015). While the authors conclude that DFO infusion elevated middle cerebral artery velocity, compared to saline placebo, the DFO condition appeared mostly comparable to baseline in both studies. For example, middle cerebral artery velocity increased by ~1 cm·s-1 (level of significance not provided) from pre- to 4 hrs post-DFO in older adults (Sorond et al., 2009) and by 2 cm·s-1 (p>0.05) and 4 cm·s-1 (p<0.05) at 3 hrs post-DFO in young and older adults, respectively (Sorond et al., 2015). These extremely small changes in middle cerebral artery velocity are unlikely to be of physiological significance – especially if there is constriction/dilation of the middle cerebral artery (Ainslie & Hoiland, 2014). Studies of volumetric blood flow to the brain during iron manipulation (via iron and chelator infusion) have not been performed during conditions where the HIF pathways are up-regulated (i.e., hypoxia). Tibetan Sherpa have been reported to present with a unique phenotypic adaptation to high altitude characterized by a blunted ventilatory response to hypoxia (Beall et al., 1997), a more efficient plasma volume-hemoglobin ratio to aid exercise capacity (Stembridge et al., 2019), a reduced prevalence of excessive erythrocytosis (Simonson et al., 2010; Gassmann et al., 2019), exhibit less pronounced pulmonary hypertension, and higher lung diffusing capacity [reviewed in:(Gilbert-Kawai et al., 2014)]. Cerebral O2 delivery is also lower in Sherpa – and thus, potentially suggestive that Sherpa experience less deleterious cerebral consequences to hypoxia compared to lowlanders at high altitude (Garrido et al., 1996; Hoiland et al., 2019). Healthy high-altitude adapted Andeans demonstrate numerous attributes that enhance their hypoxic tolerance compared to lowlanders including: elevated birth weights, increased exhaled NO concentrations, larger lungs, improved aerobic capacity and genotypic adaptations (Lenfant & Sullivan, 1971; Beall et al., 2001; Moore 127 et al., 2004; Frisancho, 2013; Storz, 2021), and display preserved endothelial function at high altitude (Tymko et al., 2019). While both Sherpa and Andean highlanders display adaptive characteristics to hypoxia, both also display positive selection for HIF pathway candidate genes (Bigham et al., 2010; Foll et al., 2014). It seems plausible, therefore, that iron manipulation may also differentially impact cerebrovascular function in healthy Andeans and Sherpas who have many naturally selective traits to high altitude. To explore these possibilities, we examined the hypothesis that elevations in CBF would occur in response to acute iron depletion (i.e. increasing HIF activity) and repletion (i.e. decreasing HIF activity). Furthermore, we hypothesized that during exaggerated hypoxia at high altitude, the increase in CBF would be amplified by chelation and attenuated by iron infusion. Finally, by assessing high altitude populations with ancestral adaptation to hypoxic exposure, we sought to explore whether the genotypic advantages of Andeans and Sherpas, related to iron metabolism, would manifest through the modulation of CBF. 6.2. Materials and Methods 6.2.1. Ethical Approval All experimental procedures were approved by the University of British Columbia Research Ethics Board (H16-01028, H17-02687 and H18-01404), the Nepal Health Research Council (no. 586), the Universidad Peruana Cayetano Heredia Comité de Ética (no. 101686), and conformed to the Declaration of Helsinki, except for registration in a database. All participants received both written and oral information about the study and provided written informed consent. All highlander (Sherpa and Andean) participants read an in-depth translated study information form and had the study explained to them in their local language, and gave written informed consent prior to participating. 6.2.2. Experimental Design Data collected during this study have previously been published as part of an investigation that focused exclusively on the pulmonary and peripheral vascular responses to iron manipulation (Patrician et al., 2021a; Patrician et al., 2021b; Willie et al., 2021). Thus, although the present 128 study adopted an identical experimental design, it constitutes an entirely separate question complemented by de novo experimental measures constrained to the cerebral vasculature. 6.2.3. Participants A total of 82 volunteers participated in the study, which was conducted across two high altitude research expeditions (Willie et al., 2018; Tymko et al., 2020a). Participants consisted of 38 lowlanders (30 ± 8 years, 176 ± 7 cm, 73 ± 9 kg, 23 ± 2 kg·m-2), 20 Sherpa (25 ± 7 years, 169 ± 6 cm, 64 ± 11 kg, 22 ± 3 kg·m-2) and 24 Andeans (29 ± 11 years, 162 ± 5 cm, 63 ± 7 kg, 24 ± 3 kg·m-2). Andeans were not considered to suffer from CMS (León-Velarde et al., 2005)], as they did not exhibit excessive erythrocytosis (Hb 19 g·dl-1 for females and 21 g·dl-1 for males) and had a Qinghai CMS questionnaire score of 0.5±0.8. 6.2.3.1. Study 1 – Lowlanders and Sherpa at 5,050 m At 5,050 m in the Nepalese Himalaya (EV-K2-CNR Pyramid Research laboratory), 7 male lowlanders and 8 male Sherpa received an infusion of DFO (7 mg/kg/hour over 4 hr) and 7 male lowlanders and 12 male Sherpa received an infusion of iron [iron (III)-hydroxide sucrose; 200 mg over 30 min followed by 3.5 hours of slow-drip saline (0.9% NaCl); the total infusion time for iron was 4 hr to mirror the time-course of the DFO infusion]. Lowlanders receiving DFO and iron were tested after 13±3 days and 12±4 days, respectively at 5,050 m. There were 8 Sherpa who ascended from Kathmandu and tested following 7±3 days at 5,050 m, and then 12 Sherpa who ascended from 3,800-4,200 m, and were tested 1-2 days following arrival to 5,050 m. 6.2.3.2. Study 2 – Lowlanders and Andeans at 4300 m At 4,300 m in the Peruvian Andes (Cerro de Pasco), 11 lowlanders (2 female) and 12 Andeans (1 female) received an infusion of saline (250 ml of 0.9% NaCl saline) and 11 lowlanders (4 female) and 12 Andeans (2 female) received an infusion iron sucrose [iron (III)-hydroxide sucrose; 200 mg in 250 ml 0.9% NaCl saline] over 30 min. Lowlanders receiving saline and iron were tested after 95 and 63 days, respectively. 129 6.2.3.3. Infusion randomization For Studies 1 & 2, block-randomization of participants was performed for two primary purposes: 1) it enabled the optimization of logistics and coordination with other ongoing studies (hence why DFO was not feasible during Study 2), and 2) ensured that lowlanders allocated to iron and saline or DFO conditions were appropriately weighted, in terms of the number of days at altitude, to limit any potentially confounding influence of hypobaric exposure on iron stores. 6.2.4. Changes in PIO2 Measures of CBF, blood pressure, SpO2 and ventilation (via Wright spirometer) were collected during resting supine breathing of: 1) ambient air (PIO2 of 87 mmHg at 5,050 m and PIO2 of 96 mmHg at 4,300 m), and 2) exaggerated hypoxia (PIO2 of 67 mmHg at 5,050 m and PIO2 of 73 mmHg at 4,300 m; simulating an additional elevation gain of ~2000 m each), both before and after the infusion. The partial pressure of end-tidal CO2 (PETCO2) was assessed using capnography (EMMA, Masimo); however, we were unable to collect PETCO2, whilst Douglas bag breathing (i.e., exaggerated hypoxia) due to increased apparatus deadspace. Consequently, PETCO2 during exaggerated hypoxia was calculated, based on the change in ventilation. 6.2.5. Experimental measures 6.2.5.1. Cerebral blood flow Internal carotid artery (ICA) and vertebral artery (VA) diameter with synchronous measurements of blood velocity were performed using a 10-MHz multifrequency linear array probe with high-resolution duplex ultrasound (Terason t3200 and Terason uSmart 3300, Teratech). The ICA was measured at least 2 cm from the carotid bifurcation, whilst avoiding turbulent or retrograde flow. The VA was measured approximately at the transverse process of C4 and the subclavian artery. Each ICA and VA measures were conducted at the same location within each subject. Images of blood vessel diameter and blood velocity were recorded as video files, which were analyzed offline using an automated edge-detection software [FMD/Blood Flow Software version 5.1, Reed C, Australia;(Woodman et al., 2001)]. All data are based on imaging of >15 cardiac cycles, with stable and repeated angle of insonation. Volumetric blood flow (Q) was quantified using the following Equation 5.1: 130 Q={(peak envelope blood velocity 2⁄ )×[(π×(vessel diameter 2⁄ )2]}×60 Global CBF (gCBF) was estimated as twice the sum of unilateral ICA and VA flows (QICA and QVA, respectively). Cerebrovascular conductance was estimated using MAP. Automated blood pressure was collected in duplicate using brachial oscillometry. The absolute change in blood flow (∆Q) from room air to exaggerated hypoxia was used to assess hypoxic reactivity of a particular vessel (e.g. ∆QVA) and the bulk flow to the brain (i.e. ∆gCBF). 6.2.5.2. Iron status Venous blood samples were separated by microcentrifugation, with serum samples frozen in liquid nitrogen at -196 C for analysis. Serum iron was analysed according to clinical laboratory standards (Samyak Diagnostic Pvt. Ltd., Kathmandu, Nepal and Medlab Clinical Laboratories, Lima, Peru). 6.2.6. Data analysis PETCO2 during poikilocapnic hypoxia was calculated (Equation 6.1) using a mean slope of PETCO2 [derived from 15 min of poikilocapnic hypoxia in 22 healthy individuals (Rispen et al., 2017)] per change in VE from room air breathing to hypoxia: Equation 6.1. PETCO2 calculation. [(VEhypox-VEnormox) ×(-0.401591258383796)] +PETCO2normox 6.2.7. Statistical analysis Data were analyzed using a linear mixed effects model with a compound symmetry repeated measure co-variance structure (SPSS v24, IBM Statistics). The fixed factors for the model were ancestry and time (i.e., pre to post infusion) – with the latter being a repeated factor with a compound symmetry repeated covariance structure. When a significant interaction effect (e.g., ancestrytime) was detected, Bonferroni adjusted post-hoc tests were utilized to test pairwise comparisons. Pre- and post-infusion iron markers were assessed using paired samples t-test. Correlations of iron status with gCBF were assessed using Pearson correlation. A one-way ANOVA was used to assess the absolute change in serum iron across groups. All results are reported as mean ± SD and significance was set at P<0.05. 131 6.3. Results 6.3.1. Baseline iron status on global cerebral blood flow When data from lowlanders and highlanders were pooled across both expeditions, baseline iron status was not associated with gCBF during room air breathing (R2=0.001, P=0.766; Figure 6.1 Panel A); however, during exaggerated hypoxia, iron status was significantly correlated with gCBF (R2=0.082, p=0.019; Figure 6.1 Panel B). Consequently, as illustrated in Figure 6.1 Panel C, variations in baseline and physiologically normal iron levels appears to influence hypoxic reactivity (R2=0.174, P<0.001). 6.3.2. Between study infusion comparability The effectiveness of iron infusion to increase serum iron was comparable across lowlanders and Andeans at 4,300 m and lowlanders and Sherpa 5,050 m (P=0.819; Figure 6.1 Panel D). Saline infusion did not alter serum iron in either lowlanders or Andeans (main effect of time P=0.759), whereas DFO decreased serum iron by ~60%, to nearly undetectable levels of 2.4±0.9 µmol·l-1 in lowlanders and 2±0 µmol·l-1 in Sherpa (main effect of time P<0.001). 132 Figure 6.1. Influence of baseline iron status on gCBF in pooled lowlanders and pooled highlanders at high altitude. Panels A and B illustrate the relationship between gCBF during room air breathing and exaggerated hypoxia. Panel C indicates that the change in hypoxic reactivity of gCBF is greater in those with lower iron levels, compared to those with more elevated iron levels. Panel D highlights serum iron levels in each group [lowlanders (LL), Andeans (AN) and Sherpas (SH) at their respective altitudes] at baseline (i.e. pre-infusion), and the absolute increase in serum iron following iron infusion (i.e. ∆post-infusion). 6.3.3. Study 1: DFO and iron at 5,050 m In both lowlanders and Sherpa, VE, PETCO2, SpO2 and MAP were unaltered by either DFO or iron. Ultimately, there was no change in QICA, QVA, gCBF or CVC following either DFO or iron, in lowlanders and Sherpa (Table 6.1). Moreover, and in contrast to our hypotheses, during exaggerated hypoxia (PIO2=67 mmHg), DFO did not increase gCBF and iron did not lower gCBF in either lowlanders or Sherpa (Table 6.2). When normalized to the change in SpO2, the reactivity of gCBF (or QICA and QVA) during hypoxia was also not altered by iron or DFO manipulation (Table 6.2). 133 6.3.4. Study 2: Saline and iron at 4,300 m In both lowlanders and Andeans, VE, PETCO2, SpO2 and MAP were unaltered by saline and iron infusions (Table 6.3). While saline infusion did not alter gCBF, iron infusion led to a 4±10 % reduction in gCBF (main effect of time p=0.043) and a 7±13 % decrease in CVC (main effect of time p=0.011) in both lowlanders and Andeans (Table 6.3). During exaggerated hypoxia (PIO2=73 mmHg), however, neither saline or iron, altered the rise in gCBF. The reactivity of gCBF during hypoxia was attenuated following iron infusion (main effect of time P=0.019; 134 Table 6.4). In only lowlanders at 4,300 m, the change in VA blood flow from room air to hypoxia (i.e. ∆QVA) increased from 4±13 ml·min-1 pre-iron, to 21±19 ml·min-1 post-iron infusion (p=0.023; Figure 6.2). Figure 6.2. The change of blood flow to the posterior part of the brain (i.e., ∆QVA) from baseline to hypoxia). Lowlanders (black) and Sherpa (green) or Andeans (blue) following iron, DFO or saline infusions (Panels A, C, B and D respectively *P<0.05 from pre—infusion, †P<0.05 from Andean group post-infusion. 135 Table 6.1. During room air breathing (PIO2=87 mmHg) at pre- and post-infusion at 5,050 m in lowlanders and Sherpa. Variable Group Pre-DFO Post-DFO Ancestry Time Inter Pre-Iron Post-Iron Ancestry Time Inter QICA (ml·min-1) Lowlander 245 ± 59 276 ± 46 [6] 0.270 0.326 0.179 259 ± 59 [7] 264 ± 59 [8] 0.046 0.909 0.441 Sherpa 216 ± 82 [7] 218 ± 69 [6] 209 ± 53 202 ± 64 QVA (ml·min-1) Lowlander 74 ± 13 82 ± 23 [6] 0.313 0.880 0.130 63 ± 27 62 ± 26 0.261 0.233 0.393 Sherpa 72 ± 29 [7] 64 ± 19 52 ± 24 [11] 51 ± 22 [9] gCBF (ml·min-1) Lowlander 639 ± 135 718 ± 125 [6] 0.280 0.433 0.148 655 ± 99 [7] 652 ± 111 [8] 0.010 0.929 0.592 Sherpa 577 ± 214 [7] 568 ± 175 [6] 512 ± 125 [11] 515 ± 110 [9] CVC (ml·min-1mmHg-1) Lowlander 7.23 ± 1.65 8.11 ± 1.52 [6] 0.516 0.136 0.668 6.78 ± 1.2 [7] 6.74 ± 1.39 [8] 0.106 0.785 0.585 Sherpa 6.64 ± 2.6 [7] 7.12 ± 2.25 [6] 5.87 ± 1.33 [11] 5.81 ± 1.25 [9] Systolic (mmHg) Lowlander 120 ± 4 122 ± 8 0.976 0.190 0.010 130 ± 7 129 ± 9 0.018 0.974 0.685 Sherpa 124 ± 11 118 ± 11* 117 ± 11 118 ± 16 Diastolic (mmHg) Lowlander 74 ± 10 74 ± 8 0.549 0.452 0.311 82 ± 9 83 ± 7 0.019 0.811 0.784 Sherpa 73 ± 9 69 ± 14 73 ± 9 73 ± 12 MAP (mmHg) Lowlander 89 ± 7 90 ± 7 0.644 0.277 0.078 98 ± 8 98 ± 7 0.014 0.849 0.956 Sherpa 90 ± 9 85 ± 12 88 ± 9 88 ± 12 VE (l·min-1) Lowlander 10.1 ± 1.4 12.1 ± 2.6 0.006 0.091 0.705 11.7 ± 1.9 12.9 ± 2.1 0.821 0.048 0.905 Sherpa 14.8 ± 3.5 16 ± 3.8 11.4 ± 3.5 12.7 ± 3.5 PETCO2 (mmHg) Lowlander 28 ± 2 29 ± 3 0.027 0.626 0.788 28 ± 2 29 ± 3 0.248 0.874 0.447 Sherpa 31 ± 2 32 ± 3 30 ± 3 30 ± 3 SpO2 (%) Lowlander 87 ± 2 87 ± 2 0.482 0.542 0.838 88 ± 3 86 ± 2 0.374 0.321 0.059 Sherpa 86 ± 3 86 ± 3 86 ± 4 86 ± 3 HR (bpm) Lowlander 60 ± 7 65 ± 10 0.030 0.275 0.265 66 ± 12 61 ± 9 0.024 0.158 0.103 Sherpa 72 ± 9 72 ± 9 74 ± 9 74 ± 12 Serum iron (µmol·l-1) Lowlander 11.7 ± 7.5 2.4 ± 0.9 0.206 <0.001 0.243 10.8 ± 7.5 53.8 ± 22.6 0.606 <0.001 0.936 Sherpa 7.6 ± 4.7 2 ± 0 8.9 ± 7.1 51.2 ± 15.7 [11] Mean ± SD in 7 lowlanders and 8 Sherpa receiving DFO and 9 lowlanders and 12 Sherpa receiving iron, unless otherwise noted [n]. 136 Table 6.2. Absolute change from room air (PIO2=87mmHg) to hypoxia (PIO2=67mmHg) in lowlanders and Sherpa. Variable Group Pre-DFO Post-DFO Ancestry Time Inter Pre-Iron Post-Iron Ancestry Time Inter ∆QICA (ml·min-1) Lowlander 47 ± 26 [6] 47 ± 35 [6] 0.651 0.550 0.559 7 ± 59 [7] 63 ± 98 [8] 0.384 0.212 0.095 Sherpa 69 ± 61 [7] 49 ± 79 [6] 57 ± 36 [8] 52 ± 55 [8] ∆QVA (ml·min-1) Lowlander 15 ± 16 [5] 30 ± 18 [6] 0.778 0.823 0.047 20 ± 23 [8] 26 ± 24 0.738 0.816 0.123 Sherpa 27 ± 29 [6] 13 ± 15 [7] 20 ± 17 [10] 16 ± 23 [9] ∆gCBF (ml·min-1) Lowlander 138 ± 62 [5] 154 ± 91 [6] 0.608 0.309 0.179 73 ± 169 [6] 174 ± 222 [8] 0.777 0.502 0.085 Sherpa 226 ± 141 [6] 133 ± 184 [5] 157 ± 93 [7] 124 ± 85 [5] ∆CVC (ml·min-1mmHg-1) Lowlander 0.7 ± 0.69 [5] 1.91 ± 0.74 [6] 0.436 0.839 0.045 0.54 ± 1.65 [6] 1.55 ± 2.32 [8] 0.402 0.886 0.030 Sherpa 2.7 ± 1.94 [6] 1.39 ± 2.61 [5] 2.12 ± 1.53 [7] 0.96 ± 0.84 [5] ∆gCBF/∆SpO2 (ml·min-1%-1) Lowlander -12.4 ± 7.2 [5] -7 ± 4.2 [4] 0.895 0.071 0.575 -0.1 ± 25.7 [6] -16 ± 24.9 [7] 0.540 0.165 0.241 Sherpa -11.3 ± 7.1 [6] -7.4 ± 8.4 [5] -0.6 ± 18.3 [7] -6.1 ± 3.3 [4] ∆MAP (mmHg) Lowlander 8 ± 5 [6] -2 ± 10 [6]* 0.435 0.181 0.002 1 ± 6 2 ± 5 0.862 0.091 0.156 Sherpa -2 ± 6† 3 ± 8 -2 ± 8 6 ± 6 ∆PETCO2 (mmHg) Lowlander -3 ± 1 [6] -2 ± 2 [6] 0.260 0.973 0.213 -4 ± 3 -2 ± 1 0.176 0.016 0.673 Sherpa -1 ± 2 -2 ± 3 -2 ± 2 -1 ± 2 ∆VE (l·min-1) Lowlander 7 ± 3 [6] 5 ± 4 [6] 0.260 0.973 0.213 9 ± 8 5 ± 2 0.176 0.016 0.673 Sherpa 2 ± 6 5 ± 7 6 ± 5 3 ± 5 ∆SpO2 (%) Lowlander -11 ± 3 [6] -16 ± 12 [6] 0.137 0.634 0.441 -13 ± 5 -12 ± 8 0.049 0.838 0.706 Sherpa -19 ± 6 -18 ± 10 -18 ± 10 -19 ± 9 [11] Mean ± SD in 7 lowlanders and 8 Sherpa receiving DFO and 9 lowlanders and 12 Sherpa receiving iron, unless otherwise noted [n]. PETCO2 during hypoxia was predicted. 137 Table 6.3. During room air breathing (PIO2=96 mmHg) at pre- and post-infusion at 4,300 m in lowlanders and healthy Andeans. Variable Group Pre-Saline Post-Saline Ancestry Time Inter Pre-Iron Post-Iron Ancestry Time Inter QICA (ml·min-1) Lowlander 260 ± 122 265 ± 119 0.148 0.905 0.560 257 ± 53 [10] 234 ± 59 0.028 0.043 0.922 Andean 211 ± 45 197 ± 51 [11] 202 ± 34 188 ± 31 QVA (ml·min-1) Lowlander 57 ± 27 63 ± 34 0.408 0.202 0.408 74 ± 21 76 ± 24 [10] 0.014 0.606 0.939 Andean 51 ± 19 52 ± 19 53 ± 15 52 ± 18 gCBF (ml·min-1) Lowlander 635 ± 240 657 ± 221 0.090 0.761 0.389 661 ± 86 [10] 611 ± 100 [10] <0.001 0.043 0.992 Andean 525 ± 87 502 ± 113 [11] 511 ± 65 480 ± 54 CVC (ml·min-1mmHg-1) Lowlander 6.98 ± 3.06 7.27 ± 2.6 0.441 0.753 0.474 7.54 ± 0.66 [10] 6.8 ± 1.35 [10] 0.246 0.011 0.592 Andean 6.51 ± 0.92 6.27 ± 1.25 [11] 6.92 ± 1.23 6.2 ± 1.22 Systolic (mmHg) Lowlander 123 ± 12 124 ± 12 0.008 0.546 0.613 120 ± 10 120 ± 10 <0.001 0.415 0.469 Andean 109 ± 7 111 ± 16 103 ± 8 106 ± 11 Diastolic (mmHg) Lowlander 76 ± 7 75 ± 9 0.037 0.881 0.405 73 ± 8 76 ± 6 0.002 0.011 0.447 Andean 66 ± 9 69 ± 13 61 ± 7 66 ± 11 MAP (mmHg) Lowlander 92 ± 8 91 ± 10 0.015 0.738 0.438 89 ± 8 91 ± 7 <0.001 0.038 0.406 Andean 81 ± 8 83 ± 14 75 ± 6 79 ± 11 VE (l·min-1) Lowlander 11 ± 2 [9] 10 ± 2 [9] 0.498 0.506 0.606 8.2 ± 1.6 [10] 7.8 ± 1.6 [9] <0.001 0.811 0.313 Andean 12 ± 4 12 ± 4 12.6 ± 2.5 13.4 ± 4.3 PETCO2 (mmHg) Lowlander 30 ± 3 31 ± 3 0.357 0.756 0.653 33 ± 3 [8] 32 ± 2 [8] 0.326 0.264 0.792 Andean 32 ± 3 32 ± 3 31 ± 3 31 ± 4 SpO2 (%) Lowlander 89 ± 3 88 ± 4 0.694 0.596 0.596 88 ± 3 88 ± 3 [10] 0.453 0.110 0.218 Andean 88 ± 2 88 ± 2 88 ± 4 87 ± 3 HR (bpm) Lowlander 68 ± 11 62 ± 14 0.758 0.027 0.852 71 ± 14 66 ± 16 0.421 <0.001 0.295 Andean 66 ± 12 61 ± 9 69 ± 9 60 ± 8 Serum iron (µmol·l-1) Lowlander 13.7 ± 6.3 13.4 ± 6 0.238 0.759 0.641 15.6 ± 8.4 63.2 ± 7.7 [10] 0.355 <0.001 0.894 Andean 16.2 ± 5.6 17.3 ± 9.8 17.6 ± 5.1 [11] 65.7 ± 9.9 Mean ± SD in 11 lowlanders and 12 Andeans receiving saline or iron, unless otherwise noted [n]. 138 Table 6.4. Absolute change from room air (PIO2=96 mmHg) to hypoxia (PIO2=73 mmHg) in lowlanders and healthy Andeans. Variable Group Pre-Saline Post-Saline Ancestry Time Inter Pre-Iron Post-Iron Ancestry Time Inter ∆QICA (ml·min-1) Lowlander 5 ± 61 19 ± 78 [9] 0.699 0.687 0.508 19 ± 33 [10] 39 ± 30 [10] 0.717 0.299 0.589 Andean 7 ± 43 4 ± 30 [10] 18 ± 59 21 ± 57 [11] ∆QVA (ml·min-1) Lowlander 9 ± 12 2 ± 9 [10] 0.831 0.257 0.271 4 ± 13 [9] 21 ± 19 [9]* 0.090 0.387 0.014 Andean 6 ± 11 [11] 6 ± 9 [9] 7 ± 12 [10] -2 ± 17 [10]† ∆gCBF (ml·min-1) Lowlander 27 ± 121 52 ± 151 [8] 0.604 0.902 0.521 35 ± 92 [8] 116 ± 87 [9] 0.572 0.267 0.221 Andean 24 ± 103 [11] 15 ± 72 [8] 51 ± 141 [10] 39 ± 157 [9] ∆CVC (ml·min-1mmHg-1) Lowlander -0.14 ± 1.32 0.04 ± 1.4 [8] 0.835 0.905 0.63 0.1 ± 1.01 [8] 1.16 ± 1.05 [9] 0.956 0.136 0.475 Andean -0.07 ± 1.27 [11] -0.03 ± 0.61 [8] 0.44 ± 1.99 [10] 0.69 ± 2.09 [9] ∆gCBF/∆SpO2 (ml·min-1%-1) Lowlander 0.4 ± 9.1 [11] -10.8 ± 27 [8] 0.849 0.400 0.208 -1.5 ± 5.2 [8] -6.8 ± 5.7 [8] 0.863 0.019 0.593 Andean -5.4 ± 12.9 [11] -3.1 ± 9.7 [8] -1.5 ± 8.2 [10] -5 ± 8.7 [8] ∆Systolic (mmHg) Lowlander 3 ± 12 7 ± 7 0.761 0.185 0.625 -1 ± 9 2 ± 6 0.951 0.850 0.345 Andean 3 ± 8 5 ± 8 2 ± 11 -1 ± 6 ∆Diastolic (mmHg) Lowlander 6 ± 8 5 ± 6 0.461 0.701 0.919 4 ± 10 1 ± 7 0.614 0.038 0.661 Andean 4 ± 9 4 ± 5 6 ± 6 1 ± 6 ∆MAP (mmHg) Lowlander 5 ± 7 6 ± 6 0.495 0.760 0.888 3 ± 9 1 ± 6 0.718 0.145 0.468 Andean 4 ± 8 4 ± 5 5 ± 7 1 ± 6 ∆PETCO2 (mmHg) Lowlander -1 ± 1 [9] -1 ± 1 [9] 0.070 0.248 0.103 -1 ± 3 [7] -2 ± 0.4 [7] 0.036 0.472 0.422 Andean -1 ± 1 0.3 ± 1 0.1 ± 1 [11] 0.1 ± 2 ∆VE (l·min-1) Lowlander 2 ± 3 [9] 3 ± 2 [9] 0.070 0.248 0.103 3 ± 5 [10] 5 ± 2 [10] 0.015 0.350 0.252 Andean 1 ± 3 -1 ± 3 -0.2 ± 4 [11] -0.2 ± 5 ∆SpO2 (%) Lowlander -15 ± 7 -15 ± 7 0.886 0.772 0.944 -18 ± 3 -18 ± 4 [10] 0.238 0.966 0.749 Andean -15 ± 10 -15 ± 8 -15 ± 8 -15 ± 10 Mean ± SD in 11 lowlanders and 12 Andeans receiving saline or iron, unless otherwise noted [n]. PETCO2 during hypoxia was predicted. 139 6.4. Discussion Our primary finding was iron status may provide a novel, albeit subtle, influence on CBF that is potentially dependant on the severity and length-of-stay at high altitude. These data also highlight the comparable partial reliance of iron on cerebral blood flow responses between lowlanders and highlanders. A secondary preliminary observation was that that acute elevations in iron (and hence HIF downregulation) may lead to a preferential elevation in posterior cerebral blood flow in both lowlanders and Andean highlanders. The following discussion outlines the implications and experimental considerations that underpin these findings. 6.4.1. Cerebral blood flow, HIF expression, and iron status at high altitude During initial ascent and arrival to high altitude, it is well established that CBF increases in proportion to the reduction in arterial O2 content, in order to maintain cerebral O2 delivery. Over the following 1-2 weeks at a given altitude, CBF gradually normalizes to slightly above sea level values as erythropoiesis increases the O2 carry capacity of the blood and metabolic compensation occurs to partially correct the initial respiratory alkalosis [reviewed in: (Hoiland et al., 2018)]. As illustrated in Figure 6.3, it is noteworthy that the kinetics of cellular HIF expression also follows a similar trajectory during hypoxic exposure – in rodent models, HIF DNA binding activity reached 77% of maximal levels within one minute, and by 15 minutes HIF activity was detectable (Wang & Semenza, 1993a). In human endothelial cell culture models, HIF-1 and HIF-2 levels peaked after 4-6 hrs and 13 hours of hypoxic exposure, respectively (Bartoszewski et al., 2019). Interestingly, HIF-1 protein expression within the brain is more prevalent (versus HIF-2) (Wiesener et al., 2003) and HIF-1 sensitivity to hypoxia is greater than other tissues (Stroka et al., 2001) – for example, an FIO2 of 0.18 was sufficient to induce HIF-1a protein expression in the brain, whereas FIO2 of 0.06 was needed for hepatic and renal tissue cells (Stroka et al., 2001). In mouse cortical tissue, HIF-1 expression in the brain peaks at 6-12 hours and normalizes within ~3 weeks (Chávez et al., 2000). 140 Figure 6.3. Summary of global CBF during prolonged stay at high altitude. Altitudes of 5,050 m and 4,300 m refer to Studies 1 and 2, respectively. Cortical HIF-1a expression adapted from (Chávez et al., 2000) and CBF during early exposure to 5,050 in lowlanders adapted from (Hoiland et al., 2019). Since most of the lowlanders tested during Study 1 of the current study, were also included in the study by Hoiland and colleagues, the dashed line is included to illustrated the change in CBF across time. Iron – due to its constituent role in HIF stabilization, via PHD activity (Knowles et al., 2003) – can influence the coordination of HIF-mediated responses designed to maintain O2 delivery (Gassmann & Muckenthaler, 2015). However, because iron deficiency commonly occurs with sojourn to high altitude (Robach et al., 2007; Gassmann & Muckenthaler, 2015; Patrician et al., 2021b; Willie et al., 2021), the responsiveness at which downstream HIF responses are stimulated might be influenced by the individuals’ prevailing iron status. This notion is based on our finding that iron infusion attenuated gCBF in lowlanders and Andeans at 4,300 m, but not in lowlanders and Sherpa at 5,050 m. To reconcile this discrepancy, the impacts of iron manipulation on the cerebral vasculature, between Study 1 & 2, should be interpreted within the context of degree and duration at altitude where the iron status of participants between Studies 1 & 2 were not identical. In Study 1 (5,050 m), 65% of lowlanders and 9% of Sherpa had ferritin levels of <15 ng·ml-1, while in Study 2, (4,300 m) only 14% of lowlanders and 4% of Andeans had ferritin levels <15 ng·ml-1. 141 While we would postulate that a greater prevalence of iron depletion at pre-infusion, would enhance the potency of iron infusion on altering cerebral vascular function, our findings that 1) iron infusion did not attenuate gCBF at 5,050 m, and 2) DFO did not markedly increase gCBF at 5,050 m. Together these data indicate that at least for the brain at this high altitude, the characteristic rise in arterial O2 content (Howe et al., 2019) plays a likely more potent role in CBF regulation since HIF expression would be expected to be declining over this timeframe (Chávez et al., 2000). 6.4.2. Exaggerated vertebral artery blood flow during hypoxia Since the VA provides blood flow to the posterior region of the brain, including the brainstem – a key site of cardiorespiratory control – some studies have shown that posterior regions of the brain demonstrate a preferential blood flow distribution during hypoxia (Binks et al., 2008; Willie et al., 2012; Subudhi et al., 2014). Therefore, our finding of greater ∆QVA distribution (i.e. the elevation in VA flow from room air to exaggerated hypoxia) with iron in lowlanders at 4,300 m, suggests HIF activity, may aid in the regulation of flow to these highly homeostatic functioning regions of the brain. 6.4.3. Role of high-altitude ancestry While high altitude Shera and Andeans display adaptive characteristics to hypoxia, including positive selection for HIF pathway candidate genes (Bigham et al., 2010; Foll et al., 2014), there appears to be little evidence from the current study that iron manipulation differentially impacts cerebrovascular reactivity to hypoxia between partially acclimatized lowlanders and healthy highlanders. While this contrasts with our hypothesis, we acknowledge that we were only able to assess the acute (i.e., 4 hours) impact of HIF up-/down-regulation (via iron manipulation) on CBF. Since many of the downstream constituents of HIF are only evident after days-weeks (e.g. changes in cerebral microvascular density and hematocrit (Benderro & LaManna, 2014), neurovascular angiogenesis and pericyte proliferation (Urrutia et al., 2020)], the potential for cerebrovascular differences between high altitude residents and lowlanders to emerge over time warrants further investigation. 142 6.5. Conclusion In both healthy lowlanders and highlanders at high altitude, prevailing iron status appears to contribute to the variability in cerebral hypoxic reactivity. However, acute manipulation of iron status only minimally influences cerebral blood flow and function, that is potentially dependant on the severity and length of high-altitude exposure. Given the variable and progressive depletion of iron stores at high altitude, broadening the scope of iron metabolic assessments to >24 hours post-infusion, may shed insightful into potential implications between the reversal/exaggeration of iron deficiency cerebral blood flow control during hypoxia. 143 Chapter 7. Conclusion 7.1. Overview of research objectives and study findings The two overarching objectives of the current thesis were to 1) determine the role of iron status manipulation on pulmonary, peripheral and cerebral responses to high-altitude; 2) and examine if high-altitude populations (i.e. Sherpa and Andeans), with ancestral adaptation to their hypoxic environment, exhibit iron-regulatory enhancements. In Study 1, lowlanders and partially de-acclimatized Sherpa ascended over 9-10 days from 1400 m to 5050 m, and then resided at 5050 m for ~3 weeks. The objective of the study was two-fold: 1) track and assess iron status, and examine the relationship to pulmonary pressure, during ascent to high altitude, and 2) whether manipulating iron levels at 5050 m, via infusion of iron or a chelator, would divergently alter pulmonary pressure during exaggerated hypoxia and exercise. Aligning with previous studies in lowlanders (Mairbäurl et al., 1990; Bonfichi et al., 2000; Robach et al., 2004; Robach et al., 2007; Smith et al., 2009; Piperno et al., 2011; Talbot et al., 2012; Goetze et al., 2013; Ryan et al., 2014; Altamura et al., 2015; Holdsworth et al., 2020), iron status (i.e. serum iron levels and Tf sat) decreased during ascent in lowlanders and Sherpa (albeit to lesser degree in Sherpa). Interestingly, during ascent Sherpa demonstrated a greater recruitment capacity for iron compared to lowlanders (based on the rate of change in hepcidin and indices of body iron stores (via sTfR and ferritin) across ascent; termed the iron recruitment index). Perhaps because iron status in lowlanders and Sherpa were close-to or within range of iron-deficiency, chelation of iron did not exaggerate PASP during exercise or exaggerated hypoxia. Iron infusion reduced PASP and PVR at baseline, but did not limit the rise of either to exaggerated hypoxia or exercise. In Study 2, 3 & 4, lowlanders ascended from sea level to 4300 m over 8 hours, where they then resided for ~3 weeks. Only Andeans resident of 4300 m, who were not considered to have CMS, were recruited. At baseline, Andeans had similar iron metrics as lowlanders, except Andeans had elevated serum ferritin. While, ferritin is used clinically an indicator of iron status (low ferritin values indicating iron deficiency), it also used as an indicator of pro-inflammatory status. In contrast to the saline infusion, the infusion of iron attenuated pulmonary pressure (Study 2) and increased peripheral blood flow and microvascular function (Study 3) in both lowlanders and Andeans at 4300 m. 144 In Study 4, which consisted of the analyses of all participants (n=83), across both expeditions to Nepal and Peru, provided an opportunity for an exploratory de novo focus on the brain. The influence of iron infusion on CBF at high altitude was subtle, and potentially dependant on the severity and length-of-stay at high altitude. A novel finding was that baseline iron levels in both lowlanders and highlanders, contributed to statistical variability in hypoxic cerebral vasodilation. 7.2. Overall interpretation – Is iron supplementation useful? With the constituent role played by iron in the O2 sensing pathway of HIF, the consequences of low iron levels on HIF activation is fairly well established – iron chelation induces HIF-1 expression (Wang & Semenza, 1993b; Salceda et al., 1997), extends HIF-1𝛼 half-life (Huang et al., 1998), and increases propagation of downstream HIF-related processes (Semenza, 2004). Under normal conditions, IRP and HIF-2𝛼 expression during hypoxia should augment dietary iron uptake by enterocytes (Shah et al., 2009; Anderson et al., 2011). Thus, more dietary iron will be absorbed. However, given 1) gastrointestinal distress and reductions in appetite usually accompany ascent to high altitude (which contributes to weight loss and reductions in dietary iron intake (Rose et al., 1988; Westerterp, 2001), and 2) in less accessible regions, there is often a shift in reliance to nonheme iron sources – means that the replenishment of iron stores occurs at a much slower rate (Figure 2.4). Although by no means extensively studied, ensuring an iron replete status prior to ascent has reportedly proffered some benefits in limited AMS severity (Talbot et al., 2011) and improving SpO2 and indices of right heart function (Holdsworth et al., 2020) that seem independent of any differences in Hb or Hct. While headache scores were not reported in Talbot and colleagues (Talbot et al., 2011), our finding of iron status contributing to the magnitude of hypoxic cerebral vasodilation may hold particular relevance, as there is no available data on iron status in individuals with high altitude cerebral edema. However, although interesting in view of the postulated link between high altitude headache and high altitude cerebral edema (Wilson & Imray, 2016; Turner et al., 2021), many well-controlled studies have reported no relationship between changes in CBF and incidence of high altitude headache or AMS [reviewed in: (Roach & Hackett, 2001; Ainslie & Subudhi, 2014)]. 145 Aligning with the iron infusion study in lowlanders by Smith and colleagues (2009) – who showed a 6 mmHg drop in PASP within 3 days of arrival at high altitude – data from this thesis (Chapter 3 and 4) extend the efficacy of iron infusion to reduce PASP by ~3 mmHg at ~1 week and by ~4 mmHg at ~2 weeks at 4300 and 5050 m, respectively. Similarly, in Chapter 5 it was also demonstrated that iron infusion can elevate brachial blood flow and microvascular function at 4300 m. Therefore, the findings of this thesis bring a new evidence and perspective to the early report by Wanner and Jasinski (1953), that low iron stores can compromise our ability to acclimatize to high altitude. 146 Figure 7.1. Summary of the role of iron (Fe) infusion on pulmonary, peripheral and cerebral responses at high-altitude. There is an inverse decline in iron status with ascent to (and prolonged stay at) high altitude. The effect of 200 mg of iron (III)-hydroxide sucrose, is illustrated in red. Given the shared main effect of iron infusion across lowlander and highlander groups, pooled data is presented, as percent change from baseline, with significant measures (*P<0.05). The putative role of iron on pulmonary, peripheral and cerebral HIF regulation is conceptually included, with green line denoting activation and purple line denoting inhibition. CVC, cerebrovascular conductance; Q, blood flow (global cerebral blood flow, cardiac output, and brachial blood flow); PVR, peripheral vascular conductance; FMD, flow mediated dilation; RH, reactive hyperemia; VE, ventilation; MAP, mean arterial pressure; PETCO2, partial pressure end-tidal carbon dioxide; PIO2, partial pressure inspired oxygen; SpO2, peripheral arterial oxygen saturation; PASP, pulmonary artery systolic pressure; PVR, pulmonary vascular resistance; ↓, decrease; ↑, increase; NS, not significant. 147 7.3. Overall interpretation – Is there a facet of iron metabolism that can be considered to be an attribute of successful high-altitude adaptation? To date, the only iron manipulation study performed in high altitude highlanders, was in Andeans at 4300 m that display maladaptive characteristics of high altitude exposure – CMS (Smith et al., 2009). In that study, venesection-induced iron deficiency worsened PASP by ~10 mmHg (to 37 mmHg), thus highlighting the negative consequence of iron deficiency at high altitude. Certainly, there are large timeline and methodological differences between the study by Smith and colleagues (2009) and Chapter 3 (PASP tracked for ~2 weeks post-venesection, versus 4 hours post-chelation, respectively). Therefore, it is difficult to directly compare the exaggerated PASP response in CMS to venesection-induced iron deficiency (Smith et al., 2009), with Study 3 that showed chelation did not influence PASP in Sherpa. Conversely, what is more reconcilable, is that there does appear to be divergent responses between healthy adapted Andeans (who demonstrated reductions in PASP with iron infusion) and Andeans with CMS (who did not demonstrated reductions in PASP with iron infusion). It is likely that the observation of iron not influencing PASP in Andeans with CMS, is consistent with the adverse remodelling of the pulmonary vasculature in Andeans with CMS (Penaloza & Arias-Stella, 2007) and an otherwise ‘healthy’ pulmonary circulation in healthy Andeans. The potential for iron regulatory adaptations in high altitude highlanders nevertheless still exists. Bigham and Lee (2014) identified a number of unique genes that relate to HIF/hypoxia with genetic signatures specific to Andean and Tibetan populations that may play a role in iron regulation. Of the 40 Andean- and 39 Tibetan-nominated genes, there were: 2 genes specific to Andeans (TF, EGLN2), 7 genes specific to Tibetans (EPAS1, EGLN3, EPO, FLT, HBB, HIF1A, HMOX), and 6 genes that are conserved across both populations (EGLN1, IL1B, IL6, MDM2, TNF, VEGFA), that have a mechanistic role in the regulation if iron (Hentze et al., 2010; Chepelev & Willmore, 2011; Muckenthaler et al., 2017; Hirota, 2019). However, how the expression specifics of these transcription factors may relate to phenotypical outcomes such as pulmonary pressure or iron metabolic differences (e.g. iron recruitment capacity in Figure 3.3), remains to established. Likewise, whether these observations are broadly consistent with a genetic mechanism(s) and thus attributable to an ancestral lineage of high-altitude exposure, remains to be explored. 148 7.3.1. Potentially confounding influence of heavy metal pollutants at high altitude The anthropogeographic extremes of Himalayan highland villages to Peruvian mining cities should also be acknowledged. Most notable is the contrasting role that the environment plays as a source of either dietary iron or pollutants to potentially occlude iron uptake. For example, it has been demonstrated that more than half of the soil sample sites evaluated in Cerro de Pasco, had Pb (lead) levels exceeding 1200mg/kg (Van Geen et al., 2012), which is ~10-fold higher the health recommendations in Canada [140 mg/kg(Health Canada, 2013)]. Recently, and not surprisingly, whole blood Pb concentrations in residents of Cerro de Pasco (with Hb 10-25 g/dl) were >2-fold greater than Pb concentrations in sea-level residents of Lima [4.751.53 ug/dl, range: 1.5-9.8 ug/dl versus 2.030.62 ug/dl, range: 1.0-4.6 ug/dl, respectively (Childebayeva et al., 2021)]. While Pb toxicity may not be prevalent in our cohort [due to the lack of iron deficiency and anemia (reviewed in: (Kwong et al., 2004))], consequence(s) of Pb exposure on upstream inflammatory factors and ferroportin activity, have been demonstrated in animal and cell-preparation models. For example, in mice, Pb exposure increased the expression of Nrf2 [nuclear factor (erythroid-derived 2)-like 2], nuclear factor-kappaB (NF-B) and inflammatory cytokines (Hosseinirad et al., 2021). NrF2 is a transcription factor, which when expressed, has been linked to increased activity during erythrophagocytosis (Harada et al., 2011). Since red blood cells are the primary sink of iron, and ferroportin is an iron exporter, increased ferroportin activation could culminate in an enhanced efflux of iron from senescent red blood cells and mimic a state of iron overload (i.e. a rise in serum ferritin). In this thesis, serum iron in healthy Andeans was not different from lowlanders, so the rise in ferritin may stem from more inflammatory-related processes. However, because a ferroportin-mediated release of iron can vary across different macrophage types (Recalcati et al., 2010), the influence of Pb on ferroportin activity could still be occurring in some circumstances, and should be considered. NF-B is a transcription factor that plays a regulatory role in pro-inflammatory function, including TNF𝛼, (Baldwin, 2001), which is one of the pro-inflammatory cytokines linked to ferritin regulation via transcription transduction (Torti & Torti, 2002). Interestingly, in an endothelial cell culture model, NF-B activation has been implicated in the pro-inflammatory state of Andeans with excessive erythrocytosis (Brewster et al., 2021). Pb exposure can also alter endocrine function, by blunting many the hormones derived from the 149 hypothalamic-pituitary axis (Doumouchtsis et al., 2009), and could make the hypothalamic-pituitary axis less capable of mediating inflammatory stimuli (Chrousos, 1995) – which are elevated following Pb exposure (Hosseinirad et al., 2021). Further work on this topic is warranted and it remains to be determined whether Pb exposure could extend beyond iron deficiency/anemia and potentially contribute to the cascade of downstream consequences potentially accounting for the elevated ferritin levels in Andeans. The city of Cerro de Pasco, also starkly contrasts to those of small towns/villages in the Himalayan highlands, in terms of lifestyle (e.g. diet, physical activity) and level of industrialization (e.g. drive-in and mining versus hike-in and agriculture/tourism, respectively). Nevertheless, it has been recently demonstrated that the Khumbu valley is not immune to the accumulation of pollutants, despite its less industrious environment. For example, the chemical composition of stream water and snow from areas ranging from Everest Basecamp to Pheriche – showed stream water quality degradation, with detectible heavy metal concentrations (including Pb), that in some locations exceeds safety level guidelines by World Health Organization (Clifford et al., 2021). Ultimately, environmental heavy metal pollution has the potential to confound physiological responses, and thus should be explored in future investigations. 7.4. Field research at high altitude and the challenges As eloquently detailed in John West’s textbook the “High Life” (West, 2013), high-altitude research has a rich history. While field research continues to inspire topics of high-altitude physiology and medicine, there are restrictions to time, logistics and personnel that inevitably shape and limit the methodological design. 7.4.1. Temporal assessments of physiological outcomes A notable hurdle associated with iron manipulation studies, is that an intravenous infusion of iron affects systemic iron levels and the pulmonary vascular response to acute hypoxia for at least 40 days (Bart et al., 2016). Therefore, for both Nepal and Peru expeditions, data collection for the studies in this thesis were relegated to the latter portion of the expedition, to accommodate the other studies being run. Certainly, simply assessing prevailing iron status and relating it to phenotypical responses could provide valuable information, however performing an interventional 150 iron manipulation protocol provides more mechanistic insight. This comes at the cost of a lengthy protocol duration (DFO requires a 4-hour infusion). Ultimately, such constraints did not proffer the opportunity to extend physiological measures beyond ~4 hours post-infusion (e.g. to ~24+ hours). An aspect for future research, as initial explored in Holdsworth and colleagues (2020), is to allow new iron status to fully unfold to propagate downstream HIF-related processes. These time course studies, especially when combined with tissue sampling of HIF-related metabolic pathways, may shed additional light on iron-mediated modulation at high altitude, and potentially expose novel highlander-unique differences in iron and HIF regulation. 7.4.2. Ethiopia Originally, plans were made for a third expedition in 2019, on this occasion to the Simien Mountains, in the northern highlands of Ethiopia. An iron status and manipulation protocol in Ethiopian highlanders was set and ethical approval had been received. However, the anticipated November 2019 departure came to an abrupt halt when shifting government approvals forced the expedition to delay 6 months. Unfortunately, along with COVID, the political climate then destabilized and the trip eventually was suspended. The objective of this trip was to provide insight into the pulmonary circulation and iron metabolism in Ethiopian highlanders and to ultimately enable comparisons with the Andean and Sherpa populations. The work is planned for whenever it is possible to travel and work in Ethiopia again. 7.5. Future directions 7.5.1. Role of sex in iron metabolism at high altitude Given the greater prevalence of iron deficiency in females (Pasricha et al., 2021), it is probably not too surprising that iron levels (via serum ferritin) were lower in females, compared to males, before ascent to- and after 16 days at 5260 m (Ryan et al., 2014). However, as highlighted in Figure 2.4: across all 12 studies included, only 14% of participants were female and only study (Ryan et al., 2014) aggregated their results by sex. The important and physiologically relevant discussion surrounding biological sex as an significant factor in physiology has deservedly increased (Arnegard et al., 2020). For example, in a recent Journal of Applied Physiology point-counterpoint commentary, on whether menstrual cycle phase 151 should (or should not) be controlled for when performing studies of vascular control, 26 commentaries were published in the series (Giersch et al., 2020). One of the commentaries (by Tremblay and colleagues) postulates that one factor contributing to the lack of contraception and menstrual considerations (and exclusion of female participants) at high altitude, is primarily because of the inflexible time/logistical constraints, inherent of field research. For example, for each of the latest high altitude expeditions to Nepal and Peru, there were >15 studies vying for ~2-4 weeks of data collection (Willie et al., 2018; Tymko et al., 2020a); therefore, controlling contraception and menstrual cycle becomes highly difficult, if not impossible, especially since high altitude can also disrupt normal menstruation (Keyes, 2015). However, due to contrasting hormonal profile to males, there may well be a differential influence of female sex hormones at altitude vs. sea level. With respect to exercise performance at altitude, it is well established that with ascent to high altitude 1) sub-maximal and peak heart rates increase and decrease, respectively, and 2) O2 uptake declines (Pugh, 1969). At sea level, evidence suggests that a state of iron depletion (often investigated in females due to higher prevalence of reduced iron status) can impair exercise performance [in healthy humans (Woodson et al., 1978), rat models (Perkkiö et al., 1985), and patients with chronic heart failure (Jankowska et al., 2011) and pulmonary hypertension (Ruiter et al., 2011)] and in more severe stages of iron deficiency, can restrain the improvements of aerobic training on maximal O2 uptake (Hinton et al., 2000; Brownlie et al., 2002). Consequently, over the course of 4-week altitude training camp, utilizing a model of live high-train low (live at 2500 m and train at 1250-1300 m), in both males and females with low iron status, there were no changes in maximal O2 uptake and a less responsive erythropoietic response, which contrasted to the favourable responses in iron replete athletes (Okazaki et al., 2019). While many altitude training studies are inclusive of iron metrics, and often include both sexes [albeit without aggregation (Robach et al., 2006; Wehrlin et al., 2006; Govus et al., 2017; Heikura et al., 2018; Hall et al., 2019)], the sheer diversity of exercise modalities and intensities, diets, measurements and hypoxic protocols make exploring sex-specific questions complex. Certainly, the consensus of the efficacy of iron therapy in iron-deficient athletes for exercise performance remains mixed (Rubeor et al., 2018; Kuwabara et al., 2022). Therefore, as highlighted in a recent review, better understanding the relevance of iron and sex status, as it pertains to exercise performance is certainly warranted 152 (Ryan et al., 2021). Moreover, the implications of this thesis are particularly relevant to females – therefore, exploring iron deficiency in females during ascent to- and upon arrival at high altitude is a logical gap that should be explored. 7.5.2. Microgravity and space While space has always intrigued humans, human physiology has not evolved to manage the physiological consequences of weightlessness. From body fluid shifts that reduce blood volume, transient motion sickness, muscle atrophy, bone demineralization, immune dysregulation and psychosocial effects, space exerts a powerful influence that has required on-board strategies to minimize (with some success) these impacts (Thirsk et al., 2009; Williams et al., 2009; Demontis et al., 2017). The occurrence of anemia has also been a notable phenomenon during space travel. Since the early American multi-day space flights (1965-1974), red blood cell mass has been reported to decrease by 10±5 % across thirteen missions, ranging in duration from 4-84 days [reviewed in: (Tavassoli, 1982)]. More recently, following 157 days aboard the international space station, Hb decreased by 9% (14.3 g/dl at pre-departure to 13.0 g/dl after 4 days back on Earth) – which was attributed to hemolysis (Trudel et al., 2022). Interestingly, the reduction of Hb (and re-allocation of iron) during space flight seems to mirror the reduction in Hb concentration during de-acclimatization from high altitude (Ryan et al., 2014). Therefore, investigating strategies aimed at slowing the assimilation of red blood cell mass during high altitude de-acclimatization, may provide useful insight into prophylactic iron interventions for prolonged space flight. Since red blood cells are produced in bone marrow, it would be interesting to establish whether there is a bone marrow shift in astronauts, and if so, 1) does duration in space travel correlate to red-yellow metamorphosis, and 2) if so, does such a transition hinder the erythropoietic response. There exists some data in rats, that spent 20 days aboard the Cosmos space shuttle that suggests this does occur (Ahlers et al., 1980). However, at least in available human data (n=4 astronauts) following 17 days in space, there were no marked changes in functional cellular or fat fractions shortly upon arrival (LeBlanc et al., 1999). In contrast, over the next 5-6 months, there appeared to be changes in T2 (a measure of signal decay that results from the loss of phase coherence of the hydrogen nuclei and relates to osteoblastic activity) after the flight (LeBlanc et al., 1999). However, as the authors state, longer duration space flights may give rise to different results. 153 Further research should investigate whether certain strategies can be employed to overcome space anemia – for example, by stimulating EPO. With the recent technological advancements by private enterprises (e.g. SpaceX, Virgin Galactic, and Blue Origin) that wish to make space travel more accessible, highlights the importance of understanding the decline in iron status with prolonged microgravity – especially if humans begin traveling to Mars. 7.6. Conclusion Iron is an essential constituent of life. While there exists many regulatory pathways to maintain iron homeostasis, iron deficiency is highly prevalent across the world. Sojourn to high altitude is characterized by an increase in the demand for iron, without a concomitant replenishment of iron stores, that results in a decline in iron bioavailability with time at altitude. This thesis provides new evidence that supports iron status influences blood flow regulation in the pulmonary, peripheral and cerebral circulations during exposure to high-altitude. Ultimately, highlighting the value of being iron replete at high altitude. 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Thesis/Dissertation
eng
Interdisciplinary Studies
Vancouver : University of British Columbia Library
Attribution-NonCommercial-ShareAlike 4.0 International
Graduate
Ainslie, Philip N., 1974-
Physiological aspects of iron status and manipulation in lowlander and adapted highlanders at high altitude
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