Health and Social Development, Faculty of (Okanagan)
Health and Exercise Sciences, School of (Okanagan)
DSpace
UBCO
Steele, Andrew
Doctor of Philosophy - PhD
University of British Columbia
Blood rheology properties-including whole blood viscosity, plasma viscosity, and red blood cell (RBC) deformability and aggregation—are key regulators of blood flow and vascular function. Blood viscosity increases blood flow resistance but also contributes to shear stress, a key determinant of vascular resistance and diameter. RBC deformability and aggregation underlie the shear-thinning behavior of blood, allowing blood viscosity to adjust with changing shear rates. However, the extent to which blood rheology regulates blood flow in humans remains poorly understood. This dissertation aimed to examine how acute elevations in blood viscosity (Study 1) and RBC deformability (Studies 2 and 3) influence vascular perfusion and reactivity under distinct physiological and environmental conditions, including plasmapheresis, plasma contraction, exercise, and chronic hypoxia. In separate trials, study 1 induced hemoconcentration through plasma donation (via plasmapheresis) and diuresis (via oral furosemide) to assess its effects on brachial artery blood flow and reactive hyperemia flow-mediated dilation (RH-FMD). While increased blood viscosity did not alter brachial artery blood flow or RH-FMD, plasma donation and not diuresis enhanced microvascular function, as assessed by reactive hyperemia. Study 2 examined the impact of handgrip exercise and phosphodiesterase-5 inhibition (PDE5) with sildenafil citrate on RBC deformability, brachial artery blood flow, and cerebral blood flow (CBF). Exercise increased RBC deformability, and PDE5-i prolonged this elevated deformation; however, these changes did not affect basal CBF, brachial artery blood flow, or handgrip-induced exercise hyperemia. Study 3 evaluated the effects of pentoxifylline, a non-specific PDE inhibitor, on blood rheology, cardiac output, and CBF at high altitude. Chronic hypoxia increased blood viscosity and reduced RBC deformability, but pentoxifylline had no significant effect on blood rheology, CBF or cardiac output. In summary, while physiological and environmental stressors altered blood rheology, these changes did not significantly influence blood flow or vascular reactivity in healthy individuals.
Blood rheology on blood flow during plasmapheresis, plasma contraction, exercise, and chronic hypoxia in humans. by Andrew Steele B.Sc., University of Alberta, 2017 M.Sc., University of Alberta, 2019 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy in THE COLLEGE OF GRADUATE STUDIES (Kinesiology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) August 2025 © Andrew Steele, 2025 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies, the dissertation entitled: Blood rheology on blood flow during plasmapheresis, plasma contraction, exercise, and chronic hypoxia in humans. submitted by Andrew Steele in partial fulfilment of the requirements for the degree of Doctor of Philosophy Examining Committee: Dr. Philip Ainslie – School of Health and Exercise Sciences Supervisor Dr. Glen Foster – School of Health and Exercise Sciences Supervisory Committee Member Dr. Ryan Hoiland – Department of Cellular and Physiological Sciences Supervisory Committee Member Dr. Christopher West – Department of Cellular and Physiological Sciences University Examiner Dr. Michael Simmonds – Griffith University External Examiner iii Abstract Blood rheology properties-including whole blood viscosity, plasma viscosity, and red blood cell (RBC) deformability and aggregation—are key regulators of blood flow and vascular function. Blood viscosity increases blood flow resistance but also contributes to shear stress, a key determinant of vascular resistance and diameter. RBC deformability and aggregation underlie the shear-thinning behavior of blood, allowing blood viscosity to adjust with changing shear rates. However, the extent to which blood rheology regulates blood flow in humans remains poorly understood. This dissertation aimed to examine how acute elevations in blood viscosity (Study 1) and RBC deformability (Studies 2 and 3) influence vascular perfusion and reactivity under distinct physiological and environmental conditions, including plasmapheresis, plasma contraction, exercise, and chronic hypoxia. In separate trials, study 1 induced hemoconcentration through plasma donation (via plasmapheresis) and diuresis (via oral furosemide) to assess its effects on brachial artery blood flow and reactive hyperemia flow-mediated dilation (RH-FMD). While increased blood viscosity did not alter brachial artery blood flow or RH-FMD, plasma donation and not diuresis enhanced microvascular function, as assessed by reactive hyperemia. Study 2 examined the impact of handgrip exercise and phosphodiesterase-5 inhibition (PDE5) with sildenafil citrate on RBC deformability, brachial artery blood flow, and cerebral blood flow (CBF). Exercise increased RBC deformability, and PDE5-i prolonged this elevated deformation; however, these changes did not affect basal CBF, brachial artery blood flow, or handgrip-induced exercise hyperemia. Study 3 evaluated the effects of pentoxifylline, a non-specific PDE inhibitor, on blood rheology, cardiac output, and CBF at high altitude. Chronic hypoxia increased blood viscosity and reduced RBC deformability, but pentoxifylline had no significant effect on blood rheology, CBF or cardiac output. In summary, while physiological and environmental stressors altered blood rheology, these changes did not significantly influence blood flow or vascular reactivity in healthy individuals. iv Lay Summary Blood flow through the body is influenced not only by the heart and blood vessels, but also by the physical properties of the blood itself. Important factors include blood viscosity, which describes how thick the blood is, and RBC deformability, which refers to the ability of RBCs to change shape and move through small vessels. This dissertation explored how changes in these properties affect blood flow regulation during physiological stressors such as plasma donation, dehydration, exercise, and high-altitude exposure. Across three experimental studies, it was examined tested whether increasing blood viscosity or enhancing RBC deformability would affect blood flow regulation in healthy individuals. Study 1 increased blood viscosity, while studies 2 and 3 elevated RBC deformability, these changes had little impact on blood flow or vascular reactivity. These findings suggest that in healthy individuals, the vascular system compensates well for short-term changes in blood rheology. v Preface The research included in this dissertation was conducted at the Okanagan campus of The University of British Columbia, located on the traditional, ancestral, and unceded territory of the Syilx Okanagan Nation, and during a high-altitude research expedition to the White Mountain Research Center (Barcroft Field Station) in Bishop, California, situated on the traditional lands of the Nüümü (Northern Paiute) and Newe (Western Shoshone) peoples. The University of British Columbia Clinical Research Ethics Board (CREB) approved all the studies included in this dissertation (CREB ID#s: H20-03708, H24-03340, H21-02780, and H22-01091). No generative artificial intelligence was used in any aspect of this dissertation. Chapter 1. Written by Andrew Steele, and edited by Prof. Philip Ainslie. Chapter 2. Written by Andrew Steele, and edited by Prof. Philip Ainslie. Chapter 3. In preparation for submission to the Journal of Physiology Chapter 4. In preparation for submission to the Clinical Hemorheology and Microcirculation Chapter 5. Accepted in American Journal of Physiology-Heart and Circulatory Physiology at the time of dissertation submission. Author contributions: A.R.S., C.A.H., T.D.G., K.F., A.M.W., H.G.C., L.M.B., J.D., J.A.M., P.S., J.D.A., M.S., P.N.A., and J.C.T. were involved in data collection. A.R.S., T.D.G., P.N.A., and J.C.T. were involved in data analyses, subsequent interpretation, and drafting the article. All authors reviewed and accepted the final version of this article. Chapter 6. Written by Andrew Steele, and edited by Prof. Philip Ainslie. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ ix List of Figures .................................................................................................................................x List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiv Chapter 1: Introduction ................................................................................................................1 1.1 Thesis objectives ............................................................................................................. 2 1.2 Individual study aims and hypotheses: ........................................................................... 2 1.2.1 Study 1 - The impact of plasmapheresis on blood rheology, and peripheral vascular function ..................................................................................................................... 3 1.2.2 Study 2 - Exercise-induced enhancements in RBC deformability are prolonged by oral phosphodiesterase-5 inhibition ........................................................................................ 3 1.2.3 Study 3 - Hemorheological, cardiorespiratory, and cerebrovascular effects of pentoxifylline following acclimatization to 3,800 m .............................................................. 3 Chapter 2: Literature Review .......................................................................................................5 2.1 Origins of blood rheology ............................................................................................... 5 2.2 Constituents of blood .................................................................................................... 10 2.2.1 Plasma composition and function ......................................................................... 13 2.2.2 Red blood cell structure and function ................................................................... 13 2.3 Rheological determinants of blood flow ....................................................................... 16 2.3.1 Plasma viscosity and microvascular dynamics ..................................................... 17 2.3.2 Red blood cell deformability ................................................................................ 17 2.3.2.1 Influence of red blood cell deformability on blood flow .................................. 18 2.3.2.2 Vasodilator signaling mediated by red blood cell deformability ...................... 20 2.3.2.3 Mechanisms of red blood cell deformability .................................................... 21 2.3.3 Influence of red blood cell aggregation in blood flow dynamics ......................... 22 vii 2.3.4 Influence of shear rate on apparent viscosity ........................................................ 25 2.3.5 Hematocrit and blood viscosity ............................................................................ 26 2.3.6 Vessel diameter-blood rheology interactions........................................................ 28 2.4 Blood flow regulation: shear stress, blood viscosity and oxygen content .................... 29 2.4.1 Shear stress-mediated control of blood flow ........................................................ 29 2.4.2 Shear stress patterns and magnitude in flow-mediated dilation............................ 31 2.4.3 Blood viscosity and vascular resistance ................................................................ 32 2.4.4 Blood viscosity as a determinant of flow-mediated dilation ................................ 34 2.4.5 Oxygen demand the master regulator ................................................................... 34 2.5 Blood rheology in applied and clinical contexts ........................................................... 36 2.5.1 High altitude exposure and blood rheology .......................................................... 36 2.5.1.1 The relationship between blood rheology and blood flow at high altitude ....... 37 2.5.2 Plasmapheresis-induced changes in blood rheology ............................................. 37 2.5.2.1 Effects of plasmapheresis on blood flow .......................................................... 42 2.5.3 Blood rheology in cardiovascular disease and health ........................................... 43 2.5.3.1 Blood rheology alterations in disease ............................................................... 43 2.5.3.2 Blood rheology in endurance trained athletes ................................................... 45 Chapter 3: Study 1 - Effects of plasmapheresis on blood rheology and peripheral vascular function. ........................................................................................................................................46 Chapter 4: Study 2 - Exercise-induced enhancements in RBC deformability are prolonged by oral phosphodiesterase-5 inhibition. .....................................................................................75 Chapter 5: Study 3 - Hemorheological, cardiorespiratory, and cerebrovascular effects of pentoxifylline following acclimatization to 3,800 m ................................................................100 Chapter 6: Conclusion ...............................................................................................................128 6.1 Main findings: ............................................................................................................. 128 6.1.1 Study 1 - Effects of plasmapheresis on blood rheology and peripheral vascular function 128 6.1.2 Study 2 – Exercise-induced enhancements in RBC deformability are prolonged by oral phosphodiesterase-5 inhibition. ................................................................................... 128 viii 6.1.3 Study 3 - Hemorheological, cardiorespiratory, and cerebrovascular effects of pentoxifylline following acclimatization to 3,800 m .......................................................... 128 6.2 Contributions to the field ............................................................................................ 129 6.3 Integrated summary .................................................................................................... 131 6.4 Strengths of the dissertation: ....................................................................................... 132 6.5 Weakness .................................................................................................................... 133 6.6 Future directions ......................................................................................................... 136 6.6.1 Anticoagulants on vascular function and longitudinal influence of plasma donation on cardiovascular health ...................................................................................... 136 6.6.2 Independent effect of PDE5-i on RBC deformability......................................... 138 6.6.3 Therapeutic potential of PDE5-i in diseases with impaired RBC deformability 138 6.6.4 Pentoxifylline in high altitude related disorders ................................................. 139 6.7 Conclusion .................................................................................................................. 139 References ...................................................................................................................................140 ix List of Tables Table 3-1. Hematology parameters, whole blood viscosity, and plasma viscosity at discrete shear rates before, immediately following and 24 hours following plasma donation. ........................... 57 Table 3-2. Hematology parameters, whole blood viscosity, and plasma viscosity at discrete shear rates before, immediately following and 24 hours following plasma donation. ........................... 58 Table 3-3. Whole blood viscosity, RBC deformation, hemodynamics, brachial artery reactive hyperemia flow-mediated dilation parameters before and after plasm contraction. ..................... 59 Table 3-4. Time control participants (n = 5; 2F). ......................................................................... 68 Table 4-1. Comparison of blood parameters across days. ............................................................ 86 Table 4-2. Partial pressure of oxygen and carbon dioxide, and cerebral blood flow. .................. 88 Table 4-3. Systemic hemodynamics, hemoglobin concentration, hematocrit, apparent viscosity, and BA parameters during progressive handgrip exercise. .......................................................... 89 Table 5-1. Arterial blood gases, electrolytes and venous blood inflammation ........................... 111 Table 5-2. Whole blood viscosity, plasma viscosity, red blood cell deformability and aggregation. ................................................................................................................................. 112 Table 5-3 Hemrheology parameters measured at low altitude and during the placebo day of testing at high altitude. (n = 14). ................................................................................................. 114 Table 5-4 Cardiorespiratory parameters during room-air and isocapnic hypoxia (40 mmHg). . 116 x List of Figures Figure 2-1. Antoni Van Leeuwenhoek drawing of “globules” (erythrocytes) in fish blood. ......... 7 Figure 2-2. A drawing of Poiseuille's experimental apparatus. ...................................................... 9 Figure 2-3. Components of blood. ................................................................................................ 12 Figure 2-4. RBC deformability in the splenic cords and ex vivo. ................................................ 14 Figure 2-5. Schematic of the RBC discocyte shape, and membrane structure. ............................ 16 Figure 2-6. RBC elongation to increasing shear stress. ................................................................ 18 Figure 2-7. RBC elongation during incremental handgrip exercise. * Significantly different from baseline (p < 0.05). ....................................................................................................................... 19 Figure 2-8. Rouleaux formations caused by RBC aggregation. ................................................... 23 Figure 2-9. Shear rate-viscosity curves depicting RBC deformability and aggregation. ............. 26 Figure 2-10. The hematocrit-blood viscosity relationship. ........................................................... 27 Figure 2-12. Schematic of plasmapheresis (left) and real-world photo (right). ............................ 41 Figure 3-1. Elongation curves from plasma donation participants ............................................... 61 Figure 3-2. RBC deformability and aggregation characteristics plasma donation participants ... 62 Figure 3-3. FMD and blood flow parameters from plasma donation participants ........................ 65 Figure 3-4. FMD, exercise hyperemia and blood flow parameters from plasma contraction participants .................................................................................................................................... 66 Figure 4-1. Schematic of the study. .............................................................................................. 79 Figure 4-2. EImax during BL, 27.5% and 55% of MVC with and without PDE5-i ..................... 91 Figure 4-3. SS₁/₂ during BL, 27.5% and 55% of MVC with and without PDE5-i ....................... 92 Figure 4-4. Brachial artery specific erythrocyte elongation index during BL, 27.5% and 55% of MVC with and without PDE5-i .................................................................................................... 93 xi Figure 4-5. Erythrocyte elongation index curves during BL, 27.5% and 55% of MVC with and without PDE5-i ............................................................................................................................. 94 Figure 5-1. Schematic of the experimental design ...................................................................... 105 Figure 5-2. Pentoxifylline on cerebral blood flow ...................................................................... 119 Figure 5-3. Pentoxifylline on carotid body tonic activity and tonic control ............................... 120 Figure 5-4. Pentoxifylline on pulmonary artery systolic pressure .............................................. 121 Figure 5-5. Pentoxifylline on pulmonary artery systolic pressure reactivity to isocapnia hypoxia..................................................................................................................................................... 122 Figure 5-6. Sex differences to pentoxifylline and pulmonary artery systolic pressure .............. 123 Figure 6-1. EDTA on endothelium dependent-vasodilation via intrabrachial infusion of acetylcholine. .............................................................................................................................. 137 xii List of Abbreviations AMP – Amplitude AI –Aggregation index BA – Brachial artery CaO₂ – Arterial oxygen content CBF – Cerebral blood flow cAMP – 3',5'-cyclic adenosine monophosphate cGMP – Guanosine 3',5'-cyclic monophosphate CO – Cardiac output Cond – Conductance DO₂ – Oxygen delivery eNOS – Endothelial nitric oxide synthase EI – Elongation index FMD – Flow-mediated dilation gCBF – Global cerebral blood flow ICA – Internal carotid artery LORRCA – Laser-assisted rotational red cell analyser NO – Nitric Oxide Pa – Pascal PASP – Pulmonary artery systolic pressure PDE – Phosphodiesterase PDE5 – Phosphodiesterase-5 PDE5-i – Phosphodiesterase-5 inhibitor PetCO2 – end-tidal partial pressure of carbon dioxide PetO2 – end-tidal partial pressure of oxygen PTX – Pentoxifylline PV – Plasma viscosity Q – Blood flow RBC – Red blood cell RH-FMD – Reactive hyperemia-flow-mediated dilation xiii SaO2 – arterial oxygen saturation of hemoglobin SpO2 – peripheral oxygen saturation of hemoglobin SR – Shear rate SS – Shear stress SRAUC – shear rate area-under-the-curve SS½ – Shear stress half-maximal deformability T½ – aggregation half-time VA – Vertebral artery V̇E – minute ventilation WBV – Whole blood viscosity xiv Acknowledgements I would first like to apologize to Professor Phil Ainslie. I cannot imagine the headaches I have caused you… (it does not stop here though) You had the foresight to let me choose my own adventure, even if it meant failing more often than not. Thank you for having confidence in me and always pushing me forward. That freedom allowed me to grow into a more complete and (hopefully) competent scientist. Your ability to see the big picture, invest in others, and your passions for the great outdoors is truly inspiring. Thank you. Thank you to my committee members, Professor Glen Foster and Dr Ryan Hoiland, for taking the time to teach and mentor me. Thank you to my external examiner, Professor Michael Simmonds, and university examiner, Professor Chris West, for your time and insight. And thank you Dr. Jodie Koep for babysitting. Dr Travis Gibbons, you are the most uncompromising scientist I have ever met. You taught me scientific rigor, design, and passion. I miss beers on the stoop. Please add white flour to your bread. Dr Josh Tremblay, you are a tremendous researcher and friend. Your mentorship and support truly made my PhD possible. Your passion for science and adventures is contagious. Thank you for bringing me on adventures. Dr. Jay MJR Carr, I was afraid of you at first. But once I got past the chest tattoo, I realized you're a deeply considerate guy with thought-provoking insights like how crabs can be used as a supercomputer. Thank you for your candor. Dr. Madden Brewster, it’s inspiring to watch how much you have accomplished after switching fields. Your support and passion are always appreciated. xv Drs. Hannah and Connor Caldwell, you both taught me a lot. Most importantly, thank you for being my friends. Please respond to my emails, Connor. Future Dr. Jordan Bird, your coding and stats knowledge has saved my butt so many times. But more importantly, you have always been a source of support and a willing drinking partner. To the Ainslie Lab, thank you for everything. My PhD has been the hardest thing I have ever done, but also the most fulfilling. It’s made me a fuller, more complete person, mostly because of all the incredible people (minus maybe Tymko). To my parents, sorry I’m still in school. Please don’t expect me to host Christmas anytime soon. Thank you for always pushing me to pursue my dreams. 1 Chapter 1: Introduction This dissertation will focus on mechanisms that regulate blood rheology - the study of the physical properties of blood and its behavior within the vascular system - and their influence on blood flow. Chapter 1 provides a brief introduction to blood rheology and emphasizes the relevance of rheological properties on perfusion. Chapter 2 provides a comprehensive overview of the relevant literature related to this dissertation. Chapters 3 through 5 are the individual experimental studies. Finally, Chapter 6 offers an overarching synthesis of the preceding individual studies highlighting key strengths, limitations, and potential areas of future research. Understanding the properties of blood rheology and their contributions to blood flow regulation is critical for the identification of various hematological disorders (i.e., sickle cell disease) and advancement of cardiovascular technologies, such as blood pumps, heart valves, and stents (Paul et al., 2003). Substantial evidence has linked altered blood rheology to a range of pathological conditions, including sickle cell disease (Alapan et al., 2016), cerebrovascular disease (Ernst et al., 1988), arterial hypertension (Letcher et al., 1981), and diabetes mellitus (Turpin et al., 2023; Ebenuwa et al., 2024). Blood rheology could therefore be used as an additional and important metric to monitor disease progression and severity. In particular, red blood cell (RBC) deformability has been directly linked to microvascular function and the development of microcirculatory diseases, underscoring the intricate relationship between blood rheology and perfusion (Sakuta, 1981; Bowton et al., 1989; Turpin et al., 2023). Blood flow regulation is inherently complex and influenced by various physiological (Doshi et al., 2001; Nyberg et al., 2013) and mechanical factors (Celermajer et al., 1992), determined by the pressure gradient across the arterial and venous circulations, and opposed by vascular resistance (Mc, 1955). Vascular resistance is determined by vessel length, radius, and whole blood viscosity, the latter being influenced by shear rate, hematocrit, plasma viscosity, and RBC deformability and aggregation (Rand et al., 1964; Rand, 1964; La Celle, 1969; Chien et al., 1970). Further complicating rheology, as vessel diameter decreases (below ~500 µm), shear rates increase markedly and radial migration distances shorten, augmenting the Fahraeus effect. This leads to a local reduction in hematocrit and, consequently, apparent viscosity compared to upstream vessels. 2 As a result, hematocrit can vary by up to threefold between conduit and microvascular vessels. Overall, blood rheology differs markedly between the macro- and microvascular circulation(Pries et al., 1992) Blood rheology can significantly influence blood flow through numerous interconnected and sometimes opposing mechanisms: (i) blood viscosity is directly proportional to flow resistance, and thus impedes flow (Gustafsson et al., 1980); (ii) conversely blood viscosity contributes to shear stress, a key stimulus for vasodilation and a regulator of vascular diameter; and (Gnasso et al., 2001) (iii) RBC deformability and aggregation cause the shear-thinning behavior of blood, dynamically altering blood viscosity during states of altered shear rate (e.g., exercise hyperemia and clinical conditions such as stenosis) (Chien et al., 1967; Chien et al., 1970). The complex interaction between these factors make it challenging to isolate their individual contributions to blood flow regulation, particularly in humans. Nevertheless, understanding the rheological properties of blood is critical as these properties can both resist and facilitate blood flow depending on physiological context. 1.1 Thesis objectives Thus, this dissertation aimed to advance understanding of the mechanisms regulating blood rheology by specifically examining the effects of increased blood viscosity and enhanced RBC deformability on blood flow. Two key insights emerged: 1. A detailed characterization of blood rheology under specific stressors (e.g., plasmapheresis, plasma contraction, exercise, and chronic hypoxia). 2. Novel evidence that acute alterations in blood rheology can influence blood flow and vascular reactivity. 1.2 Individual study aims and hypotheses: 3 1.2.1 Study 1 - The impact of plasmapheresis on blood rheology, and peripheral vascular function This study employed two distinct methods (e.g., plasma donation and diuresis) to induce hypovolemic hemoconcentration and assessed the effects of elevated whole blood viscosity on brachial blood flow and vascular reactivity. It was hypothesized that these methods would yield different responses, as plasmapheresis, being an extracorporeal procedure, exposes RBCs to mechanical stressors, artificial surfaces, and anticoagulants. Accordingly, we expected that brachial blood flow and vascular reactivity would be lower following plasmapheresis, due to rheological alterations, compared to diuresis. Specifically, it was anticipated plasmapheresis to reduce RBC deformability and FMD immediately post-procedure, with these effects resolving by the 24-hour follow-up. 1.2.2 Study 2 - Exercise-induced enhancements in RBC deformability are prolonged by oral phosphodiesterase-5 inhibition The purpose of this study was to determine whether the phosphodiesterase-5 (PDE5) inhibitor – sildenafil citrate could: (i) enhance RBC deformability and, in turn, (ii) increase resting and exercise brachial blood flow. A secondary outcome was to assess whether PDE5 inhibition would increase cerebral blood flow (CBF). It was hypothesized that enhanced RBC deformability would augment resting CBF, brachial blood flow and exercise hyperemia. 1.2.3 Study 3 - Hemorheological, cardiorespiratory, and cerebrovascular effects of pentoxifylline following acclimatization to 3,800 m Chronic hypoxia increases blood viscosity through hemoconcentration and reduced RBC deformability, changes that could decrease microcirculatory flow and bulk blood flow. Pentoxifylline, a non-specific PDE inhibitor with rheological properties, reduces blood viscosity, induces vasodilation, and thus may enhance blood flow at high altitude. It was hypothesized that 4 pentoxifylline would enhance RBC deformability and vasodilation, increasing cardiac output and CBF. 5 Chapter 2: Literature Review This chapter provides foundational context for the studies presented in this dissertation. Section 2.1 offers a brief historical overview of blood rheology. Section 2.2 introduces the components of blood, including plasma and the RBC. Section 2.3 outlines the key determinants of blood viscosity, such as hematocrit, plasma viscosity, RBC deformability, and aggregation and their individual influence on blood flow. Section 2.4 will build on this foundational knowledge from section 2.3 by examining how shear stress, blood rheology and arterial oxygen content (CaO2) interact. Finally, section 2.5 will highlight the unique physiological stimuli examined in this dissertation namely plasmapheresis, and high altitude providing background on their relevance to blood rheology and blood flow. The section will conclude with a discussion on blood rheology in the context of health and disease. 2.1 Origins of blood rheology Humanity has long been fascinated by the function and power of blood. Across numerous ancient civilizations, blood has had spiritual and divine properties. The Mesopotamian Babylonians believed that the god Marduk created humanity from the blood of a slain god, Qingu. Babylonians used bloodletting to communicate with these gods (George, 2003). In traditional Chinese medicine, (Huang Di Nei Jing ~200 BC), blood (Xue) is described as a nourishing and moistening fluid, that supports vitality and emotional wellbeing. Imbalances in Xue cause dizziness, pale skin and emotional instability (Zhang, 2013). Mesoamerican Aztecs believed that the sun god – Huitzilopochtli - required human blood sacrifice, as nourishment to move across the sky. Ritual sacrifices, both involuntary and voluntary, were a core practice and considered a great honor (Jansen & Jiménez, 2022). For millennia, blood was a symbol of life, power and connection to the gods. The ancient Greeks theorized about the role of blood in health and disease. The father of clinical medicine, Hippocrates of Kos (~460–370 BC) (Pappas et al., 2008), proposed humans had four fundamental humors: blood, phlegm, black bile and yellow bile (Cairus, 2005). Each humor originated from a specific organ: the brain, lung, spleen and gall bladder, respectively. Illness was not attributed to infection or disease, but a humor imbalance. To restore balance, humors had to be 6 removed, either through purgatives (vomiting, diuretics, and laxatives), sweating, cupping (i.e., placing a hot cup on the skin to create a vacuum), enemas and famously, bloodletting (Bhikha & Glynn, 2017). Hippocrates believed that blood was created in the liver and then circulated to the rest of the body to be consumed (Meletis & Konstantopoulos, 2010). This view persisted until the Renaissance, when Andreas Vesalius produced remarkable drawings of the heart and circulatory system, from human autopsies (Vesalius, 1543), laying the groundwork for William Harvey. In 1628, Harvey published Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus, where he made two significant discoveries: the circulatory system was a continuous loop driven by the heart, not the liver, and the blood in the arteries and veins is the same fluid (Harvey, 1628). Exercise Concerning the Motion of the Heart and Blood in Animals”), first published in 1628. In 1661, Marcello Malpighi, using the newly developed microscope, observed the “transition of blood from arteries to veins through tiny, hair-like vessels,” marking the first recorded observation of the capillary (Malpighi, 1661). Antoni Van Leeuwenhoek, the Father of Microbiology, came soon after in 1675, probably unaware of Malpighi’s work (Martins e Silva, 2009) began studying the erythrocyte. Van Leeuwenhoek provided the first clear and detailed description of the erythrocyte in Philosophical Transactions of the Royal Society: “I have divers times endeavoured to see and to know, what parts the Blood consists of, and at length I have observed taking some blood of my own hand, that it consists of small round globules driven through a crystalline humidity or water (Leeuwenhoek, 1674)” 7 Figure 2-1. Antoni Van Leeuwenhoek drawing of “globules” (erythrocytes) in fish blood. Experimenta & Contemplationes page 51 (Leeuwenhoek, 1722). This image is in the public domain. Van Leeuwenhoek later expanded on his observations, describing not only these “globules” but also noted two key phenomena: erythrocyte sedimentation and aggregation, both of which occur gradually after blood is extracted: “…the blood is let out of the veins, those globules by little and little subside towards the bottom; and being made of soft fluid corpuscles, and many lying upon one another (Leeuwenhoek, 1674).” Years later, he described that tadpole RBCs change shape in response to increased blood flow velocity: ...”these particles [of blood], though in this creature they were of a flat and oval shape, yet sometimes by reason of the smallness of the artery, assumed a kind of oblong round figure, and when the animal, by being taken out of the water, grew languid, the blood in these very minute arteries began to stagnate, and when it again acquired motion, many of the globules appeared twice as long as broad, and also pointed at their extremities. (Leeuwenhoek, 1722).” 8 Van Leeuwenhoek’s astonishing observations identified two key erythrocyte properties: deformability, globules become “twice as long as broad” in high flow, and aggregation “lying upon one another” when flow stops (Leeuwenhoek, 1674, 1722). The history of blood rheology would be incomplete without acknowledging the pivotal contributions of Jean-Marie Léonard Poiseuille. Having received his medical doctorate from the École de Médecine de Paris. His dissertation focused on force generation in the heart and pulse wave propagation, setting the groundwork for his future scientific contributions. Early on, Poiseuille was interested in studying blood flow in the frog mesenteric circulation. However, he realized that the uncontrolled environment of an amphibian gut was not suitable for formulating the laws governing fluid flow. He instead conducted careful and extensive studies of liquid flow (e.g., water and wine) through small diameter glass tubes (Sutera & Skalak, 1993) (Figure 2-2). 9 Figure 2-2. A drawing of Poiseuille's experimental apparatus. Designed to study fluid flow through a small tube. The system was pressurized using a hand-operated pumped (labelled water jacket), which raised fluid levels in the manometers to set the pressure. After pressure stabilized, a valve near the viscometer-controlled flow through the test tube, allowing precise measurements of flow rate at different pressures. Image from Recherches experimentales sur le mouvement des liquides dans les tubes de tres-petits diametres (Poiseuille, 1844). This image is in the public domain. 10 In these experiments, Poiseuille examined the effects of pressure drop, tube length, tube diameter, and temperature on fluid flow (Sutera & Skalak, 1993). This work, along with the research completed by Gotthilf Heinrich Ludwig Hagen (Hagen, 1839), described the relationship between pressure, flow, and resistance in cylindrical tubes, now known as the Hagen-Poiseuille equation: Q̇ = (Π ⋅ r4 ⋅ ΔP) / (8 ⋅ n ⋅ L) Equation 2.1.1 Where: Q̇ (m³/s) = volumetric flow rate; Π = pi; n (Pa·s) = apparent viscosity; L (meters) = length; R (meters) = radius; ΔP (Pa) = pressure The Hagen-Poiseuille equation states that increasing pressure increases fluid flow by providing a greater driving force. In contrast, a reduction in tube diameter substantially decreases fluid flow, as fluid rate is proportional to the fourth power of the radius, meaning even small changes in diameter has a large impact. Greater tube length also decreases flow by increasing resistance through more fluid-wall interaction. Lastly, higher viscosity, the internal resistance of a fluid to flow, also decreases fluid flow (Hagen, 1839; Poiseuille, 1844; Secomb, 2016). Poiseuille and Hagens’ research laid the foundation for fluid dynamics, and their work set the stage for pivotal studies by August Krogh, Robin Fåhræus, and Torsten Lindqvist, leading to the development of blood rheology and the study of blood flow through vessels (Krogh, 1919; Krogh, 1921; Fåhræus & Lindqvist, 1931). 2.2 Constituents of blood This section will briefly highlight the components of blood with an emphasis on RBC morphology. Blood is a connective tissue, which is meant to bind, support or protect organs, alongside bone and cartilage. Like all connective tissues, blood is a suspension of corpuscles (e.g., erythrocytes, leukocytes, and thrombocytes) in an extracellular matrix composed of a network of proteins and other molecules supporting the corpuscles (Figure 2-3). Blood accounts for 7-8% of body weight (Oberholzer et al., 2024), and is composed of plasma (i.e., the extracellular matrix), which makes up about 55% of total volume, and cellular components, accounting for the remaining approximate 45%. Centrifugation of a blood sample, as shown in Figure 2-3, separates blood components by 11 density. Plasma collects at the top, followed by a thin buffy coat composed of leukocytes and thrombocytes, with erythrocytes settling at the bottom. This separation is driven by differences in cellular density rather than mass. Erythrocytes are especially dense due to high hemoglobin concentrations (Norouzi et al., 2017). 12 Figure 2-3. Components of blood. Blood is a composite fluid with cellular components including erythrocytes, leukocytes, and thrombocytes suspended in plasma. Cellular components equate to ~45%, while the remaining ~55% is composed of plasma. By courtesy of Encyclopædia Britannica, Inc., copyright 2017; modified with permission. 13 2.2.1 Plasma composition and function Plasma is composed of approximately 92% water, serving as the primary solvent for various substances (Benjamin & McLaughlin, 2012). Roughly 7% of plasma consists of proteins, mainly albumin (~60%), globulins (~35%), fibrinogen (~4%), along with numerous low-abundance proteins (Gordeeva et al., 2024). The remaining 1% includes electrolytes (e.g., sodium, potassium, chloride, and bicarbonate) and organic molecules (e.g., glucose, urea and amino acids) (Feldman & Dickson, 2017). Plasma supports a wide range of physiological functions, including perfusion, osmotic pressure, nutrient transport, waste removal, and immune function (Powers, 1999; Pérez-Castillo Í et al., 2023), 2.2.2 Red blood cell structure and function Erythrocytes are the most abundant host cells, comprising ~83% of all cells, and equating to 25 trillion in circulation (Nemkov et al., 2018), with approximately 2 to 3 million being produced and broken down every second. (Nemkov et al., 2018; Sender & Milo, 2021). To maintain normal RBC function, the body relies on ongoing production and clearance, replacing dysfunctional or aging cells with new ones produced in the bone marrow (Lew & Tiffert, 2017). Unlike most cells, mature erythrocytes lack many intracellular organelles typically present in other cell types, including mitochondria and nuclei. The absence of a nucleus maximizes hemoglobin concentration, enhances the surface area-to-volume ratio, minimizes diffusion distances, and, together with a flexible cytoskeleton, facilitates cellular deformation (Li et al., 2005; Li et al., 2007). Mature RBCs have a lifespan of approximately 120 days (Li et al., 2019), during which they travel an estimated 480 kilometers through the circulation (Lasch et al., 2000). As they age, RBCs lose flexibility and accumulate cellular damage (Mykhailova et al., 2025). These senescent cells are sequestered in the spleen and liver, preventing the accumulation of dysfunctional RBCs in circulation (Cha, 2024; Mykhailova et al., 2025). The primary function of RBCs is gas transport, delivering oxygen from the lungs to tissues while also facilitating carbon dioxide removal by transporting it back to the lungs for exhalation (Kheir et al., 2012). Beyond gas exchange, RBCs play essential roles in immune modulation (Lam et al., 14 2021), vasodilation (e.g., nitric oxide [NO] and ATP release) (Sprung et al., 2002; Kleinbongard et al., 2006a), antioxidant defense (e.g., superoxide dismutase, catalase, glutathione peroxidase) (Iuchi et al., 2007), and acid-base regulation. RBC have the capacity to undergo substantial, reversible deformations (i.e., the ability to change shape in response to an external force). This flexibility is essential for traversing the microcirculation, where RBCs must squeeze through narrow capillaries (Safeukui et al., 2018). The spleen imposes the greatest mechanical constrain on RBCs, as they must pass through splenic cords of red pulp, sinusoidal slits as narrow as 2–3 micrometers, about one-third the diameter of an RBC (Figure 2-4) (Mohandas & Gallagher, 2008; Li et al., 2018). Figure 2-4. RBC deformability in the splenic cords and ex vivo. An RBC passing through the splenic cord to the venous sinus, where the cell must squeeze through the narrow interendothelial slits (left panel). The right panel depicts the ellipsoidal shape of an RBC exposed to high shear forces in an ex vivo setting. This image was obtained using Couette flow, which generates a non-turbulent, parallel-layer shear field by rotating one cylinder while keeping the other stationary. Measurements are conducted in a highly viscous, Newtonian medium, producing a linear velocity gradient and an effectively uniform shear stress with minimal radial variation due to the narrow-gap geometry. While this ellipsoidal deformation reflects the RBC 15 response to shear stress, this may differ under physiological conditions, where shear stress fluctuates with the cardiac cycle, cellular interactions occur (with leukocytes, platelets, and endothelium), and the plasma (i.e., medium) is less viscous than the RBC cytoplasm. © Mohandas N, Gallagher P Red cell membrane: past, present, and future. Blood. 2008;112(10):3939-48. Page 3940. Modified with permission from Elsevier Science & Technology Journals The deformability of RBC’s is facilitated by a highly specialized cellular membrane that allows flexibility, while maintaining structural integrity. The membrane is highly elastic, responding to shear stress within 100 milliseconds, with remarkable mechanical strength (Pesen et al., 2025). To maintain its function, the RBC has developed a composite structure consisting of a 2-dimensional spectrin lattice (α- and β-spectrins) cytoskeleton, connected to a phospholipid bilayer, via tethering sites on transmembrane proteins (e.g., ankyria and band 4.2) (Pesen et al., 2025). Structure integrity is maintained by the spectrin cytoskeleton, while the phospholipid bilayer enables membrane bending (Figure 2-5) (Shen et al., 1986). 16 Figure 2-5. Schematic of the RBC discocyte shape, and membrane structure. Inset shows the cross-sectional view of the RBC membrane with vertical and horizontal interactions. © Gallagher P Red blood cell membrane disorders Hematology American Society of Hematology Education Program. 2005:13-8. Page 14. Used with permission from John Wiley & Sons 2.3 Rheological determinants of blood flow This section will examine the primary determinants of blood viscosity (e.g., plasma viscosity, RBC deformability and aggregation, shear rate, and hematocrit) and their individual effects on blood 17 flow. These factors will be progressively integrated to highlight their interaction and connection to blood flow control. Section 2.4 will expand on this foundational knowledge by exploring the interplay between blood viscosity, shear stress, and CaO2. 2.3.1 Plasma viscosity and microvascular dynamics Plasma viscosity is primarily determined by protein composition and concentration, with fibrinogen, immunoglobulins, and albumin being the most abundant contributors (Gordeeva et al., 2024). Although plasma has traditionally been classified as a Newtonian fluid, plasma may exhibit viscoelastic properties under high shear conditions (Brust et al., 2013; Varchanis et al., 2018). Plasma has a significant role in blood rheology and flow regulation by contributing to the formation of the cell-poor layer (i.e., a thin plasma-rich region that forms along the inner wall of blood vessels). This area of reduced cellular concentration, results from axial migration of RBCs. While axial migration occurs in vessels of all sizes, it becomes particularly pronounced in the microcirculation and contributes to the Fåhræus effect, which is discussed in greater detail in Section 2.3.6. The cell-poor layer also plays a central role in the transmission of shear stress to the endothelium, as RBCs typically do not make direct contact with the vessel wall under physiological flow conditions (Namgung et al., 2011; Yalcin et al., 2015). 2.3.2 Red blood cell deformability Deformability is a defining feature of soft matter, including RBCs. RBC deformability is determined by the membrane surface-to-area ratio (Huisjes et al., 2018), cytoplasmic viscosity (Yokoyama et al., 2011), and cytoskeletal flexibility (Pesen et al., 2025). Cytoskeleton flexibility enables RBCs to dynamically deform in the circulation. This deformation serves three key functions: (i) reduces flow resistance, (ii) allows passage through narrow microvessels, and (iii) promotes the release of vasodilatory molecules (i.e., NO and ATP). These features of RBC deformability are important regulators of blood flow. This section will first examine the role of RBC deformability on blood flow, followed by a discussion of its key modulators. 18 2.3.2.1 Influence of red blood cell deformability on blood flow Under elevated shear stress, RBCs elongate, becoming more fluid dynamic, similar to the streamlined shape of a torpedo. RBCs flowing in a vessel align with the direction of movement and elongate into an asymmetric “slipper” shape that minimizes cross-sectional area. This streamlined shape reduces blood viscosity by lowering internal fluid resistance (Wang et al., 2022). In the microcirculation, where shear forces are high and vessel diameters are small, RBCs instead morph into a parachute-like shape, which also minimizes cross-sectional area and improves flow efficiency (Figure 2-6) (Trejo-Soto et al., 2022). Figure 2-6. RBC elongation to increasing shear stress. As velocity increases, the RBC aligns with the axis of flow, as indicated by the dashed lines, and morphs into an asymmetrical slipper shape. With further increases in velocity and decreases in vessel diameter, RBCs transition to a parachute-like shape. RBC elongation provides a streamlined advantage and reduces blood viscosity. Modified from (Trejo-Soto et al., 2022). No special permission is required to reuse all or part of article published by MDPI, including figures and tables. This mechanism was demonstrated by Leo et al., (2020) (Figure 2-7) where participants completed four stages of graded rhythmic handgrip exercise at 20, 40, 60 and 80% of their maximal workload. Exercise-induced hyperemia increased shear rate, elongating RBCs in a dose dependent manner, leading to a reduction in blood viscosity. RBC deformation causes this elongation contributing to a more streamlined shape, causing apparent viscosity to be reduced during incremental exercise (Leo et al., 2020; Tremblay et al., 2020). Decreased apparent viscosity lowers blood flow resistance and facilitates blood flow. 19 Figure 2-7. RBC elongation during incremental handgrip exercise. * Significantly different from baseline (p < 0.05). Reductions in apparent viscosity during incremental exercise are driven by shear-dependent erythrocyte deformation. Increased RBC elongation (i.e., elongation index, left) reduces apparent viscosity (right) © Leo J, Simmonds M, Sabapathy S. Shear-thinning behaviour of blood in response to active hyperaemia: Implications for the assessment of arterial shear stress-mediated dilatation. Experimental Physiology. 2020;105(2):244-257. Pages 248 and 240. Modified with permission from John Wiley & Sons The RBC deformability can influence blood flow independent of blood viscosity. Reduced RBC deformability impairs capillary transit, leading to increased microvascular resistance (Pantely et al., 1988). This was investigated by Simchon et al., (1987), who partially stiffened native RBCs using 0.025% glutaraldehyde, which doubled their filtration resistance through 5-μm pores (Simchon et al., 1987). Radiolabeled native and rigidified RBCs were infused into rats. Approximately 70% of rigid RBCs were cleared from circulation within 25 minutes, whereas native RBCs remained stable over the same period. The stiffened cells were preferentially trapped 20 in the spleen, lungs, bones, and liver, with minimal accumulation in the heart, kidneys, and brain. Regions with greater RBCs trapping exhibited corresponding reductions in regional blood flow (Simchon et al., 1987). These regional differences in blood flow may reflect variations in microvascular architecture, which impairs capillary transit when RBC deformability is reduced (Parthasarathi & Lipowsky, 1999; Kelch et al., 2015). These findings suggest that RBC deformability plays a critical role in facilitating tissue perfusion, independent of blood viscosity. 2.3.2.2 Vasodilator signaling mediated by red blood cell deformability It is known that RBCs contribute to vasodilation through the release of ATP and NO (Ulker et al., 2010; Racine & Dinenno, 2019; Leo et al., 2023), a process that depends on cellular deformation. Racine and Dinenno (2019) demonstrated that treating RBCs from older adults with a Rho-kinase inhibitor restored hypoxia-induced ATP release to levels observed in young adults (Racine & Dinenno, 2019). ATP has a significant role in exercise hyperemia (Nyberg et al., 2013) and sympatholysis – the attenuation of sympathetic vasoconstriction during exercise (Hearon et al., 2020). Sympatholysis is blunted with aging (Mortensen et al., 2012; Kruse et al., 2018), and contributes to reduced exercise capacity in older adults (Dinenno et al., 2005) Impaired sympatholysis appears to stem from diminished functional signaling (i.e., reduced ATP availability), rather than from intrinsic vascular dysfunction (Hearon et al., 2020). Thus, reduced RBC deformability may limit ATP availability in older adults, impairing sympatholysis and contributing to diminished exercise capacity. Red blood cells (RBCs) produce nitric oxide (NO) through a functional form of endothelial nitric oxide synthase (eNOS), which is activated by shear stress. Mechanical stimulation opens mechanosensitive calcium channels, triggering eNOS activation and increasing NO production (Ulker et al., 2011; Grau et al., 2013). Consistent with this mechanism, Suhr et al., (2012) demonstrated that one hour of moderate-intensity running (at 70% of lactate threshold) enhanced RBC deformability, increased eNOS phosphorylation at Ser1177 (an indicator of eNOS activation), and upregulated the PI3-kinase/Akt signaling pathway (a key upstream eNOS regulator) (Suhr et al., 2012). Even brief rhythmic handgrip at 60% of maximal workload has been shown to increase eNOS phosphorylation at Ser1177, highlighting the sensitivity of RBC eNOS to mechanical stress 21 (Leo et al., 2023). RBC deformation contributes to NO production and likely plays a role in vascular regulation (Leo et al., 2021). 2.3.2.3 Mechanisms of red blood cell deformability The discoid shape of RBCs provides a unique physiological advantage by allowing reversible deformation. Despite a relatively small volume (~90 fL), RBCs possess a surface area of approximately 135 μm² substantially exceeding the ~97 μm² required to enclose a sphere of this volume. This surplus membrane area enables RBCs to deform extensively without significantly altering their surface area-to-volume ratio, allowing them to withstand substantial shear stresses during circulation (Takakuwa & Mohandas, 1988). RBC deformability is not a passive property but is actively regulated through a range of biochemical and cellular mechanisms that modulate cytoskeletal organization and ion channel activity (Bookchin & Lew, 1980; Betz et al., 2009). Cellular osmolarity (~295 mOsm/kg) plays a critical role in this process, as substantial changes in osmotic balance can significantly alter RBC volume and, consequently, deformability (Nemeth et al., 2015). Deviations from normal osmolarity reduce deformability by inducing volume changes that mechanically strain the membrane (Nemeth et al., 2015; Kuck et al., 2020a). To maintain cell volume homeostasis, RBCs tightly regulate their ion and water content via membrane transport systems, including ion pumps, channels, symporters, and antiporters (Bookchin & Lew, 1980; Gallagher, 2013). These systems allow RBCs to adapt to transient osmotic challenges, such as those that occur during exercise (Takamata et al., 1998). Importantly, the energy required for ion transport is supplied entirely by glycolysis, as RBCs lack mitochondria (Park et al., 2010). Beyond its role in ion regulation, ATP directly contributes to maintaining RBC membrane flexibility. It facilitates the dissociation of spectrin from actin within the cytoskeleton, preventing excessive cross-linking that would otherwise stiffen the membrane and impair deformability (Betz et al., 2009). ATP also plays a central role in regulating intracellular calcium concentrations through the activity of Ca²⁺-ATPases, which actively export Ca²⁺ to maintain homeostasis. Elevated intracellular Ca²⁺ impairs deformability and can trigger hemolytic pathways. If ATP levels are insufficient or intracellular Ca²⁺ becomes excessive, it can lead to cellular swelling and eventual lysis (Kuck et al., 2024). 22 RBC deformations are sensed by Piezo1, a mechanosensitive ion channel that opens in response to membrane stretch, allowing an influx of Ca²⁺ (Dyrda et al., 2010). This rise in intracellular calcium activates Ca²⁺-sensitive Gárdos channels, leading to K⁺ efflux, followed by Cl⁻ and water loss, which results in cellular dehydration and a reduction in cell volume (Cahalan et al., 2015; Danielczok et al., 2017). Decreased intracellular volume reduces RBC deformability (Kuck et al., 2020a), though it may facilitate capillary recruitment by allowing smaller, more compact cells to traverse narrow vessels more efficiently (Danielczok et al., 2017). Additionally, Piezo1-mediated Ca²⁺ influx has been shown to stimulate eNOS phosphorylation at Ser1177, promoting NO production, which may partially counteract the dehydration-induced stiffening of RBCs by modulating RBC mechanics (Kuck et al., 2022c). NO binds to cytoskeletal proteins (e.g., α- and β-spectrins) via S-nitrosylation, inducing conformational changes and altering spectrin–actin interactions (Grau et al., 2013). Indirectly, RBCs, similar to endothelium, are equipped with the enzyme soluble guanylate cyclase (sGC) (Adderley et al., 2011). NO activates sGC increasing intracellular cGMP concentrations, which likely prolongs cAMP-dependent kinase by inhibiting phosphodiesterase-3 (PDE) (Muravyov & Tikhomirova, 2013). It is therefore unsurprising that cGMP inhibition decreases RBC deformability (Ziegler, 2017), while PDE-5 inhibition enhances deformation (Hagley et al., 2011; Ziegler, 2017). However, sodium nitroprusside can completely reverse impairments in RBC deformability following cGMP inhibition, suggesting cGMP is not obligatory for NO-mediated increases in RBC deformability (Bor-Kucukatay et al., 2003). Thus, NO likely contributes to blood flow regulation not only via vasodilation but also by modulating RBC deformability. 2.3.3 Influence of red blood cell aggregation in blood flow dynamics RBCs suspended in plasma spontaneously form three-dimensional, coin like structures known as rouleaux formations during low shear rates, increasing blood viscosity (Figure 2-8) (Chien et al., 1967). This process is reversible, disaggregating as shear rates increase (Weber‐Fishkin et al., 2022). RBC aggregation is determined by the presence of high-molecular-weight polymers (e.g., fibrinogen) (Armstrong et al., 2001; Hardeman et al., 2001), hematocrit concentration (Chien et al., 1966), and RBC surface properties (e.g., surface charge and glycocalyx depth) (Meiselman et 23 al., 2007). Two theories have been proposed to explain RBC aggregation – the bridging model and the depletion model (Neu et al., 2008; Bosek et al., 2022). The bridging model proposes that adjacent RBCs are physically linked by fibrinogen or other large plasma proteins (Chen et al., 1996). In contrast, the depletion model suggests that RBC aggregation arises from an osmotic imbalance. When large macromolecules are excluded from the thin plasma layer near the RBC membrane, a region of lower osmotic pressure is created between adjacent RBCs overcoming their repulsive forces (Bäumler et al., 1996). Figure 2-8. Rouleaux formations caused by RBC aggregation. Note that the stacked coin-like appearance, indicating RBC greater RBC-to-RBC interaction. RBCs form coin like structures called rouleaux formations during low shear forces. © Baskurt O, Meiselman H Erythrocyte aggregation: basic aspects and clinical importance. Clinical Hemorheology and Microcirculation. 2013;53(1-2):23-37. Page 24. Modified with permission from SAGE Publications Ltd. Journals 24 The impact of RBC aggregation on blood flow is complex and context-dependent. RBC aggregation increases blood viscosity by promoting cell cohesion (Hardeman et al., 2001), but it also enhances axial migration (Cokelet & Goldsmith, 1991; Reinhart et al., 2017). This axial migration promotes plasma skimming at vessel bifurcations, which can reduce blood viscosity, as observed in rat arterioles (Ong et al., 2011; Reinhart et al., 2017). Consequently, predicating the net effect of RBC aggregation on blood flow remains challenging. To explore this, Yalcin et al., (2005) used an isolated guinea pig perfused heart model with either native blood (Hematocrit 40%) or pluronic-coated RBCs suspended in autologous plasma. The pluronic-coated RBCs, mimic increased aggregation. No significant differences in flow resistance were observed between the two perfusates at high perfusion pressures. However, at low perfusion pressures (≤ 40 mmHg), vascular resistance was elevated in the pluronic-coated RBC model. These findings suggest that at low perfusion pressures, weaker shear forces are insufficient to disrupt rouleaux formations, resulting in increased vascular resistance (Hardeman et al., 2001; Yalcin et al., 2005). Thus, elevated RBC aggregation may decrease blood flow at low perfusion pressures. The effect of RBC aggregation on human blood flow is difficult to isolate, as it rarely occurs in isolation. Elevated RBC aggregation often coincides with impaired RBC deformability (Ebenuwa et al., 2024), increased plasma viscosity (Wiewiora et al., 2007; Jung et al., 2011), and microvascular dysfunction (Tan et al., 2020) Despite this, Jung et al., (2011) observed an inverse relationship between RBC aggregation and RBC velocity in nailfold capillaries among 133 patients with diabetes mellitus, suggesting that increased RBC aggregation might enhances microcirculatory flow. However, a similar inverse relationship was also observed between plasma viscosity and nailfold RBC velocity, making it difficult to determine whether RBC aggregation, plasma viscosity or both contributed to this increase in microcirculatory flow (Jung et al., 2011). RBC aggregation is probably less likely to significantly influence nailfold capillary blood flow due to the high shear forces present (Hardeman et al., 2001). Therefore, it is more plausible that plasma viscosity was the primary factor increasing RBC velocity. RBC aggregation likely plays a more prominent role under low shear and high hematocrit conditions (Rand et al., 1964). 25 2.3.4 Influence of shear rate on apparent viscosity Liquids are broadly classified as Newtonian or non-Newtonian based on whether variations in shear rate influences viscosity. Newtonian fluids exhibit a constant viscosity regardless of shear rate, whereas non-Newtonian fluids display shear-dependent viscosity (i.e., apparent viscosity) (Chhabra & Richardson, 2008). The apparent viscosity of a non-Newtonian fluid may decrease (shear-thinning behavior) or increase (shear-thickening behavior) as shear rates increase. Blood is non-Newtonian, a shear-thinning fluid, where blood viscosity decreases with increasing shear rate and increases with decreasing shear rate (Hess, 1915). Consequently, blood viscosity is not accurately represented by a single value, but is better depicted as a two-phase exponential decay curve (Figure 2-9), varying with shear rate (Leo et al., 2020). Blood viscosity increases sharply at shear rates below 100 s⁻¹ and rising exponentially as shear rates decline further. For example, at a shear rate of 0.1 s⁻¹, blood viscosity can reach approximately 100 cP, whereas at 200 s⁻¹, it decreases to 4–5 cP (Rand et al., 1964). The shear-thinning behavior of blood results from the biophysical properties of RBCs, specifically RBC deformability and aggregation (Rand & Burton, 1964; Chien et al., 1967; Chien et al., 1970). This is illustrated in Figure 2-9, where chemically rigidified RBCs suspended in an 11% albumin buffer (i.e., a medium that prevents RBC aggregation) do not exhibit the characteristic exponential relationship, due to the absence of RBC aggregation and deformability, respectively. 26 Figure 2-9. Shear rate-viscosity curves depicting RBC deformability and aggregation. Shear rate–viscosity curves for normal RBCs in plasma, RBCs suspended in an 11% albumin buffer (preventing aggregation), chemically rigidified RBCs (non-deforming), and chemically rigidified RBCs in an 11% albumin buffer illustrates the distinct contributions of RBC aggregation and deformability. Variations in viscosity at low and high shear rates highlight the respective influences of these properties. © Secomb T Hemodynamics. Comprehensive Physiology. 2016;6(2):975-1003. Page 991. Modified with permission from John Wiley & Sons 2.3.5 Hematocrit and blood viscosity Hematocrit and mean corpuscular hemoglobin concentration are the primary determinants of blood viscosity, reflecting the number and internal viscosity of the RBCs. As a result, blood viscosity generally increases with hematocrit (Rand et al., 1964; Chien et al., 1966). However, this relationship is nonlinear becoming more pronounced at low shear rates with high hematocrits due to increased RBC aggregation (Figure 2-10) (Chien, 1975). High hematocrits enhance cell-to-cell 27 interaction, creating large rouleaux formations augmenting blood viscosity, which requires greater shear forces to disrupt (Hardeman et al., 2001; Mehri et al., 2018). In contrast, at high shear rates (>100 s-1), cohesive forces between RBCs are overcome, and the influence of hematocrit on blood viscosity is reduced (Takeishi et al., 2021). For example, Thomas et al., (1977) found that venesection in polycythemic patients reduces hematocrit by ~15%, lowering whole blood viscosity by 30.9% at a low shear rate (0.67 s⁻¹), but only by 11.8% at a high shear rate (91 s⁻¹)(Thomas et al., 1977). Shear rate is therefore a key determinant of blood viscosity (Pearson et al., 1980). Accordingly, blood viscosity varies widely between vessel types: shear rates can be as low as ~1.0 s⁻¹ in veins and exceed 400 s⁻¹ in arteries (Roux et al., 1991; Dammers et al., 2003). Even among arteries, significant variations exist. At rest, the shear rate in the common carotid artery is approximately 3.5 times higher than in the brachial artery (Dammers et al., 2003). Thus, apparent viscosity within a vessel is primarily governed by hematocrit and shear rate. Figure 2-10. The hematocrit-blood viscosity relationship. High hematocrits increase blood viscosity, particularly at low shear rates, due to enhanced RBC aggregation. © Chien S The red blood cells: v.2 - Chapter 26 - Biophysics Behavior of Red Blood Cells in Suspensions. 1975: 1031-1133. Page 1045. Modified with permission from Elsevier Science & Technology Journals 28 Even at high shear rates (>100 s⁻¹) a 1% increase in hematocrit can elevate blood viscosity by 4% (Rand et al., 1964; Chien et al., 1966). This has important implications during exercise, where hemoconcentration occurs increasing blood viscosity (Bouix et al., 1998; Connes et al., 2004; Connes et al., 2009). For example, following 30 minutes of exercise in young healthy men, hematocrit increased by ~9%, while whole blood viscosity measured at a shear rate of 1000 s-1 rose by ~21% (Bouix et al., 1998). This rise in whole blood viscosity during exercise is driven by both hemoconcentration (Ploutz-Snyder et al., 1995), and increased plasma viscosity (Bouix et al., 1998). Theoretically, an increase in blood viscosity could reduce blood flow by raising vascular resistance (see Equation 1: Hagen-Poiseuille equation). However, because blood is a shear-thinning fluid, hemoconcentration during exercise is offset by disproportionately greater increases in shear rate, resulting in a net decrease in apparent viscosity (Leo et al., 2020; Tremblay et al., 2020). This reduction in apparent viscosity may be beneficial, as hemoconcentration increases CaO₂, allowing more oxygen to be delivered with less internal fluid resistance (Leo et al., 2020). 2.3.6 Vessel diameter-blood rheology interactions The circulatory system spans an extraordinary range of vessel diameters from the 25,000 μm aorta to 4–8 μm capillaries—a difference of over 3,000-fold (Ellsworth et al., 1987; Masri et al., 2016), comparable to the size contrast between a basketball (~243,000 µm) and a human hair (~75 µm). This extreme variation profoundly influences blood rheology and flow dynamics. In large vessels (diameters > 300 μm), blood behaves as a continuous, shear-thinning fluid (Pries et al., 1992; Cokelet & Meiselman, 2007), as the 8 μm RBCs are much smaller than the vessel lumen (Pries et al., 1992; Yalcin et al., 2015). In contrast, within the microcirculation (e.g., capillaries, arterioles, venules), vessel diameters approach the size of RBCs, making flow behavior more akin to drinking bubble tea through a straw than to conventional fluid dynamics. As a result, blood exhibits fundamentally different flow properties in the micro- versus macrocirculation. In 1929, Robin Fåhræus and Torsten Lindqvist identified two key properties of microcirculatory blood flow. Blood flowing through a long narrow glass tube, the hematocrit within the tube is lower than the hematocrit in the discharge reservoir. This phenomenon, now known as the Fåhræus effect (Fåhræus & Lindqvist, 1931; Chebbi, 2015), occurs because RBCs migrate toward the center 29 of the vessel (i.e., axial migration), creating a cell-poor layer near the wall (Pries et al., 1992; Yalcin et al., 2015). RBC migration is primarily driven by shear-induced deformation arising from the parabolic velocity profile of laminar flow. Shear rates are highest near the vessel wall, causing RBCs to experience asymmetric shear stress, which generates hydrodynamic lift forces that push RBCs from the wall toward the centerline (Namgung et al., 2017). This behavior results in a concentrated stream of RBCs along the vessel centerline, with a plasma-rich (i.e., cell poor) layer forming near the vessel wall. This axial migration is also a key contributor to plasma skimming at vascular bifurcations, where RBCs preferentially remain in the central flow and enter larger daughter branches, leaving smaller branches relatively depleted of cells and thus with a lower local hematocrit (Yalcin et al., 2015). As a result of these biophysical mechanisms, apparent hematocrit and viscosity are reduced in small vessels compared to larger ones (Krogh, 1921; Fåhræus & Lindqvist, 1931; Namgung et al., 2017). The Fåhræus–Lindqvist effect is critical for microcirculatory flow, as it provides a lubricating cell-free layer near the vessel wall, facilitating efficient RBC movement through a fast-moving central stream. Impairments in RBC deformability decreases axial migration, as demonstrated in vivo rat arterioles, leading to abnormal microvascular flow (Namgung et al., 2017). However, the Fåhræus–Lindqvist effect, reaches a minimum at vessel diameters of approximately 6 to 8 μm (corresponding to the approximate diameter of capillaries), and then blood viscosity increases sharply as diameters decreases further (Pries et al., 1992). 2.4 Blood flow regulation: shear stress, blood viscosity and oxygen content This section will integrate shear stress, and blood viscosity as interconnected factors in blood flow regulation, concluding with a discussion on the influence of CaO2. 2.4.1 Shear stress-mediated control of blood flow Blood flow regulation is an intergraded process, involving a range of neural, local, mechanical, and hormonal mechanisms, as reviewed in (Joyner & Casey, 2015). Vascular control dynamically adjusts blood flow to match metabolic demand by combing sympathetic input (Fairfax et al., 2013), local vasodilators (Nyberg et al., 2010), and mechanical forces like shear stress (Doshi et 30 al., 2001). Shear stress is the tangential force exerted by blood moving across the endothelial surface and is determined by the product of apparent viscosity and shear rate. Shear rate (s-1) = (4 ⋅ [peak envelope velocity / D]) Equation 2.4.1.1 Where: SR (s-1) = shear rate; 4 = location of sampling gate correction (Thijssen et al., 2019) D (cm) = diameter SS=SR x n Equation 2.4.1.2 where: SS (Pa) = shear stress; SR (s⁻¹) = shear rate; n (mPa·s) = apparent viscosity (viscosity of blood at the given shear rate) (Leo et al., 2020). Accordingly, shear stress is proportional to blood velocity and viscosity, and inversely proportional to vessel diameter (Leo et al., 2020). However, this calculation reflects mean shear stress across the lumen, as the calibrator samples ~80% of the vessel. This approach underestimates the peak shear stress experienced at the vessel wall, particularly in large arteries and during pulsatile flow across the cardiac cycle due to high velocity gradients (Thijssen et al., 2019). Importantly, shear stress is maximal near the vessel wall, as this region exhibits the steepest velocity gradient (Reneman & Hoeks, 2008). The local shear rate at the wall not only determines wall shear stress but also strongly influences RBC aggregation, deformation, and axial migration (i.e., Fåhræus effect), all of which play essential roles in regulating vascular blood flow and tissue perfusion (Yalcin et al., 2005; Yalcin et al., 2015). Elevated shear stress is sensed by mechanosensitive structures on the endothelial surface (Ando & Yamamoto, 2013), triggering the releasing of endothelium-derived hyperpolarizing factors such as NO, prostaglandins and epoxyeicosatrienoic acids, (Doshi et al., 2001; Fischer et al., 2007; Petterson et al., 2021) ultimately leading to vasodilation (i.e., flow-mediated vasodilation [FMD]) (Celermajer et al., 1992). Shear stress is dynamically regulated through adjustments in vascular 31 diameter, playing a critical role in local blood flow regulation (Pyke et al., 2008). Shear stress has a considerable influence on both micro- and macrovasculature diameter (Koller et al., 1995; Huang et al., 2001). For example, in rats, arterioles can dilate up to three times basal diameter during supraphysiological saline infusion, largely mediated by through NO and prostaglandins pathways (Koller et al., 1995). Shear stress is also a key determinant of conduit artery diameter. For example, Pyke et al., (2008, demonstrated this by applying distal compression to the brachial artery during rhythmic handgrip exercise to control shear stress. Compression reduced blood velocity, while decompression restored it. The authors found that maintaining baseline shear stress during exercise hyperemia abolished the brachial artery FMD. This finding indicates that shear stress, and not muscle activation causes conduit artery vasodilation (Pyke et al., 2008). It also suggests that conductive vasodilation alone is insufficient to dilate the brachial artery during exercise (Segal et al., 1989). Similar findings were observed in the radial artery (Jazuli & Pyke, 2011) and during localized forearm heating (Pyke et al., 2008). It seems clear, therefore, that shear stress is a key regulator of vascular diameter in both the micro- and macrocirculation. 2.4.2 Shear stress patterns and magnitude in flow-mediated dilation Flow-mediated dilation is a widely used measure of endothelium-dependent vasodilation, assessed through various stimuli including reactive hyperemia (Green et al., 2013), exercise hyperemia (Padilla et al., 2006), pharmacological induced distal vasodilation (Mullen et al., 2001), and localized limb heating (Pyke et al., 2008). FMD occurs because microvascular vasodilation lowers downstream resistance, increasing blood flow in the upstream artery. The resulting rise in shear stress causes vasodilation (as reviewed in (Thijssen et al., 2011). The most common method for assessing FMD is brachial artery reactive hyperemia, which involves temporarily occluding the proximal forearm to induce ischemia, followed by a surge of blood flow upon cuff release (i.e., reactive hyperemia [RH]) (Thijssen et al., 2011). Shear stress patterns strongly influence the FMD response. Laminar flow, characterized by predominantly forward (antegrade) and minimal backward (retrograde) flow, promotes vascular health and exerts anti-atherogenic effects (Caro et al., 1969; Asakura & Karino, 1990). In contrast, regions with high retrograde flow (e.g., bifurcations) are more prone to atherosclerotic plaque 32 development (Caro et al., 1971). Human vasculature has adapted to function optimally in high mean shear stress conditions (Thijssen et al., 2009b). Even brief reductions in mean shear stress from inactivity (Thosar et al., 2015), or increased oscillatory shear patterns (Thijssen et al., 2009b; Tremblay et al., 2019c), can impair FMD. However, interventions that increase shear stress, such as hypoxia (Hanson et al., 2022), limb heating (Restaino et al., 2016; Coombs et al., 2021), or exercise (Thosar et al., 2015) can counteract impairments in endothelial function. The magnitude of the shear stress stimulus is a key determinant of the FMD response. For example, Pyke and Tschakovsky (2007) demonstrated that longer cuff occlusion durations (10, 20, 30, 40 50 seconds and 5-minutes) during RH-FMD caused dose-dependent increases in shear rate (a surrogate for shear stress). Authors found a strong positive association between shear rate and FMD, with shear rate explaining 56% of the variability in the RH-FMD response (Pyke & Tschakovsky, 2007). However, RH varies widely between individuals. Male sex, older age, smoking, elevated BMI, high fasting glucose, and increased pulse pressure are all associated with reduced RH (Mitchell et al., 2004). In addition, shear stress transduction differs significantly across individuals due to structural variations in conduit arteries and microvascular architecture (James et al., 1995), differences in the rate of shear stress propagation following occlusion (e.g., time to peak), and variations in NO bioavailability across health and disease states (Green et al., 2011). As result, the same shear stress can produce markedly different FMD responses between different individuals. For example, in a study of 51 children, 57 young adults, and 27 older adults, Thijssen et al., (2009) found that shear rate explained only 15% of the variability in the RH-FMD response across participants (Thijssen et al., 2009a). In contrast, a larger study found that shear rate accounted for 33.5% of the inter-individual variability in the RH-FMD response. Both the magnitude of shear stress (i.e., generated through RH) and the propagation of that mechanical force are critical determinants of FMD, and should be considered in analysis. Since blood viscosity directly influences shear stress generation, it too has a pivotal role in shaping the FMD response. 2.4.3 Blood viscosity and vascular resistance Elevated blood viscosity can paradoxically reduce vascular resistance by increasing shear stress, causing vasodilation. Animal studies provide strong evidence that both resistance and conduit 33 arteries dilate in response to acute increases in plasma and whole blood viscosity (Melkumyants et al., 1989; Melkumyants & Balashov, 1990; Martini et al., 2005; Tsai et al., 2005). In hamsters, isovolumic hemoconcentration initially lowers systemic vascular resistance as hematocrit rises, but only up to a ~12% increase. Beyond this point, further increases in hematocrit cause systemic vascular resistance to rise. However, this threshold can extend to ~20% when RBCs lack NO scavenging capacity, such as with methemoglobin-containing RBCs (Salazar Vázquez et al., 2008). These findings suggest that hemoglobin is a strong NO scavenger (Azarov et al., 2005), and this limits the vasodilatory effect of NO. Although NO has a very short half-life (~2 milliseconds) and must cross the RBC membrane and cytosol to reach hemoglobin, the NO scavenging by intact RBCs is still significant. Notably, this vasodilatory response is abolished by NO inhibition (Martini et al., 2005; Salazar Vázquez et al., 2008), indicating that increased blood viscosity reduces SVR via NO-dependent mechanisms. However, once blood viscosity exceeds a critical threshold, this response is no longer sufficient, and vascular resistance increases regardless of NO availability. In humans, Connes et al., (2012) found that 15 minutes of moderate exercise (105% of the first ventilatory threshold) increased whole blood viscosity by ~27% at a shear rate of 225 s -1. Increased blood viscosity was inversely associated with systemic vascular resistance and positively correlated with stable nitric oxide end-products (e.g., nitrite and nitrate). However, isovolumic hemodilution also decreased systemic vascular resistance, reducing whole blood viscosity by ~41% as measured using a porous-type disposable viscometer in healthy participants (Tu & Liu, 1996). Together, these findings suggest there is an inverted U-shaped relationship between hematocrit and systemic vascular resistance (Tu & Liu, 1996; Connes et al., 2012), at least within moderate hematocrit ranges in humans (Fan et al., 1980). Elevated blood viscosity enhances shear stress, promoting vasodilation and reducing vascular resistance, whereas decreased hematocrit may reduce vascular resistance by decreasing NO scavenging (Azarov et al., 2005), despite the lower shear stress. Thus, changes in hemoglobin can influence vascular resistance through blood viscosity and NO bioavailability leading to opposing, mechanisms. 34 2.4.4 Blood viscosity as a determinant of flow-mediated dilation Elevated blood viscosity may improve endothelium function by increasing shear stress. For example, Tinken et al., (2009) demonstrated that increasing mean shear stress via local exercise hyperemia, limb heating or indirectly from lower limb exercise improves brachial RH-FMD (Tinken et al., 2009). Accordingly, increasing blood viscosity may similarly enhance FMD by elevating shear stress. Supporting this, Verbeke et al., (2007) found that in seven patients with end-stage renal disease, anemia correction through three months of erythropoietin therapy (targeting hemoglobin > 110 g/dL) led to a 300% increase in limb-heating–induced FMD, potentially driven by enhanced shear stress (Verbeke et al., 2007). However, potential confounders such as reduced uremic toxins, oxidative stress, and inflammation may also have contributed to this improvement in vascular function (Hausberg et al., 1999; Verbeke et al., 2011; Iwamoto et al., 2018). Conversely, individuals with excessive erythrocytosis (Hb ≥21 g/dL), exhibit impaired FMD and larger brachial and common carotid artery diameters, suggesting chronic hyper-viscosity causes arterial remodeling (Rimoldi et al., 2012; Tremblay et al., 2019b). Acute and chronic hemoconcentration likely have distinct physiological effects. Acute reductions in hemoglobin has been show to increase RH-FMD in both healthy individuals (Hoiland et al., 2020b) and clinical populations (Keklik et al., 2017; Tremblay et al., 2019b), although this is not always the case (Gnasso et al., 2019). Whether acute increases in hemoglobin—and thus blood viscosity—enhances FMD remains unclear. 2.4.5 Oxygen demand the master regulator A key caveat in this discussion of shear stress, blood viscosity and hemoglobin is the neglect of oxygen demand and the role of oxygen delivery (DO₂) in regulating blood flow. DO₂, the amount of oxygen delivered per minute, is calculated as the product of blood flow and CaO₂ (Fick, 1870). Oxygen delivery and consumption are tightly coupled (González-Alonso et al., 2006), changes in CaO₂ are compensated by adjustments in blood flow (Tu & Liu, 1996). CaO2 = (1.34 ⋅ [Hb] ⋅ SaO2) + (0.0031 ⋅ PaO2) Equation 2.4.5.1 35 Where: CaO₂ (ml O2 per 100 ml of blood) = arterial oxygen content; 1.34 mL/g = oxygen-carrying capacity of hemoglobin; [Hb] (g/dL) = hemoglobin concentration; SaO₂ (%) = arterial oxygen saturation; 0.0031 = solubility coefficient of oxygen in plasma and PaO₂(mmHg) = partial pressure of oxygen in arterial blood ḊO2 = CaO2 ⋅ Q̇ Equation 2.4.5.2 Where: ḊO₂ (ml O₂/min) = oxygen delivery; CaO₂ (ml O2 per 100 ml of blood) = arterial oxygen content and Q̇(ml/min) = blood flow Dissociating the effects of CaO₂ from blood viscosity remains a significant challenge. For example, isovolumic hemodilution increases the post-occlusion blood flow response. Assuming that downstream signaling (i.e., ischemic induced microvascular vasodilation) is comparable between pre- and post-hemodilution, the greater post-occlusion blood flow could be attributed to either: (i) lower blood viscosity facilitating greater blood flow or (ii) a compensatory increase in blood flow to offset the reduction in CaO₂. The latter explanation is likely more plausible, given the central role of oxygen in regulating blood flow (González-Alonso et al., 2006). Moreover, a higher blood flow response would increase shear rate, potentially preserving shear stress despite a lower blood viscosity (Hoiland et al., 2020b). Conversely, during hemoconcentration, reduced blood flow (i.e., reduced shear rate) may counterbalance increased blood viscosity, preserving net shear stress. Thus, the dynamic interaction between shear rate and CaO₂ may offset changes in shear stress that would otherwise result from altered blood viscosity. The independent effect of blood viscosity, separate from CaO₂, remains unclear. Lindenfeld et al., (2005) demonstrated that in polycythemic dogs, raising hematocrit by ~42% using methemoglobin RBCs (which do not carry oxygen), resting and exercise cardiac output did not decrease as hypothesized. In contrast, converting methemoglobin to oxyhemoglobin with methylene blue infusion increased CaO₂ by ~43%, without changing hematocrit or blood viscosity, resulting in a 36 reduction in cardiac output during exercise. Similarly, individuals with excessive erythrocytosis show no reduction in DO₂ during handgrip exercise compared to healthy controls (Tremblay et al., 2019a), nor is maximal oxygen uptake impaired (Hansen et al., 2021). These findings suggest that elevated blood viscosity, even in polycythemic conditions, does not limit DO₂. Shear stress and blood viscosity are likely secondary to oxygen demand. Although blood rheology may not directly dictate blood flow, it remains essential for regulation. It buffers blood viscosity increases during exercise-induced hemoconcentration, promotes the release of vasodilators from RBCs, and transmits shear stress to the endothelium to induce vasodilation. 2.5 Blood rheology in applied and clinical contexts Blood rheology is highly dynamic responding to physiological and environmental stressors. This final section will highlight the influence of high altitude and plasmapheresis on blood rheology, while connecting hemorheology to blood flow. The section will conclude with a discussion on blood rheology in the context of disease and health. 2.5.1 High altitude exposure and blood rheology Hypoxic exposure, whether from reduced barometric pressure (e.g., high-altitude) or normobaric hypoxia (i.e O2 13%), reduces CaO2. CaO2 is fiercely defended through a series of integrated acute and chronic acclimatization responses. Acutely, (~seconds to minutes), ventilation (Cymerman et al., 1989) and cardiac output (Vogel & Harris, 1967) increase to counter the decline in CaO2. After ~24 hours, plasma volume contraction begins, leading to hemoconcentration (Schlittler et al., 2021). With continued exposure, RBC volume increases through erythropoiesis (Rasmussen et al., 2013; Siebenmann et al., 2015). Blood viscosity increases at high altitude due to elevated hematocrit and is further exacerbated by hypoxia-induced reductions in RBC deformability (Wood & Appenzeller, 1988; Gilbert-Kawai et al., 2017; Emeric, 2021; Salvi et al., 2023; Steele et al., 2024). Salvi et al., (2023) observed a progressive decline in RBC deformability with ascent to 5050 meters. In contrast, Grau et al., (2016) found that severe hypoxia (10% O2) increased RBC deformability, while moderate normobaric hypoxia (16.21% O2) decreased it (Grau et al., 2016). One possible explanation is that 37 hypoxia elevates RBC oxidative stress (Härtel et al., 2022), impairing RBC deformability. However, under severe hypoxia, increased blood flow and shear stress may enhance RBC eNOS activity, promoting NO production and compensating for elevated oxidative stress (Leo et al., 2023). Hypoxia also increases the release of circulating reticulocytes (i.e., immature RBCs) (Emeric, 2021; Härtel et al., 2022), which are less deformable than mature RBCs (Chasis et al., 1989). Thus, both elevated oxidative stress and a higher reticulocyte proportion in blood likely contribute to reduced RBC deformability during hypoxia. 2.5.1.1 The relationship between blood rheology and blood flow at high altitude Reduced RBC deformability combined with hemoconcentration may impair blood flow at high altitude. Lowlanders ascending to 5050 meters exhibit impairments in sublingual capillary blood flow with progressive ascent (Gilbert-Kawai et al., 2017; Salvi et al., 2023), decreasing the number of perfused capillaries per area, and attenuating RBC velocity. RBC deformability was also found to be reduced, suggesting that slower RBC velocities may be driven, at least in part, by impaired deformation (Salvi et al., 2023). In contrast, RBC deformability and sublingual capillary blood flow was not decreased in indigenous Nepalese highlanders (i.e., Sherpa) during ascent to 5050 meters, indicating that evolutionary pressures may preserve microcirculatory flow and RBC flexibility in this population (Gilbert-Kawai et al., 2017; Salvi et al., 2023). However, high altitude also increases sympathetic nerve activity decreasing vascular function (Tremblay et al., 2018). After 14–21 days at 4,440 meters, endothelial-dependent vasodilation to acetylcholine is reduced, but this impairment was abolished with adrenergic blockade (Tymko et al., 2020). This suggests that elevated sympathetic activity, rather than impaired RBC deformability, may contribute to microvascular dysfunction at high altitude, although these mechanisms may not be mutually exclusive. Together, these findings suggest that both impaired RBC deformability and heightened sympathetic activity may independently or synergistically limit microvascular perfusion at high altitude. 2.5.2 Plasmapheresis-induced changes in blood rheology In 1914, Abel, Rowntree, and Turner developed a technique that could remove large volumes of plasma without harming the subject, provided RBCs were returned. A dog weighting only 11 38 kilograms had 1705 cc of plasma removed over two days, equivalent to ~15% of its weight or twice its total blood volume. Remarkably, the dog was reported to be: “…. very well and lively, runs about, eats meat (Abel et al., 1914).” Decades later, Kliman et al., (1964) similarly removed 5 liters of plasma over five days from healthy human volunteers and found that participants had no change in appetite or sense of well-being — very similar to dog (Kliman et al., 1964) Since its inception, plasmapheresis – the removal, treatment, and return of blood plasma or components to the blood circulation – has become a prominent medical procedure. Historically, plasmapheresis has been a therapeutic intervention, with plasma removed and replaced to eliminate pathogenic elements such as autoantibodies, toxins or infectious agents (Palmer et al., 1989; Chaidemenos et al., 1997; Busund et al., 2002). More recently, there has been growing excitement around the development of plasma-derived medications (e.g. immunoglobulins, clotting factors and albumin) for therapeutic uses. As a result, plasma demand has surged five-fold over the past two decades, with nearly 40 million liters of plasma required in the United states alone (Hartmann & Klein, 2020). Plasmapheresis typically uses centrifugation to separate blood components based on specific gravity (Gurland et al., 1984; Waitz et al., 2022). Blood is drawn into an apheresis machine, where plasma is separated from denser components. Plasma is collected and stored for future use, while the remaining cellular components are returned to the donor through the same venous access site (Lee & Arepally, 2012). This cycle of blood extraction, plasma separation, and reinfusion is repeated multiple times during a session, typically lasting 60-90 minutes (Figure 2-12). Plasmapheresis decreases protein concentration, (Schooneman et al., 1997), blood volume (Roberts et al., 2000), and exposes RBCs to elevated mechanical shear stress, artificial surfaces (Hirayama et al., 1985) and anticoagulants (Das et al., 2005; Lee & Arepally, 2012). Despite these stressors, little is known about their effects on blood rheology, particularly on RBC mechanics, which may be significantly altered. 39 Artificial circulatory systems such as hemodialysis machines (Ene-Iordache & Remuzzi, 2012), heart valves (Hirayama et al., 1985), cardiopulmonary bypass (Kameneva et al., 1999), and apheresis machines (Bor-Kucukatay et al., 2008) can expose RBCs to high mechanical stress. Even brief exposure to shear forces exceeding 150 Pa, such as those encountered during cardiopulmonary bypass (Leverett et al., 1972), is sufficient to cause hemolysis. While RBCs may initially benefit from moderate shear stress below 10 Pa by enhancing flexibility (Horobin et al., 2020), mechanical stress beyond physiological thresholds leads to a marked decline in cellular function. Hemolysis occurs at shear stresses near 150 Pa, but even shear stress as low as 30 Pa, have been shown to impair RBC deformability (Ekeström et al., 1983; Simmonds & Meiselman, 2016). There has been an emerging emphasis on the impact of this sublethal trauma on RBC mechanics and integrity (see (McNamee & Simmonds, 2023) for a comprehensive review). Prolonged exposure to mechanical forces found in circulatory support devices (e.g., ventricular assist devices) has been linked to secondary complications including multi-organ failure, latent infection, and neurological events like stroke, which may arise independent of the patient's initial condition. Persistent blood trauma is believed to drive these outcomes, though whether similar effects occur during procedures such as plasmapheresis remains unclear. Sublethal shear stress (50 – 100 Pa) for five minutes increases the elastic shear modulus of RBCs without altering bulk deformability. These changes are thought to result from cytoskeletal remodeling, protein denaturation, and glycoprotein cleavage. Beyond affecting mechanical properties, elevated shear stress has also been shown to alter RBC surface charge (electronegativity), a key determinant of cell aggregability (McNamee et al., 2018b). These changes may promote RBC aggregation and increase blood viscosity at low shear rates, such as in the venous circulation (McNamee et al., 2018b). Interestingly, repeated shear stress (e.g., 64 Pa × 3 for 10 minutes) demonstrate an initial decline in RBC deformability followed by a recovery then a secondary decline (McNamee et al., 2016). This pattern may reflect the selective hemolysis of older, more fragile RBCs during early exposure, leaving behind a population of more resilient, younger cells that temporarily maintain deformability. However, with continued exposure, even these young robust cells may incur damage, resulting in a subsequent decline in function (Kuck et al., 2020a). It is possible that plasmapheresis selectively removes less deformable RBCs without 40 significantly affecting younger, more flexible cells. Conversely, this may cause anemia longterm, which is a consequence of chronic and frequent plasma donations (Rosa-Bray et al., 2013). Few studies have investigated blood rheology following plasmapheresis; however, thrombocytapheresis has been shown to impair RBC deformability in healthy donors, a defect that was reversible with administration of the NO donor sodium nitroprusside (Bor-Kucukatay et al., 2008). Piezo1 channels, which regulate mechanosensitive signaling in RBCs, are directly involved in eNOS activation (Kuck et al., 2022b). This may imply that impaired NO signaling may contribute to reduced RBC deformability via reduced mechanotransduction following apheresis, alongside other potential mechanical insults. Thus, plasmapheresis may impair RBC function by limiting NO bioavailability, further underscoring the need to investigate the blood rheology consequences of plasmapheresis. The implications of altered blood rheology in plasmapheresis on blood flow will be discussed next 41 Figure 2-11. Schematic of plasmapheresis (left) and real-world photo (right). Blood is draw from the donor, spun to separate components, the plasma is collected, and the remaining cellular components are infused with anticoagulant © Hong G, Lee E, Yerigeri K, Raina R Advances in Apheresis Techniques and Therapies in the Pediatric Setting. Current Pediatrics Reports (2022);10(2) 214–226. Page 216. Modified with permission from Springer Nature BV 42 2.5.2.1 Effects of plasmapheresis on blood flow Plasmapheresis may alter blood flow and reactivity through increased shear stress following hemoconcentration (see section 2.4.3 and section 2.4.4), decreased blood pressure, increased microvascular flow, and the independent effects of anticoagulants. Plasmapheresis reduces plasma volume and protein concentration leading to a decrease in blood pressure (Schooneman et al., 1997; Yeh et al., 2002; Tran-Mi et al., 2004). Hypoproteinemia, even in the absence of hypovolemia, can lower blood pressure. Yeh et al., (2002) reported a positive association between the amount of globulin removed and the decreases in both systolic and diastolic blood pressure (Yeh et al., 2002). This is supported by animal studies demonstrating that hypoproteinemia alone can reduce arterial pressure by lowering plasma colloid osmotic pressure (Manning & Guyton, 1983). Both hypovolemia and hypoproteinemia likely contribute to the hypotensive effects of plasmapheresis. Reinhart et al (1992) and Mansouri Taleghani et al (1997) both observed that blood flow velocity in nail-fold capillaries increased following plasmapheresis, despite no change in RBC deformability (Reinhart et al., 1992; Mansouri Taleghani et al., 1997). Reinhart et al., (1992) further reported that, in patients with hyperviscosity, increases in capillary blood cell velocity correlated with reductions in plasma viscosity. Authors proposed that elevated plasma viscosity decreased microcirculatory flow in this population. These findings suggest that plasmapheresis may enhance microvascular perfusion by lowering plasma viscosity, even in the absence of changes in RBC deformability. Anticoagulants are required during reinfusion to prevent clotting, which may impact vascular tone. In Canada, citrate dextrose formula A is commonly used (Canada, 2023). Citrate chelates ionized calcium, reducing coagulation (Szymanski, 1978; Silberstein et al., 1986; Das et al., 2005). Ionized calcium concentrations are positively associated with arterial tone following hemodialysis (Scholze et al., 2005), and with systolic and diastolic pressure in healthy individuals (Kesteloot & Geboers, 1982) and emergency patients (Desai et al., 1988). In patients with coronary artery disease, 10 weeks of EDTA therapy to reduce ionized calcium did not enhance endothelium-43 dependent vasodilation to acetylcholine unless combined with vitamin therapy. However, EDTA alone showed a trend toward improved endothelium function, even without vitamin supplementation (Green et al., 1999). Notably, testing was conducted one week after the final treatment, so the acute effects remain unclear. It is possible anticoagulant infusion alters blood flow and reactivity flowing apheresis. Overall, plasmapheresis may alter vascular reactivity and blood flow through hemoconcentration, changes in RBC deformability and aggregation, increased microcirculatory flow, and the use of anticoagulants. 2.5.3 Blood rheology in cardiovascular disease and health Blood rheology has a significant impact on cardiovascular health, with alterations in blood viscosity, RBC deformability and aggregation increasingly recognized as risk factors for cardiovascular disease. This section concludes by briefly highlighting the role of blood rheology in both disease and health. 2.5.3.1 Blood rheology alterations in disease Altered blood rheology has been implicated as a risk factor for cardiovascular disease. The Edinburgh Artery Study (1997) demonstrated that even after adjusting for major risk factors including — age, sex, smoking, blood pressure and low-density lipoprotein cholesterol — hematocrit, blood viscosity, plasma viscosity and fibrinogen levels were all independently associated with an increased relative risk for cardiovascular events such as ischemic heart disease and stroke (Lowe et al., 1997). Several mechanisms may underline this association including elevated blood viscosity (Gustafsson et al., 1980, 1981), increased RBC aggregation, (Yu et al., 2011) and impaired RBC deformability (Tan et al., 2020). Elevated blood viscosity may increase blood pressure, particularly when vasodilatory capacity is compromised (Gustafsson et al., 1980, 1981), common to hypertension (Panza et al., 1995), heart failure (Katz et al., 1993) and coronary artery disease (Ludmer et al., 1986) That is, when blood viscosity rises without sufficient compensatory vasodilation, arterial pressure can increase 44 (Gustafsson et al., 1980, 1981). Supporting this, several clinical studies have reported an association between elevated blood viscosity and hypertension (Letcher et al., 1981; Leschke et al., 1987; Leschke et al., 1990; Khan et al., 2005; Hermida-Ameijeiras et al., 2024). These findings suggest that increased blood viscosity may contribute to the development of hypertension and the broader pathophysiology of cardiovascular disease (Leschke et al., 1987). Elevated hematocrit and increased fibrinogen levels are common findings in rheological studies of cardiovascular disease, and are frequently associated with elevated RBC aggregation (Razavian et al., 1994; Cicco & Pirrelli, 1999; Ami et al., 2001; Zorio et al., 2008). Elevated RBC aggregation has also been observed in hypertensive patients compared to healthy controls, even after controlling for hematocrit concentrations (Razavian et al., 1992). Together, elevated hematocrit and increased RBC aggregation can promote clot formation, particularly under low shear stress conditions, as demonstrated in preclinical models (Yu et al., 2011). This mechanism likely contributes to the strong associated between hematocrit and increased thrombosis risk (Tefferi, 2012; Verstovsek et al., 2023). Reduced RBC deformability is a common occurrence across a range of cardiovascular diseases including hypertension (Fornal et al., 2011), heart failure (Chimura et al., 2017) cerebrovascular disease (Bowton et al., 1989), and coronary artery disease (Porro et al., 2020). It is also seen in traditionally non-cardiovascular conditions such as chronic fatigue syndrome (Saha et al., 2018; Saha et al., 2019), multiple sclerosis (Simpson et al., 1987), and Alzheimer’s disease (Chang et al., 2007; Strijkova-Kenderova et al., 2021), suggesting that RBC deformability has a broad impact on overall health. Like endothelial dysfunction, reduced RBC deformability is driven by elevated oxidative stress (McNamee et al., 2018a) and inflammation (Jacob et al., 2024). Impaired RBC deformability reduces capillary requirement (Parthasarathi & Lipowsky, 1999), disrupts normal microcirculatory flow by reducing the cell-free layer (Sasaki et al., 2017), and promotes RBC aggregation (Chien et al., 1970), all of which elevate blood viscosity. Furthermore, RBC deformability likely has a significant role in RBC release of NO and ATP, significant for vasodilation (Ulker et al., 2010; Racine & Dinenno, 2019). The coexistence of impaired RBC 45 deformability and microvascular dysfunction likely contributes to the severity and progression of microvascular diseases such as seen in diabetic retinopathy (Tan et al., 2020). 2.5.3.2 Blood rheology in endurance trained athletes In contrast, to disease populations, athletes typically exhibit a rheology profile characterized by increased RBC deformability, lower RBC aggregation and decreased plasma viscosity. A meta-analysis found that chronic exercise training, ranging from 12 weeks to one year, led to reductions in hematocrit, blood viscosity and RBC aggregation (Romain et al., 2011). These changes are likely driven by exercise-induced plasma expansion (Oberholzer et al., 2019), where increased plasma volume dilutes plasma proteins and reduces plasma viscosity (Charm et al., 1979). Studies comparing untrained and trained individuals, both amateur (Tomschi et al., 2018) and professional (Smith et al., 1999), have shown elevated RBC deformability in trained groups, with a dose-dependent relationship between training status and RBC deformability (Tomschi et al., 2018). Exercise training may enhance RBC deformability by increasing the proportion of low-density RBCs and/or improving anti-oxidant capacity. When comparing low- and high-trained individuals, those with higher training levels possess a greater proportion of low-density RBCs (i.e., younger cells) relative to high-density RBCs (i.e., older cells), suggesting that training increases RBC genesis and subsequent turnover, leading to a more deformable RBC population (Smith et al., 1999; Tomschi et al., 2018). Additionally, intensive aerobic exercise promotes mild cellular oxidative stress and peroxidation, which may induce positive remodeling of the RBC membrane and upregulation of anti-oxidant mechanisms, to better withstand future aerobic challenges (Nemkov et al., 2021). While still not fully characterized, the blood rheology profile appears to include a greater plasma volume, reduced RBC aggregation, enhanced RBC deformability and heighted RBC anti-oxidant capacity. These blood rheology characteristics likely support improved blood flow and oxygen delivery. 46 Chapter 3: Study 1 - Effects of plasmapheresis on blood rheology and peripheral vascular function. Abstract Plasmapheresis - the removal, treatment, and return of blood plasma or components - is widely used for both therapeutic and donation purposes, yet its physiological effects on blood rheology and vascular function remain poorly understood. This study examined the effects of plasmapheresis on blood viscosity, RBC deformability and aggregation, and endothelium-dependent vasodilation, and compared them to furosemide-induced hemoconcentration to isolate the role of plasma volume contraction. To distinguish the effects of plasma contraction and hemoconcentration from those associated with hypoproteinemia and extracorporeal circulation during plasmapheresis, a second investigation was conducted using acute oral administration of furosemide (40 mg) to induce diuresis. In Investigation 1, fifteen participants (7F; 25±5 years) underwent plasma donation and were assessed at baseline, immediately after, and 24-hours later. In Investigation 2, thirteen participants (6F; 24±5 years) underwent diuresis, with measurements taken pre- and four hours post-furosemide administration. Blood rheology parameters and brachial artery FMD were assessed at each time point. Both interventions similarly increased hemoglobin (~8%) and whole blood viscosity (7.6% immediate post-donation; 13.1% post-diuresis, p<0.01), but neither altered basal shear stress, blood flow, or vascular reactivity. Plasma donation, but not diuresis, acutely reduced RBC deformability (theoretical maximum elongation index, p=0.04) and increased reactive hyperemia (~23%, p=0.04), with RBC deformability recovering (p=0.59) and RBC aggregation (aggregation index) being reduced by 24 hours (p=0.03). Systolic blood pressure decreased following plasmapheresis (p=0.01) and remained reduced at 24 hours (p=0.03), with no changes observed after diuresis. These findings indicate that plasma donation transiently impairs RBC deformability, reduces systolic pressure, and enhances microvascular reactivity without affecting FMD. The enhanced microvascular response was unlikely due to hemoconcentration and may instead reflect the influence of anticoagulants or changes in RBC deformability. 47 Introduction: Plasmapheresis - the removal, treatment, and return of blood plasma or components to the blood circulation (Gurland et al., 1984; Waitz et al., 2022) - is a critical treatment modality for various medical disease including neurological disorders (Moser et al., 2019), autoimmune diseases (Batocchi et al., 2000), and severe sepsis (Connelly-Smith et al., 2023; Kuklin et al., 2024). Beyond therapeutic applications (McNamee et al., 2018b), there is a growing interest in the use of donated plasma for the development of plasma-derived medications, (e.g. immunoglobulins, clotting factors and albumin) (Peyvandi et al., 2016; Covington et al., 2022). As a result, global demand for plasma has increased 5.3-fold over the past two decades. In the United States alone, nearly 40 million liters of plasma was collected from over 50 million donations (Hartmann & Klein, 2020). Despite this growing use, there is a paucity of research examining the effects of plasmapheresis on blood rheology and vascular health. Plasmapheresis, an extracorporeal technique, separates blood components (e.g., plasma, white blood cells, and red blood cells [RBCs]) using centrifugal forces based on their specific gravity (Gurland et al., 1984; Waitz et al., 2022). During plasmapheresis, RBCs are exposed to elevated mechanical shear stress, artificial surfaces, and anticoagulants (Schooneman et al., 1997), which may compromise RBC integrity and viability (Simmonds & Meiselman, 2016). Standards (e.g., ASTM F756-17) require that extracorporeal devices do not cause hemolysis (i.e., the destruction of RBCs) (ASTM, 2017). However, even sublethal mechanical trauma can compromise RBC integrity. While hemolysis typically occurs at shear stresses around 150 Pa, impairments in RBC deformability have been observed at shear stress, as low as 32 Pa (Simmonds & Meiselman, 2016). These mechanical alterations are thought to result from cytoskeletal remodeling, protein denaturation, and glycoprotein cleavage (Manno et al., 2002; Mizuno et al., 2002; Watanabe et al., 2007). In addition to changes in deformability, elevated shear stress can also reduce RBC surface electronegativity, a key factor in preventing aggregation (McNamee et al., 2018b). These changes may promote RBC aggregation and increase blood viscosity, particularly at low shear rates such as those found in the venous circulation. Given the potential for elevated shear stress in 48 extracorporeal circuits, the effects of plasmapheresis on blood rheology and RBC function are clinically significant (Hardeman et al., 2001). Recent evidence has demonstrated that RBCs possess functional endothelial nitric oxide synthase (eNOS), which is directly linked to mechanotransduction via the mechanosensitive ion channel Piezo1(Kuck et al., 2022b). Kuck et al. (2022) demonstrated that rigidifying RBCs reduces deformability and impairs eNOS activity. These findings could suggest that apheresis may impair nitric oxide (NO) production through sublethal mechanical injuries. Supporting this hypothesis, thrombocytapheresis has been shown to reduce RBC deformability in healthy donors, a defect that was reversible with administration of the NO donor sodium nitroprusside (Bor-Kucukatay et al., 2008). Thus, plasmapheresis may impair RBC function by limiting NO bioavailability, among other potential biochemical insults. Similarly, blood transfusions have been associated with decreased endothelium-dependent vasodilation (Neuman et al., 2015) and impaired microvascular function (Parviz et al., 2016), both linked to disrupted NO signaling (Alexander et al., 2013; Reynolds et al., 2013). Since apheresis may similarly reduce RBC-derived NO bioavailability, it is plausible that comparable vascular impairments could occur following plasmapheresis. However, some clinical studies have reported increased blood flow velocity in nail-fold capillaries after plasmapheresis, suggesting enhanced microcirculatory perfusion (Reinhart et al., 1992; Mansouri Taleghani et al., 1997). These seemingly contradictory findings highlight the complexity of the physiological effects of plasmapheresis and the need for further investigation into its impact on vascular function. To examine the impact of plasmapheresis on blood rheology, and endothelial-dependent vasodilation (flow-mediated dilation [FMD]), participants were tested immediately before, immediately after and 24-hours following plasmapheresis. This design allowed us to capture both the acute and residual effects of plasmapheresis on blood rheology and peripheral vascular function, while also allowing time for the effects of anticoagulants and hemoconcentration to dissipate (Evers et al., 2014). To isolate the effects of plasma contraction and hemoconcentration from those of hypoproteinemia and extracorporeal circulation (i.e., plasmapheresis), a second investigation was conducted using acute oral administration of furosemide (40 mg) to induce 49 diuresis. It was hypothesized that brachial blood flow and vascular reactivity would be lower following plasmapheresis relative to diuresis, due to rheological alterations. Specifically, it was expected plasmapheresis to reduce RBC deformability and FMD immediately post-procedure, with these effects resolving by the 24-hour follow-up. Methods Two separate investigations were conducted to examine the effects of plasma donation (Investigation 1) and plasma volume contraction (Investigation 2) on blood rheology, flow, and vascular reactivity, in order to isolate the specific effects of plasmapheresis. These investigations adhered to the Declaration of Helsinki (except registry in a database) and the Canadian Tri-council Policy Statement for Integrity in Research. The protocol was approved by the University of British Columbia’s Clinical Research Ethics Board (H24-03340 and H20-03708). Participants provided informed consent after reviewing a detailed study information form and discussing the protocol with an investigator. Investigation 1 – Plasma donation (PD) Fifteen participants (7 females, mean age: 25 ± 5 years) were recruited. Inclusion criteria included normotension (systolic blood pressure < 130 mmHg, diastolic < 90 mmHg), free of any chronic illnesses (e.g., cardiovascular, respiratory, or metabolic) or daily medication use. An additional five participants, served as time-controls. Menstrual cycle phase and oral contraceptive use were recorded but were not used as exclusion criteria. Each participant attended three lab sessions: immediately before plasma donation (pre-PD), immediately after (post immediate-PD) and 24 hours post-donation (post 24-hour-PD). All testing occurred at the University of British Columbia - Okanagan, while plasmapheresis was performed at a certified donation clinical approximately ten minutes away. The post-immediate timepoint occurred 16 ± 4 minutes after plasma donation was completed. Participants were instructed to follow their normal dietary and caffeine routines 50 but to avoid consuming calories for at least two hours prior to each session. To promote consistency, they were also asked to replicate their food and fluid intake on all testing days. The experimental protocol was consistent across all time points. All physiological assessments were conducted in the supine position following a 10-minute rest period. During plasma donation, participants were in a semi-recumbent position; however, testing posture was standardized across all time points to minimize postural influence on fluid distribution. A butterfly needle was first inserted for venous blood collection, followed by a 10-minute rest period prior to completing the remaining assessments. Blood pressure and heart rate were then measured using an automated blood pressure monitor (Omron HEM-907XL, Omron Healthcare, Kyoto, Japan), and a reactive hyperemia flow-mediated (RH-FMD) test was then performed. Plasmapheresis was performed using a centrifugal apheresis machine (NexSys PCS, Haemonetics, Boston, MA, USA). All procedures were conducted by trained medical personnel and adhered to the guidelines outlined in Health Canada's Food and Drugs Act and Blood Regulations for plasma collection and donor safety (Canada, 2023). Peripheral venous access was established using a 16-gauge peripheral catheter placed in the antecubital vein to ensure efficient plasma collection while minimizing donor discomfort. Plasma removal volumes were determined based on participant body weight and height, with 70-90 mL acid-citrate-dextrose, solution A (ACD-A) used as the anticoagulant. Participants were continuously monitored for potential adverse effects, including hypotension, vasovagal reactions, and allergic responses, with immediate medical intervention available if necessary. Post-procedure monitoring ensured hemodynamic stability, and all participants were provided with post-donation care instructions. Time-control participants (2 females, mean age: 22 ± 2 years; BMI: 23 ± 2), followed the same experimental protocol but did not donate plasma. Instead, they rested for approximately two hours between the pre- and post-immediate time points (112 ± 21 minutes), matching the timing of the experimental condition. The post–24-hour visit occurred 24 hours after the post-immediate time point. Investigation 2 – Plasma contraction 51 Thirteen young, healthy participants were recruited (female: 6; age: 24 ± 5 years; BMI: 24 ± 2 kg · m−2). Participants were instructed to avoid alcohol, caffeine, and exercise for 12 hours and to fast for at least 4 hours before testing. The experimental protocol was identical at both time points: pre-plasma contraction (Pre-PC) and post-plasma contraction (Post-PC). Upon arrival at the laboratory, participants provided a urine sample to confirm adequate hydration (urine specific gravity ≤1.020 g/ml; TS 400, Reichert Analytical Instruments, Depew, NY). An antecubital catheter was then inserted for venous blood sampling and removed immediately after collection. Participants then rested for 10 minutes while monitoring equipment was prepared. Heart rate was recorded via lead II ECG (ML138 Bio Amp, ADInstruments, Dunedin, New Zealand), and beat-by-beat blood pressure was measured using finger photoplethysmography (Finapres Medical Systems, Amsterdam, The Netherlands), calibrated against an automated brachial blood pressure reading (Omron HEM-907XL, Omron Healthcare, Kyoto, Japan). Vascular assessments were then conducted specifically RH-FMD and handgrip exercise. Hypovolemic hemoconcentration was then induced with an oral dose of furosemide (40 mg), a loop diuretic that blocks the Na⁺/K⁺/2Cl⁻ co-transporter in the thick ascending limb of the loop of Henle. Based on its pharmacokinetics, peak plasma concentration within 1–3 hours and half-life of 0.5–2 hours in healthy individuals (Van Wart et al., 2014), experimental measures were repeated 4 hours post-administration (Romero et al., 2011; Moralez et al., 2012). Total urine volume was collected over the 4-hour period to verify diuretic efficacy. Experimental Measurements: Blood rheology and blood parameters Venous blood was drawn into 6 mL EDTA-coated vacutainers, gently inverted, and mixed using an automated rotating device (The Jenna, HHH Technologies, Kelowna, BC, Canada) to ensure homogeneity prior to analysis. Whole blood viscosity was assessed using a cone-and-plate viscometer (DV2T, Brookfield Amtek, Middleboro, MA, USA) regulated at 37°C with a 52 circulating water bath (TC-150, Brookfield Amtek). Measurements were conducted using a CPA-40Z spindle. Viscosity was recorded at seven defined shear rates (45, 67.5, 90, 150, 225, 337.5, and 450 s⁻¹) to model a two-phase exponential decay curve, as described by (Leo et al., 2020). Each sample was analyzed in duplicate; if variation exceeded 0.1 mPa·s at 225 s⁻¹, a third reading was taken, and the average of the two closest values was reported to ensure a coefficient of variation <1%. Plasma viscosity was evaluated only in Investigation 1 (plasma donation). Plasma was separated by centrifugation at 600 g for 10 minutes at 4°C, then analyzed at shear rates of 150, 250, and 450 s⁻¹ RBC deformability was assessed using a laser-assisted optical rotational cell analyzer (LORRCA; RR Mechatronics, Hoorn, The Netherlands). Approximately 14 μL of whole blood was diluted into 5 mL of an isotonic polyvinylpyrrolidone solution prepared in a 0.1 mol·L⁻¹ phosphate-buffered saline (0.14 mM; 300 mOsmol/L; viscosity 29.9 mPa·s at 37°C). A 880 μL aliquot of this suspension was then loaded into the Couette system (bob configuration), where it was subjected to controlled shear stress. RBCs were exposed to nine increasing shear stresses: 0.3, 0.57, 1.08, 2.04, 3.87, 7.34, 13.94, 26.38, and 50 Pa. As the sample rotated, a laser beam passed through, generating a diffraction pattern. RBC deformability was determined by analyzing changes in this pattern, with the elongation index (EI) calculated at each shear stress using the equation: EI = (L − W) / (L + W), where L and W represent the length and width of the diffraction ellipse, respectively. The RBC aggregation dynamics were assessed in Intervention 1 using the LORRCA system, based on the syllectogram, which represents the relationship between laser backscatter intensity and time as previously described (Hardeman et al., 2001). To account for hemoconcentration following plasmapheresis, plasma was reintroduced to restore autologous hematocrit, as higher hematocrit levels are known to increase RBC aggregation (Hardeman et al., 2001). A complete blood count was performed at each time point in Investigation 1 using a hematology analyzer (DxH 520; Beckman Coulter, Mississauga, ON, Canada). Parameters included white blood cells count (WBC), RBCs count, hematocrit (HCT), mean corpuscular volume (MCV), RBC distribution width (RDW), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin 53 concentration (MCHC), and platelets (PLA). High, medium, and low calibration standards were run daily to ensure data accuracy. Investigation 2 (plasma contraction), HCT was measured using the direct micro method. Whole blood was drawn into heparinized capillary tubes and centrifuged using a PowerSpin BX Centrifuge (Unico) for 10 minutes at 11,000 rpm. After centrifugation, the tubes were placed on a hematocrit reader, a manual device with calibrated lines and numerical values, which allowed direct visual determination of HCT as the percentage of packed red cell volume relative to the total blood column. Measurements were performed in duplicate for accuracy. HGB was assessed using the photometric method (Hemocue Hb 201+ Analyzer, HemoCue, Ängelholm, Sweden Duplex ultrasound Brachial artery diameter and blood velocity was measured at the distal third of the left brachial artery using a 10-MHz multifrequency linear array Duplex ultrasound system (Terason t3000; Teratech, Burlington, MA, USA). Imaging sites were individually chosen to optimize image clarity, with a consistent insonation angle of 60° maintained across all time points. Standardized ultrasound settings (e.g., gain and dynamic range) and anatomical landmarks were used to ensure reproducible images (Thijssen et al., 2011; Thijssen et al., 2019). All recordings were captured using screen-capture software and stored for later offline analysis. Reactive hyperemia flow mediated dilation (RH-FMD) The RH-FMD was assessed in accordance with established guidelines (Thijssen et al., 2011; Thijssen et al., 2019). A one-minute baseline measurement of brachial artery diameter and blood velocity was first recorded simultaneously. A pneumatic cuff, positioned distal to the elbow (below the epicondyles), was then inflated to >220 mmHg for 5-minutes to induce arterial occlusion. Upon rapid cuff deflation, diameter and blood velocity measurements continued for an additional 3 minutes to capture the hyperemic response. Exercise hyperemia 54 Exercise hyperemia was assessed using rhythmic forearm handgrip exercise, consisting of 1-second contractions followed by 3 seconds of relaxation, performed for 3 minutes each at 5%, 15%, and 25% of maximal voluntary contraction (MVC) using feedback from researchers, a metronome (Pro Metronome, EUMLab–Xanin Technology, GmbH, Berlin, Germany ) and visual feedback from the using the handgrip force transducer (MLT004/ST Grip Force Transducer, ADInstruments, Dunedin, New Zealand). To determine target intensities, participants completed a minimum of two MVC trials, with additional attempts as needed until two MVCs were within 5% of each other. After a minimum 10-minute rest, a one-minute baseline ultrasound scan was performed to measure brachial artery diameter and blood velocity, followed by the three stages of the rhythmic handgrip exercise (MLT004/ST, ADInstruments, Dunedin, New Zealand) (Hansen et al., 2022). Data Analysis Brachial artery shear stress and blood flow To calculate shear rate and blood flow, a region of interest was placed around the highest-quality section of the B-mode longitudinal brachial artery image. Vessel diameter and blood velocity were continuously analyzed at a 30 Hz using custom edge-detection software (FMD/BloodFlow Software version 5.1, Reed C, Australia). Shear rate was calculated as (peak blood velocity x 4) divided by brachial artery diameter (Equation 3.1). To determine apparent viscosity, a two-phase exponential decay curve (GraphPad Prism 10.1.0, GraphPad Software Inc., CA, USA) was fitted to the viscosities obtained from the viscometer and viscosity was interpolated at the mean shear rate derived from duplex ultrasound. Shear stress was subsequently calculated by multiplying the mean shear rate by the apparent viscosity (Equation 3.2). Blood flow (Q) was calculated using peak envelope velocity and diameter (Equation 3.3). Conductance was calculated as Q divided by mean arterial pressure (Equation 3.4). Shear rate (s-1) = (4 ⋅ [peak envelope velocity / artery diameter]) Equation 3.1 Shear stress (dyn/cm2) = ([Mean shear rate ⋅ apparent viscosity]/100) Equation 3.2 55 Q̇ (mL/min) = (0.5 ⋅ peak envelope velocity) × (π [0.5 ⋅ diameter]2) ⋅ 60) Equation 3.3 Conductance (ml min−1 100 mmHg−1) = ([Q̇/MAP] ⋅100) Equation 4.4 RH-FMD Peak brachial artery dilation (i.e., FMD) was determined using automated edge-detection software (FMD/BloodFlow Software v5.1, Reed C, Australia), which applied a moving median filter to post-deflation diameter data to identify the maximum arterial diameter. The shear stimulus driving this dilation was quantified by calculating the shear stress area under the curve over the first 60 seconds following cuff release (SSAUC60) (Pyke et al., 2004). To assess microvascular function (i.e., ischemic vasodilation), indices of reactive hyperemia were determined. Peak hyperemic flow was defined as the highest 3-second average blood flow value post-deflation, and total reactive hyperemia was assessed as the area under the blood flow curve during the 3-minute recovery period (Limberg et al., 2020; Tremblay et al., 2020). Red blood cell deformability and aggregation RBC deformability parameters were derived using the non-linear version of the Lineweaver-Burk model (GraphPad Prism 10.1.0, GraphPad Software Inc., CA, USA), which estimates the theoretical maximum elongation index (EImax) and the shear stress required to achieve half-maximal deformation (SS½) from the nine shear stresses obtained from the ektacytometry. To account for individual differences in maximal deformability, the SS½:EImax ratio was calculated as a normalized index of shear sensitivity, as outlined by (Baskurt & Meiselman, 2013). The aggregation of the RBCs was quantified as syllectogram amplitude (AMP), aggregation index (AI) and aggregation half-time (T½). AMP represents the extent of RBC aggregation, AI reflects the overall aggregation tendency, and T½ denotes the time required for half-maximal aggregation (Hardeman et al., 2001). Statistical Analyses 56 Statistical analyses were performed in R (version 4.3.1; R Foundation for Statistical Computing, New Zealand), and figures were generated using GraphPad Prism (version 10.1.0; GraphPad Software Inc., CA, USA). Data was reported as mean ± SD, with significance set at p < 0.05. Normality and variance were evaluated via Q–Q plots, histograms, and confirmed using the Shapiro–Wilk and Brown–Forsythe tests, respectively. Paired two-tailed Student’s t-tests were used to compare pre- and post-plasma contraction variables. Plasmapheresis data were analyzed using linear mixed-effects models (lme4 package)(Bates et al., 2014), with participant as a random effect and time (pre-, immediate post-, and 24-hour post-PD) as a fixed effect. Time effects were assessed via ANOVA, with multiple comparisons conducted using Tukey-adjusted estimated marginal means. Results Hematology parameters and hemodynamics Investigation 1 Plasma donation (mean volume: 682.2 ± 182.3 mL) increased hemoglobin (+7.6%, p < 0.01) and hematocrit (+7.2%, p < 0.01) immediately, with both returning to baseline within 24 hours (hematocrit: p = 0.90; hemoglobin: p = 0.32). Whole blood viscosity (225 s⁻¹) showed a similar pattern, rising post-donation (p < 0.01) and normalizing by 24 hours (p = 0.31). In contrast, plasma viscosity decreased immediately (p < 0.01) and remained reduced at 24 hours (p = 0.04), suggesting delayed plasma protein recovery despite rapid volume restoration (Table 3-1). Heart rate was unchanged, but systolic pressure declined post-donation (p = 0.01) and remained lower at 24 hours (p = 0.03), with no change in diastolic or mean arterial pressure (Table 3-2). 57 Table 3-1. Hematology parameters, whole blood viscosity, and plasma viscosity at discrete shear rates before, immediately following and 24 hours following plasma donation. Values are presented as means ± SD unless otherwise specified. Abbreviations: WBC, white blood cells; RBC, red blood cells; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration and PLA, platelets. *Represents a significant difference between pre vs. post immediate (p < 0.05) †Represents a significant difference between pre vs. post 24-hour (p < 0.05) #Represents a significant difference between post immediate vs. post 24-hour (p < 0.05) 58 Table 3-2. Hematology parameters, whole blood viscosity, and plasma viscosity at discrete shear rates before, immediately following and 24 hours following plasma donation. Values are presented as means ± SD unless otherwise specified. Abbreviations: MAP, mean arterial pressure; Hb, hemoglobin; HCT, hematocrit and BA, brachial artery; BA Q, brachial artery blood flow; and BA Cond, brachial artery conductance *Represents a significant difference between pre vs. post immediate (p < 0.05) †Represents a significant difference between pre vs. post 24-hour (p < 0.05) Investigation 2 Furosemide-induced diuresis produced an average urine output of 1718 ± 287 mL over 4 hours. Whole blood viscosity increased by 15% (from 3.75 to 4.34 s⁻¹), while blood pressure and heart rate remained stable (Tables 3-3). Diuresis did not significantly affect RBC deformability (Table 3-3); aggregation was not assessed. 59 Table 3-3. Whole blood viscosity, RBC deformation, hemodynamics, brachial artery reactive hyperemia flow-mediated dilation parameters before and after plasm contraction. Values are presented as means ± SD unless otherwise specified. Abbreviations: MAP, mean arterial pressure; Hb, hemoglobin; HCT, hematocrit and BA, brachial artery. Red blood cell deformability and aggregation Investigation 1 60 Plasma donation had no effect on SS½, but EImax decreased by 18.2% immediately post-donation (p = 0.04) and returned to baseline within 24 hours (p = 0.59 vs. pre-donation), indicating a transient reduction (Figures 3-1 and 3-2). Immediately post-donation, AMP increased (p < 0.01), while AI (p = 0.75) and T½ (p = 0.06) were unchanged. By 24 hours post donation, AMP (p = 0.02) and AI (p = 0.03) were significantly reduced, and T½ increased (p = 0.04 vs. pre-plasma donation), indicating decreased RBC aggregation at this later time point (Figure 3-2)61 0.1 1 10 1000.00.20.40.6Shear Stress (Pa)Elogonation Index (a.u.)Pre-PDImmediatepost-PD24-hourpost-PD**** Figure 3-1. Elongation curves from plasma donation participants Elongation index (EI) of red blood cells (RBCs) at discrete shear stresses before, immediately following and 24 hours following plasma donation. *Represents a significant difference between pre vs. post immediate (p < 0.05). 62 Pre-PD Immediatepost-PD24-hourpost-PD2.02.53.03.54.0SS½:EImax(a.u.)Pre-PD Immediatepost-PD24-hourpost-PD1.01.52.02.5SS½(a.u.)Pre-PD Immediatepost-PD24-hourpost-PD0.500.550.600.650.70EImax(a.u.)✱D E FPre-PD Immediatepost-PD24-hourpost-PD051015T½(sec)†Pre-PD Immediatepost-PD24-hourpost-PD20406080AI (%)†Pre-PD Immediatepost-PD24-hourpost-PD20406080AMP(a.u.)✱#A B C Figure 3-2. RBC deformability and aggregation characteristics plasma donation participants 63 [A] Syllectogram amplitude (AMP), [B] aggregation index (AI), [C] aggregation half-time (T½), [D] shear stress (SS) required for half-maximal deformability (SS½), [E] theoretical maximal elongation index at infinite shear stress (EImax), and [F] SS½:EImax before, immediately following and 24 hours following plasma donation. *Represents a significant difference between pre vs. post immediate (p < 0.05) †Represents a significant difference between pre vs. post 24-hour (p < 0.05) #Represents a significant difference between post immediate vs. post 24-hour (p < 0.05). 64 Shear stress profiles and RH-FMD Investigation 1 Plasma donation did not alter baseline mean shear stress, apparent viscosity, blood flow, or vascular conductance across anytime point. Similar findings were observed with plasma contraction, although apparent viscosity was elevated (~ 11.6%) following diuresis (Tables 2 and 3). The shear stress stimulus for RH-FMD (SSAUC60) and total reactive hyperemia increased immediately after plasma donation (p = 0.02 and p = 0.04, respectively), but returned to baseline by 24 hours (p = 0.99 and p = 0.78 vs. pre-plasma donation). These changes did not affect RH-FMD, or allometrically scaled, covariate-adjusted SSAUC60 RH-FMD (Figure 3-3). 65 Pre-PD Immediatepost-PD24-hourpost-PD50100150200SSAUC60(a.u.)✱Pre-PD Immediatepost-PD24-hourpost-PD5101520SSAUC60-Adjusted RH-FMD (%)Pre-PD Immediatepost-PD24-hourpost-PD05101520RH-FMD(%)Pre-PD Immediatepost-PD24-hourpost-PD100200300400500Total reactive hyperemia (mL)✱CBDA Figure 3-3. FMD and blood flow parameters from plasma donation participants [A] shear stress stimulus (shear stress area under the curve 60 seconds post-deflation, (SSAUC60)) and [B] total reactive hyperemia, [C] Reactive hyperemia flow-mediated dilation (RH-FMD), and 66 [D] SSAUC60 covariate-adjusted RH-FMD before, immediately following and 24 hours following plasma donation. *Represents a significant difference between pre vs. post immediate (p < 0.05). Investigation 2 No changes were observed in SSAUC60, RH-FMD, covariate-adjusted SSAUC60 RH-FMD or exercise hyperemia following diuresis (Figure 3-4). 050100150200250Brachial vascular conductance(mL • min-1• 100 mmHg-1)BL 5% 15% 25%Intensity: p < 0.01Drug: p = 0.73Interaction: p = 0.87050100150200Brachial blood flow(mL • min-1)Intensity: p < 0.01Drug: p = 0.63Interaction: p = 0.83BL 5% 15% 25%PREPCPOSTPC0200400600800Total reactive hyperemia (mL)PREPCPOSTPC50100150200250SSAUC60(au)PREPCPOSTPC51015RH-FMD(%)PREPCPOSTPC051015SSAUC60-adjusted RH-FMD (%) Figure 3-4. FMD, exercise hyperemia and blood flow parameters from plasma contraction participants 67 [A] shear stress stimulus (shear stress area under the curve 60 seconds post-deflation, (SSAUC60)) and [B] total reactive hyperemia, [C] Reactive hyperemia flow-mediated dilation (RH-FMD), [D] log-scaled RH-FMD before, immediately following and 24 hours following plasma donation, [E] brachial artery blood flow and [F] conductance. *Represents a significant difference between pre vs. post immediate (p < 0.05). Time control data All values remained unchanged in the time control condition, except EImax. Although EImax did not differ between pre- and immediate post-control (p = 0.07), it was significantly higher at the immediate post-control time point compared to 24 hours later (p = 0.02). 68 Table 3-4. Time control participants (n = 5; 2F). 69 Values are presented as means ± SD unless otherwise specified. Abbreviations: WBC, white blood cells; RBC, red blood cells; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration and PLA, platelets. MAP, mean arterial pressure; BA, brachial artery; BA Q, brachial artery blood flow; BA Cond, brachial artery conductance; RH-FMD, reactive hyperemia flow-mediated dilation and SSAUC60, shear stress area under the curve 60 seconds post-deflation. 70 Discussion This investigation examined the effects of hemoconcentration induced by plasmapheresis and diuresis on blood rheology and peripheral vascular function. The primary findings were: 1) RBC deformability transiently decreased after plasmapheresis, returning to baseline within 24 hours, with no change following diuresis; 2) plasmapheresis increased total reactive hyperemia by ~23% without affecting RH-FMD, whereas diuresis had no effect on reactive hyperemia, RH-FMD, or exercise hyperemia; and 3) systolic blood pressure decreased after plasmapheresis but not diuresis. These results suggest that the enhanced microvascular response was not due to hemoconcentration, but may instead reflect effects of anticoagulants or altered RBC deformability. Plasmapheresis on blood rheology RBC deformability was transiently reduced following plasmapheresis, likely reflecting mechanical stress incurred during extracorporeal circulation (Kameneva et al., 1995; Baskurt et al., 2004). Sublethal shear stress impairs RBC mechanics in a dose- and duration-dependent manner under supraphysiological conditions (e.g., 40–130 Pa) (Simmonds & Meiselman, 2016), resulting in reduced deformability, increased elastic shear modulus (McNamee et al., 2020b), and elevated aggregability (Waxenberger et al., 1992; McNamee et al., 2018b). These alterations may stem from cytoskeletal remodeling, protein denaturation, and damage to the glycocalyx (Manno et al., 2002; Mizuno et al., 2002; Watanabe et al., 2007). Older, denser RBCs are particularly vulnerable to shear-induced damage (McNamee et al., 2019), likely due to impaired glycolytic capacity (Lew et al., 1982; Danielczok et al., 2017) and reduced Ca²⁺-ATPase activity (Kuck et al., 2024). This metabolic dysfunction leads to sustained elevations in intracellular Ca²⁺, chronic activation of Gárdos channels, and the emergence of a heterogeneous RBC population with increased density and cytosolic viscosity (Lew et al., 2007; Bogdanova et al., 2013). Similarly, sublethal shear stress may elevate intracellular Ca²⁺, promoting Gárdos channel activation, cellular dehydration, and reduced deformability (Kuck et al., 2020b). Beyond cellular flexibility, sublethal shear stress increases the energy required for the transition from tumbling to tank-treading motion (McNamee et al., 2021), promotes asymmetric deformation (McNamee et al., 2020a), and microparticle generation (Buerck et al., 2021). 71 McNamee et al., (2019) demonstrated that RBC deformability does not respond to shear stress in a linear fashion, but instead follows a distinct biphasic pattern under sublethal conditions: an initial decline, followed by a transient recovery toward baseline, and then a secondary decrease. In the present study, the transient reduction in deformability may reflect this biphasic pattern. It is plausible that mechanically compromised RBCs were initially responsible for the observed decline (i.e., elevated intracellular Ca²⁺), with subsequent normalization driven by the sequestration of more vulnerable, damaged senescent cells at 24-hours (Kuck et al., 2024). Although no changes were detected in RBV or RDW, these indices may lack the sensitivity to capture subtle shifts in RBC subpopulations (McNamee et al., 2020a; McNamee et al., 2021; Kuck et al., 2024). Future research should examine the effects of donation on RBC heterogeneity, additional biomechanical properties (e.g., elastic shear modulus, glycocalyx structure), and the potential contribution of enhanced RBC clearance to long-term anemia risk. Consistent with prior studies (Reinhart et al., 1992; Waxenberger et al., 1992; Mansouri Taleghani et al., 1997; Bor-Kucukatay et al., 2008), we observed reduced RBC aggregation and plasma viscosity following plasmapheresis (see Figure 3-2). This reduction is likely driven by lower plasma fibrinogen concentrations, as thrombocytapheresis has been shown to reduce total protein (Bor-Kucukatay et al., 2008), and a single therapeutic plasma exchange can lower fibrinogen by ~25% (Keller et al., 1979; Zantek et al., 2019; Wodajo et al., 2024). High sublethal shear stress (130 Pa) has been show to increase RBC aggregability, but this is likely compensated for by a larger decrease in plasma proteins (McNamee et al., 2018b). Hemoconcentration on vascular reactivity It was hypothesized that increased blood viscosity would enhance RH-FMD by elevating both baseline and reactive hyperemic shear stress. However, neither plasma donation nor diuresis altered the FMD response. Although diuresis trended toward higher basal shear stress, this was not significant and did not augment RH-FMD. Likewise, a pre-FMD shear stimulus via 10% MVC handgrip raised mean shear rate ~3.5-fold but failed to enhance FMD (Atkinson et al., 2015), whereas 15% MVC did, suggesting a threshold is needed to activate endothelial nitric oxide synthase (eNOS). Despite a 12% increase in post-occlusion shear stress following plasmapheresis, 72 FMD remained unchanged. Similarly, Pyke et al., (2007) demonstrated a ~34% increase in shear rate did not alter peak artery diameter, but greater changes in shear rate did. It is possible the mild 12% change in shear stress was insufficient to augment the endothelium vasodilation. Furthermore, pre-stimulus shear elevation (e.g., via heating local handgrip exercise or lower body exercise light exercise) enhances brachial FMD without altering post-occlusion shear rate (Tinken et al., 2009), suggesting that pre-conditioning may be more important than the post-occlusion stimulus. The lack of change in reactive hyperemia and post-occlusion shear stress after diuresis suggests that the enhancement observed with plasmapheresis reflects a distinct physiological response and may inadvertently lead to tissue hyper-perfusion. In healthy males, isovolumic hypovolemia increased reactive hyperemia (Hoiland et al., 2020b), while raising hemoglobin by ~38% through blood transfusion in patients with chronic anemia decreased reactive hyperemia (Nagy et al., 1999). A similar, though non-significant (p = 0.13), trend was observed with diuresis in this study. These findings support the idea that changes in arterial oxygen content are accompanied by alterations in blood flow (Tu & Liu, 1996). In contrast, the elevated reactive hyperemia after plasmapheresis suggests a possible uncoupling between oxygen delivery and demand. This observation aligns with earlier studies by Reinhart et al. (1992) and Mansouri Taleghani et al. (1997), who found increased capillary blood cell velocity after plasmapheresis, despite no change in RBC deformability. One potential contributing factor is the use of anticoagulants (e.g., citrate dextrose formula A) during apheresis. Citrate chelates ionized calcium (Szymanski, 1978; Silberstein et al., 1986; Das et al., 2005), thereby reducing coagulation and arterial tone (Scholze et al., 2005). In patients with coronary artery disease, 10 weeks of EDTA plus vitamin therapy enhanced endothelium-dependent vasodilation, though EDTA alone showed only a trend towards significance, possibly due to delayed testing and a small sample size (Green et al., 1999). Transient alterations in vascular reactivity following plasmapheresis may result from the short half-life of citrate (30–60 minutes); however, this remains unknown. Plasmapheresis on blood pressure 73 Systolic blood pressure decreased following plasmapheresis, consistent with prior reports during acute apheresis in healthy individuals (Schooneman et al., 1997; Yeh et al., 2002; Tran-Mi et al., 2004) and in hypertensive individuals undergoing chronic plasma donation (Rosa-Bray et al., 2015). In contrast, diuresis did not alter blood pressure, despite similar increases in hemoglobin (~8.2% vs. 8.5%), suggesting both interventions induced comparable reductions in blood volume. These findings suggest that volume contraction alone does not fully account for the reduction in systolic blood pressure. Alterations in plasma protein content may also play a role; for instance, Yeh et al. (2002) reported that decreases in systolic and diastolic pressure were positively correlated with the volume of globulin removed. Supporting this, plasma viscosity declined following plasmapheresis and remained reduced at 24 hours, indicating lower plasma protein content (Waxenberger et al., 1992). Plasmapheresis likely reduces blood pressure through a combination of plasma volume loss and decreased plasma protein concentration. Hypoproteinemia, even without hypovolemia, can lower blood pressure by reducing plasma oncotic pressure, as demonstrated in animal studies (Manning & Guyton, 1983). The drop in systolic but not diastolic pressure, with stable heart rate, suggests a reduction in stroke volume, potentially due to decreased preload or cardiac contractility, rather than changes in vascular tone (reflected by diastolic pressure). Prior work indicates that systolic pressure is particularly sensitive to changes in volume loading and stroke volume (Hopper et al., 1988). Nonetheless, future studies should include direct measures of arterial stiffness (e.g., pulse wave velocity), cardiac contractility, stroke volume and cardiac output to identify the underlying mechanism. Methodological Considerations Furosemide may induce vasoconstriction through direct effects on vascular smooth muscle (Barthelmebs et al., 1994) and by elevating circulating levels of renin and angiotensin II (Hesse & Nielsen, 1976; Johnston et al., 1983). These actions could counteract any vasodilation expected from elevated blood viscosity. However, furosemide does not directly affect forearm blood flow or arterial vasodilation in humans (Pickkers et al., 1997), suggesting that plasma contraction alone does not alter vascular function. 74 There is also evidence that furosemide may influence ion regulation in RBCs. Specifically, furosemide has been shown to increase cytosolic chloride levels, which may promote membrane depolarization (Wiley & Cooper, 1974; Brazy & Gunn, 1976). This depolarization can activate voltage-gated calcium channels, resulting in elevated intracellular calcium concentrations (Gardos, 1958). In our observations, however, furosemide did not significantly alter RBC deformability. Nevertheless, changes in calcium homeostasis could potentially affect other aspects of RBC mechanics, such as cell volume regulation through activation of Gardos channels, particularly in older RBCs (Kuck et al., 2024). While the ex vivo impact of furosemide on RBC deformability remains unclear, the absence of measurable changes suggests its influence is likely minimal, though it cannot be entirely ruled out. In Investigation 2, female participants were tested during the early follicular phase to minimize estradiol-related variability in FMD, a control not applied in Investigation 1. Although estradiol may influence FMD (Moreau et al., 2020), its effects are highly variable and unlikely to have a meaningfully impact on endothelium-dependent vasodilation in applied settings (D'Urzo et al., 2018; Liu et al., 2021; Lew et al., 2025). It was assumed that the ~2-hour interval between pre- and post-plasma donation measurements would eliminate any residual shear-induced changes in RBC deformability. However, EImax unexpectedly increased in the time control group during this period. This observation suggests that our observed post-donation decrease in EImax may actually underestimate the true reduction in RBC deformability. Conclusion Plasma donation and diuresis induced hemoconcentration and increased blood viscosity but did not enhance basal shear stress, blood flow, or vascular reactivity. Unexpectedly, plasma donation decreased RBC deformability yet augmented reactive hyperemia, suggesting that microvascular improvements may be mediated by citrate anticoagulants rather than changes in blood rheology. 75 Chapter 4: Study 2 - Exercise-induced enhancements in RBC deformability are prolonged by oral phosphodiesterase-5 inhibition. Abstract Red blood cells (RBCs) rely on their deformability to deliver oxygen and remove waste, allowing them to navigate narrow capillaries and maintain blood flow. Sildenafil citrate, a phosphodiesterase-5 inhibitor (PDE5-i), may enhance RBC deformability by modulating nitric oxide signaling. While ex vivo studies suggest potential benefits, its effects in vivo remain largely unexamined. We conducted a double-blind, placebo-controlled study to assess the effects of PDE5-i on cerebral blood flow (CBF), brachial artery (BA) blood flow (Q), and shear stress-specific RBC deformability during an incremental rhythmic handgrip exercise (baseline [BL], 27.5%, and 55% of maximal voluntary contraction [MVC]). Fourteen healthy participants (8F; age: 24±6 years) were tested on two separate days, before and 60 minutes following ingestion of sildenafil citrate (100 mg) or a placebo. CBF and BA Q were measured using duplex ultrasound, and RBC deformability was assessed with a laser-assisted optical rotational red cell analyzer. The PDE5-i had no effect on baseline cerebral or BA vascular metrics, including diameter, shear stress, Q, conductance, or steady-state exercise hyperemia (p>0.05). Prior to PDE5-i administration, exercise increased RBC deformability (EImax) in a stepwise manner: BL (0.55±0.021), 27.5% MVC (0.57±0.014), and 55% MVC (0.58±0.015 a.u.; p<0.01). Following PDE5-i, BL EImax was elevated compared to pre-PDE5-i values (0.59±0.014 vs. 0.55±0.021) and did not increase further with exercise (27.5%: 0.59±0.017; 55%: 0.59±0.015 a.u.; p>0.05), a pattern not observed with placebo. BA shear stress-specific RBC deformability also remained unchanged (p=0.66). These findings suggest that PDE5-i prolongs exercise-induced enhancements in RBC deformability, but does not increase deformability. 76 Introduction: Red blood cells (RBCs) transport oxygen to tissues and remove metabolic waste, supporting both homeostasis and vascular function. To fulfill this role, RBCs exhibit unique hemorheological properties, most notably deformability - the ability to change shape under varying shear stress conditions (Simmonds et al., 2014; Danielczok et al., 2017). This mechanical flexibility reduces flow resistance (Leo et al., 2020) and allows RBCs to navigate narrow capillaries (Simchon et al., 1987). RBC deformability is governed by both the geometric and material properties of the cell (Canham & Burton, 1968; Jay, 1975) and is actively regulated by nitric oxide (NO) (Bor-Kucukatay et al., 2003; Kleinbongard et al., 2006b). Although the exact mechanism is not fully understood, NO is believed to modulate RBC deformability by altering interactions between the cytoskeletal network and integral membrane proteins (Grau et al., 2013). Shear stress activates Piezo1 channels, leading to RBC nitric oxide synthase (RBC-NOS) activation via the PI3K/Akt pathway and resulting in increased NO production (Ulker et al., 2011; Suhr et al., 2012). NO elevates intracellular cGMP and cAMP levels, promoting phosphorylation of cytoskeletal proteins (e.g., spectrin, actin), enhancing RBC deformability (Ugurel et al., 2022). Several phosphodiesterase isoforms (e.g., PDE1, PDE3, PDE4, PDE5) regulate this pathway by degrading cAMP and cGMP. Inhibition of these enzymes prolongs cyclic nucleotide signaling and has been shown to improve RBC deformability in ex vivo models, particularly via PDE1 (Muravyov et al., 2009; Muravyov & Tikhomirova, 2013), PDE4 (Muravyov et al., 2009), and PDE5 (Hagley et al., 2011; Ziegler, 2017). However, it remains unclear whether these effects occur in vivo, or whether enhanced RBC deformability translates to augmented blood flow. Exercise increases hemoglobin in an intensity-dependent manner (González-Alonso et al., 2002); however, because shear rate rises more rapidly than hematocrit, apparent viscosity decreases despite hemoconcentration (Leo et al., 2020; Tremblay et al., 2020). This reduction is driven by enhanced RBC elongation (i.e., RBC deformability), which lowers flow resistance and may help preserve blood flow (Leo et al., 2020). Although RBC deformability likely offsets elevated 77 vascular resistance during exercise, its independent contribution to blood flow regulation in humans remains unexamined (Simchon et al., 1987). To address this question, we conducted a double-blind, placebo-controlled study examining the effects of a PDE5 inhibitor (e.g., sildenafil citrate) on brachial artery (BA) blood flow and RBC deformability during a shear-stress-inducing handgrip exercise. Duplex ultrasound and venous blood sampling were used to measure BA shear rate, whole blood viscosity, and RBC deformability, enabling calculation of shear stress-specific RBC deformability from ultrasound-derived shear stress. We hypothesized that exercise would increase RBC deformability, and that PDE5 inhibition (PDE5-i) would further enhance this response, leading to reduced apparent viscosity, lower BA resistance, and augmented blood flow compared to placebo. As a secondary objective, we compared basal BA and cerebral blood flow (CBF) following PDE5-i administration to assess tissue-specific effects. Methods Ethical approval This investigation adhered to the Declaration of Helsinki (except registry in a database) and the Canadian Tri-council Policy Statement for Integrity in Research. The protocol was approved by the University of British Columbia’s Clinical Research Ethics Board (H21-02780). Participants provided informed consent after reviewing a detailed study information form and discussing the protocol with an investigator. Participants Fourteen participants (8 females, 6 males; age 24 ± 6 years) were recruited. Inclusion criteria included normotension (systolic blood pressure < 140 mmHg, diastolic < 90 mmHg), BMI ≤ 30 kg/m² (mean BMI: 23 ± 3), free of cardiovascular, respiratory, metabolic, or other chronic illnesses. Participants were also non-smokers and did not take daily medications (excluding 78 contraceptives). Females were tested exclusively during the early follicular phase (days 2–5 following the onset of menses). Sildenafil citrate is known to metabolize differently across ethnicities. To reduce variability, all participants self-identified as being of European descent (Alwhaibi et al., 2021). Experimental design Participants attended two laboratory sessions separated by at least 48 hours. They maintained their usual caffeine and dietary habits but refrained from caloric consumption within two hours of testing. Additionally, participants avoided strenuous exercise for 12 hours before each session. To ensure consistency, they were instructed to replicate their eating and hydration patterns across testing days. Both visits followed identical protocols, differing only by the administration of sildenafil citrate (100 mg) or a placebo, in a counterbalanced order, to mitigate day-to-day and sequence effects. A schematic of the protocol is displayed in figure 4-1. Randomization (https://www.randomizer.org/) and blinding procedures were managed by an independent researcher who was not involved in data collection79 Figure 4-1. Schematic of the study. 80 Experimental protocol Participants rested in a supine position while an intravenous catheter was inserted into an antecubital vein and remained in place for the duration of the protocol. Cardiorespiratory measurements were then obtained, including heart rate (via electrocardiogram), beat-by-beat blood pressure, end-tidal carbon dioxide partial pressure (PETCO₂) and oxygen partial pressure (PETO₂) were all collected. Following instrumentation, participants rested for 10 minutes to establish a stable baseline. Cardiorespiratory variables (e.g., end-tidal gases and beat-by-beat blood pressure) were recorded continuously. Ultrasound measurements of the internal carotid (ICA) and vertebral (VA) arteries were recorded concurrently with cardiorespiratory variables but collected sequentially by the same researcher (ARS). Participants then completed a graded rhythmic handgrip exercise at two intensities (27.5 and 55% of maximal voluntary contraction [MVC] to increase shear stress-specific RBC deformation. Contractions were performed for 1 second followed by 3 seconds of relaxation, sustained for 3 minutes at each intensity using a grip force transducer (MLT004/ST, ADInstruments, Dunedin, New Zealand) (Tremblay et al., 2020). MVC was determined by at least two attempts, with additional trials as needed until two values were within 5% of each other (mean MVC: 463 ± 87 N). After a 10-minute rest, a one-minute baseline ultrasound scan was performed to assess BA diameter and blood velocity, followed by the two-stage exercise protocol. The contraction-relaxation rhythm was guided by a metronome (Pro Metronome, EUMLab–Xanin Technology, Berlin, Germany), investigator instruction, and visual feedback of handgrip force (LabChart 10, ADInstruments). Venous blood samples and central and peripheral hemodynamic measurements were collected during the final minute of baseline and each MVC stage. The two exercise intensities were performed consecutively without rest between stages. After completing the exercise protocol, participants received either sildenafil citrate in a gelatin capsule or an identical empty capsule (placebo), administered from a pre-packaged opaque bag. 81 Retesting was conducted one hour later, as sildenafil citrate reaches peak plasma concentration within approximately one hour and has a half-life of 4–5 hours (Hatzimouratidis, 2006). Experimental measurements: Cardiorespiratory metrics: Systolic and diastolic blood pressures were measured using finger photoplethysmography with the Finometer system (Finapres Medical Systems, Amsterdam, The Netherlands), which provides continuous, noninvasive arterial pressure waveforms via a finger cuff. These measurements were corrected using an automated brachial blood pressure monitor (Omron HEM-907XL, Omron Healthcare, Kyoto, Japan) to improve absolute pressure accuracy. Mean arterial pressure (MAP) was subsequently calculated using the standard weighted formula: (1/3 × systolic blood pressure) + (2/3 × diastolic blood pressure). Electrocardiogram (ECG) data were recorded continuously using a 3-lead configuration connected to a bioamplifier (ML138 Bio Amp, ADInstruments, Dunedin, New Zealand). ECG signals were sampled at 1000 Hz using a PowerLab 16/30 data acquisition system (Labchart 10.0 Pro, ADInstruments, Dunedin, New Zealand), with electrodes placed in a standard Lead II configuration (right arm, left leg, ground on right leg) to optimize R-wave detection. PETO₂ and PETCO₂ were collected continuously using a gas analysis system (ML206 Gas Analyzer, ADInstruments, Dunedin, New Zealand). The analyzer was calibrated before each session using certified gas mixtures (15.001% O2 and 5.001% CO2), and samples were obtained via a sampling line embedded within an oral mouth piece connected to the gas sampling port. PETCO₂ and PETO₂ were derived using automated peak detection algorithms applied to the respiratory waveforms, which were recorded at 1000 Hz and integrated into the PowerLab system. All signals were processed and stored for subsequent analysis using LabChart 10.0 Pro software (Dunedin, New Zealand). 82 Hemorheology and blood parameters At each time point, venous blood was collected into 6 mL EDTA vacutainers, inverted, and gently mixed using an automated rotator (The Jenna, HHH Technologies, Kelowna, BC, Canada). Whole blood viscosity was measured in duplicate within 15 minutes of collection using a cone-and-plate viscometer (Model DV2T, Brookfield Amtek, Middleboro, MA, USA) maintained at 37°C with a circulating water bath (TC-150, Brookfield Amtek). Measurements were conducted using a CPA-40Z spindle. Measurements were taken at seven shear rates: 45, 67.5, 90, 150, 225, 337.5, and 450 s⁻¹, in duplicate. If duplicate values differed by more than 0.1 mPa·s at a shear rate of 225 s⁻¹, a third measurement was taken, and the two closest values were averaged to ensure a coefficient of variation below 1%. Erythrocyte deformability was assessed using a laser-assisted optical rotational red cell analyzer (LORRCA; RR Mechatronics, Hoorn, The Netherlands). Approximately 14 μL of whole blood was diluted into 5 mL of an isotonic polyvinylpyrrolidone solution prepared in a 0.1 mol·L⁻¹ phosphate-buffered saline (0.14 mM; 300 mOsmol/L; viscosity 29.9 mPa·s at 37°C). The sample was loaded into a Couette system, where a laser beam produced a diffraction pattern as shear stress was applied. The analyzer sequentially applied nine increasing shear stresses (0.3, 0.57, 1.08, 1.65, 2.04, 3.87, 7.34, 13.94, 26.38, and 50 Pa). Changes in the diffraction pattern reflected RBC deformability, quantified by the elongation index (EI), calculated as EI = (L − W)/(L + W), where L and W represent the length and width of the elliptical diffraction pattern at each shear stress (Baskurt et al., 2009). Hematocrit (HCT) and hemoglobin (HGB) concentration were measured using microcentrifugation (PowerSpin BX Centrifuge – unico; 10 min at 11,000 rpm) and photometric analysis (Hemocue hb 201+ analyser, HemoCue, Ängelholm, Sweden), respectively. A complete blood count was performed on baseline samples using a hematology analyzer (DxH 520; Beckman Coulter, Mississauga, ON, Canada) to verify consistency in blood parameters across testing days. Hematology variables included white blood cells (WBC), red blood cells (RBCs), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) mean 83 corpuscular hemoglobin concentration (MCHC) and platelets (PLA). High, medium, and low standards were run daily to ensure accuracy and reliability of the measurements. Duplex ultrasound. CBF was assessed via extracranial measurements of blood velocity and diameter measurements from the left ICA and VA using a 10-MHz multifrequency linear array Duplex ultrasound (Terason t3000; Teratech, Burlington, MA, USA). ICA measurements were taken at least 1.5 cm distal to the carotid bifurcation to avoid retrograde flow. VA measurements were obtained at either the C4–C5 or C5–C6 vertebral level. Vessel location was determined individually to optimize image quality, and a consistent insonation angle of 60° was maintained across trials in accordance with technical guidelines (Thomas et al., 2015). BA diameter and blood velocity was measured using the distal third of the left BA. To ensure consistency, anatomical ultrasound landmarks were used to image the same arterial segment across all measurements, with gain and dynamic range settings kept constant (Woodman et al., 2001). Data were acquired via screen-capture software and saved for offline analysis. Peak diameter and velocity were analyzed at 30 Hz using custom wall-tracking software (Woodman et al., 2001). Data analysis Arterial blood flow and shear stress. Custom wall-tracking software (FMD/BloodFlow Software v5.1, Reed C, Australia) was used to automatically detect vessel diameter and blood velocity at a frequency of 30 Hz. The analyst was blinded to timepoints and condition, although blinding to exercise intensity was not possible due to the progressive nature of the handgrip protocol. Blood flow (Q) was calculated using Equation 4.1. CBF was determined was determined as twice the sum of ipsilateral ICA and VA flow (Equation 4.2). Vascular conductance was calculated by dividing Q by mean arterial pressure (Equation 4.3). Shear rate was calculated as (peak blood velocity × 4) divided by brachial artery diameter (Equation 4.4). Apparent viscosity was derived 84 by fitting a two-phase exponential decay curve (GraphPad Prism 10.1.0, GraphPad Software Inc., CA, USA) to whole blood viscosities and interpolating viscosity at the mean shear rate obtained from duplex ultrasound (Leo et al., 2020). Shear stress was then calculated by multiplying mean shear rate by apparent viscosity (Equation 4.5). Q (mL/min) = ([0.5 ⋅ peak envelope velocity] ⋅ (π [0.5 ⋅ diameter]2) ⋅ 60) Equation 4.1 CBF (mL/min) = 2 ⋅ (QICA + QVA) Equation 4.2 Conductance (mL min−1 mmHg−1) = (Q/MAP) Equation 4.3 Shear rate (s-1) = (4 ⋅ [peak blood velocity / artery diameter]) Equation 4.4 Shear stress (Pa) = ((Mean shear rate ⋅ apparent viscosity)/1000) Equation 4.5 Red blood cell deformability BA shear stress–specific RBC deformability was assessed by fitting a non-linear Lineweaver–Burk model (GraphPad Prism v10.1.0, GraphPad Software Inc., CA, USA), which yields the theoretical maximal elongation index (EImax) and the shear stress required for half-maximal deformation (SS½), as previously described (28). The elongation index was then interpolated from this model based on shear stress values obtained via ultrasound to determine vessel-specific RBC deformability (Baskurt et al., 2009). Statistical analyses Statistical analyses were conducted using R (version 4.3.1, R Foundation for Statistical Computing, New Zealand) and figures were created using GraphPad Prism (version 10.1.0, GraphPad Software Inc, CA, USA). Data are presented as mean ± SD, with statistical significance set at p < 0.05. Data normality and variance were assessed using QQ-plots and histograms and further evaluated using the Shapiro-Wilk and Brown-Forsythe tests, respectively. 85 Paired two-tailed Student’s t-tests were employed to compare PDE5-i and placebo for blood parameters. Linear mixed models (using the LMER package) were used to analyze CBF, PETCO2, and PETO2 with condition (PDE5-i and placebo), and time (pre and post) as fixed factors and participants modelled as random effects. MAP, HR, HCT, HGB, apparent viscosity, BA parameters, RBC deformability, and BA shear stress-specific RBC deformability during progressive handgrip exercise were analyzed using linear mixed models, with time and handgrip intensity (repeated measures: BL, 27.5%, and 55% MVC) specified as fixed factors and participants modeled as random effects. A two-way model was used rather than a three-way model because drug condition (PDE5-i vs placebo) was analyzed separately to reduce model complexity and avoid interactions that would be statistically underpowered due to sample size constraints. Bonferroni post hoc tests were performed when significant main effects or interactions were detected to account for multiple comparisons. Results Blood parameters, hemodynamics, and blood flow Baseline HCT, HGB, MCV, MCH, and MCHC are presented in Table 4-1. No significant changes in these parameters were observed between testing days. 86 Table 4-1. Comparison of blood parameters across days. Values are presented as means ± SD unless otherwise specified. Abbreviations: WBC, white blood cells; RBC, red blood cells; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration and PLA, platelets. 87 There were no changes in Q, conductance, diameter, mean shear rate, apparent viscosity, or mean shear stress, in the BA, VA, or ICA following PDE5-i administration (Tables 4-2 and 4-3). Additionally, no significant changes were observed in resting PETCO2, PETO2 or MAP (see Tables 4-2 and 4-3). Progressive handgrip exercise increased BA Q, conductance, diameter, mean shear rate, apparent viscosity, and mean shear stress in a dose-dependent manner (baseline [BL], [27.5%], and [55%] of MVC, main effect: p < 0.01).88 Table 4-2. Partial pressure of oxygen and carbon dioxide, and cerebral blood flow. Values are presented as means ± SD unless otherwise specified. Abbreviations: PETO2, end-tidal partial pressure of oxygen; PETCO2, end-tidal partial pressure of carbon dioxide; ICA Q, internal carotid artery flow; ICA Cond, internal carotid artery conductance; VA Q, vertebral artery flow VA Cond, vertebral artery conductance and CBF, cerebral blood flow. 89 Table 4-3. Systemic hemodynamics, hemoglobin concentration, hematocrit, apparent viscosity, and BA parameters during progressive handgrip exercise. Values are presented as means ± SD unless otherwise specified. Abbreviations: MVC, maximum voluntary contraction; MAP, mean arterial pressure; Hb, hemoglobin; HCT, hematocrit and BA, brachial artery. * p < 0.05 vs. pre-baseline;. # p < 0.05 vs. pre-27.5% MVC; † p < 0.05 vs. post-baseline ‡ p < 0.05 vs. post-27.5% MVC.90 The impact of exercise and PDE5-i on RBC deformability Prior to PDE5-i administration, exercise increased RBC deformability, as indicated by EImax: [BL] 0.55 ± 0.021; [27.5% MVC] 0.57 ± 0.014; and [55% MVC] 0.58 ± 0.015 a.u. (main effect: p < 0.01). A similar response was observed in the pre-placebo condition: [BL] 0.56 ± 0.021; [27.5% MVC] 0.58 ± 0.014; and [55% MVC] 0.58 ± 0.015 a.u. (main effect: p < 0.01) (Figure 4-2). However, following PDE5-i administration, EImax was elevated at baseline and remained unchanged throughout the handgrip protocol: [BL] 0.59 ± 0.018; [27.5% MVC] 0.59 ± 0.017; and [55% MVC] 0.59 ± 0.015 a.u. (main effect: p > 0.05). In contrast, EImax declined post-placebo, returning toward baseline values ([BL] 0.57 ± 0.016 vs. [55% MVC] 0.59 ± 0.010; p = 0.04; Figure 4-2). No significant changes were observed in SS½ across any condition (Figure 4-3). PDE5-i did not affect shear stress-specific RBC deformability in the ICA, VA, or BA (ICA: condition, p = 0.60; VA: p = 0.31; BA: p = 0.65; Table 4-2 and Figure 4-4), despite an increase in EImax at the post-PDE5-i baseline compared to pre-PDE5-i baseline. This suggests that the elevated EImax had minimal impact on shear-induced erythrocyte elongation, likely due to the low shear stress in these vessels. As shown in Figure 4-5A, mean BA shear stress at baseline reached only 0.48 Pa well below the ~7.34 Pa threshold at which significant changes in RBC deformability were observed with PDE5-i.91 Pre-PDE5-i BLPre-PDE5-i 27.5%Pre-PDE5-i 55%Post-PDE5-i BLPost-PDE5-i 27.5%Post-PDE5-i 55%0.500.550.600.65EImax(a.u.)✱✱✱✱✱Pre-Placebo BLPre-Placebo 27.5%Pre-Placebo 55%Post-Placebo BLPost-Placebo 27.5%Post-Placebo 55%0.500.550.600.65EImax(a.u.)✱✱ †† ✱ Figure 4-2. EImax during BL, 27.5% and 55% of MVC with and without PDE5-i Theoretical maximal elongation index at infinite shear stress (EImax) during progressive handgrip exercise at baseline, 27.5% maximum voluntary contraction (MVC), and 55% MVC under PDE5-i (Blue) and placebo (Red) conditions. * p < 0.05 vs. pre-baseline; † p < 0.05 vs. post-baseline.Time p = 0.17; MVC p < 0.01; Time x MVC p = 0.08. Time p < 0.01; MVC p < 0.01; Time x MVC p < 0.01. 92 Pre-PDE5-i BLPre-PDE5-i 27.5%Pre-PDE5-i 55%Post-PDE5-i BLPost-PDE5-i 27.5%Post-PDE5-i 55%0.51.01.52.02.5SS1/2(a.u.)Pre-Placebo BLPre-Placebo 27.5%Pre-Placebo 55%Post-Placebo BLPost-Placebo 27.5%Post-Placebo 55%0.51.01.52.02.5SS1/2(a.u.) Figure 4-3. SS₁/₂ during BL, 27.5% and 55% of MVC with and without PDE5-i Shear stress required for half-maximal deformability (SS₁/₂) during progressive handgrip exercise at baseline (BL), 27.5% maximum voluntary contraction (MVC), and 55% MVC under PDE5-i (Blue) and placebo (Red) conditionsTime p = 0.15; MVC p = 0.43; Time x MVC p = 0.26. Time p = 0.67; MVC p = 0.58; Time x MVC p = 0.75. 93 Figure 4-4. Brachial artery specific erythrocyte elongation index during BL, 27.5% and 55% of MVC with and without PDE5-i Brachial artery (BA) shear stress-specific erythrocyte elongation index during progressive handgrip exercise at baseline, 27.5% maximum voluntary contraction (MVC), and 55% MVC under PDE5-i (Blue) and placebo (Red) conditions. * p < 0.05, baseline vs 27.5% MVC; † p < 0.05, 27.5% vs. 55% MVC. 0.00.10.20.30.40.5Maximum Voluntary Contraction(%)Elogonation Index (a.u.)Pre-PDE5-iPost-PDE5-iPre-PlaceboPost-Placebo27.5 55BL**#Time p = 0.42; MVC p < 0.01; Time x MVC p = 0.71. Time p = 0.69; MVC p < 0.01; Time x MVC p = 0.98. 94 0.1 1 10 1000.00.20.40.6Shear Stress (Pa)Elogonation Indexat Baseline (a.u.)Pre-PDE5-iPost-PDE5-iPre-PlaceboPost-Placebo* * * *0.1 1 10 1000.00.20.40.6Shear Stress (Pa)Elogonation Indexat 27.5% MVC (a.u.)Pre-PDE5-iPost-PDE5-iPre-PlaceboPost-Placebo0.1 1 10 1000.00.20.40.6Shear Stress (Pa)Elogonation Indexat 55% MVC (a.u.)Pre-PDE5-iPost-PDE5-iPre-PlaceboPost-PlaceboABC Figure 4-5. Erythrocyte elongation index curves during BL, 27.5% and 55% of MVC with and without PDE5-i 95 Elongation index (EI) of red blood cells (RBCs) at discrete shear stresses during progressive handgrip exercise at baseline [A], 27.5% maximum voluntary contraction (MVC) [B], and 55% MVC [C] under PDE5-i (Blue) and placebo (Red) conditions. Dashed lines represent the mean brachial artery shear stress values across all conditions at each exercise intensity. * p < 0.05 vs. post- PDE5-i. 96 Discussion We examined the acute effects of PDE5-i on CBF, BA Q, and RBC deformability at rest and during progressive handgrip exercise. The key findings were: (1) resting cerebral and BA vascular metrics (e.g., diameter, shear stress, Q, and conductance) were unaffected by PDE5-i; (2) progressive handgrip exercise increased BA Q and conductance, but PDE5-i had no additional effect on exercise hyperemia; (3) handgrip exercise enhanced RBC deformability (EImax), and this increase was prolonged following PDE5-i administration; (4) however, this did not translate to greater BA shear stress-specific RBC elongation or reduced apparent viscosity. These findings suggest that acute PDE5-i administration does not directly affect vascular tone or reactivity in healthy individuals but may prolong exercise-induced enhancements in RBC deformability without enhancing deformation. The impact of PDE5-i on blood flow hemodynamics PDE5 inhibition may influence blood flow both through direct vasodilation and by altering hemorheology. Vascular smooth muscle tone is modulated by multiple vasodilatory signals, many of which converge on endothelial-derived NO. NO activates soluble guanylate cyclase in smooth muscle cells, increasing cGMP levels and promoting vasodilation (Green et al., 2005; Zhang et al., 2006; Hein et al., 2013). PDE5, which is expressed in the smooth muscle cells of both cerebral (Kruuse et al., 2005) and peripheral vasculature (Wallis et al., 1999), regulates vascular tone by degrading cGMP. However, consistent with previous studies, we observed no significant changes in resting CBF (Kruuse et al., 2009; Jahshan et al., 2017) BA Q or exercise hyperemia (Dishy et al., 2001; Limberg et al., 2017). In clinical conditions with endothelial dysfunction, such as smoking (Kimura et al., 2003), chronic heart failure (Hryniewicz et al., 2005), or cerebral small vessel disease (Webb et al., 2024), PDE5-i has been shown to enhance blood flow to both peripheral and cerebral tissues. Elevated cGMP concentrations (Kimura et al., 2003; Hryniewicz et al., 2005), likely compensate for reduced NO bioavailability or elevated PDE5 expression (Pauvert et al., 2003). Supporting the importance of NO signaling, we have previously demonstrated that NO synthase inhibition via L-NMMA reduces 97 neurovascular coupling (Hoiland et al., 2020a) and attenuates shear-mediated cerebrovascular dilation (Hoiland et al., 2022). In contrast, in healthy individuals, we and others (Limberg et al., 2017) found no effect of PDE5-i on brachial exercise hyperemia. This lack of effect may reflect a physiological ceiling, where NO–cGMP signaling is already maximally activated. Furthermore, NO only accounts for ~15% of the vasodilatory response during exercise, any additional benefit from PDE5-i would likely be minimal (Duffy et al., 1999; Rådegran & Saltin, 1999; Schrage et al., 2004). Identifying the specific contexts and vascular beds in which PDE5-i is effective remains an important area for future research (Webb et al., 2024). The impact of PDE5-i on blood rheology Exercise increased EImax in our study, consistent with the findings of Suhr et al. (2012). However, unlike their reported reduction in SS½ during prolonged whole-body exercise, we observed no change in SS½. This discrepancy may reflect differences in exercise modality and duration (e.g., single-arm versus lower-body endurance exercise). Figure 4-5A demonstrates that PDE5-i administration results in elevated erythrocyte elongation at 7.34, 13.94, 26.38, and 50 Pa at baseline, but not at 27.5% or 55% of MVC, leading to increased EImax at baseline (Figure 4-2). This pattern was not observed in the placebo condition. The washout period in the placebo condition was sufficient to return EImax to baseline, suggesting a possible interaction between exercise and PDE5-i. These findings indicate that sildenafil citrate does not directly enhance RBC deformability, but instead preserves shear stress, induced enhancements elicited by exercise. Notably, this effect did not correspond with reductions in apparent viscosity or increases in BA shear stress specific RBC elongation. Together, these results highlight the potential of PDE5-i to prolong exercise-induced hemorheological adaptations under resting conditions following exercise. PDE5-i may prolong RBC deformability either directly by elevating cGMP levels or indirectly by inhibiting PDE3, thereby sustaining cAMP signaling (Knebel et al., 2013; Zhao et al., 2016). RBC deformability was elevated at baseline when comparing pre- and post-PDE5-i at high shear stress levels (7.34 to 50 Pa), which far exceed those typically observed in large arteries at rest or during 98 exercise. For example, at baseline, brachial artery shear stress ranges from 0.48 ± 0.25 Pa and increases to 2.5 ± 0.81 Pa during exercise, with comparable values in the vertebral (0.88 ± 0.17 Pa) and internal carotid (1.27 ± 0.27 Pa) arteries (Figure 4-5, Table 4-2). Despite increased RBC deformability from pre- to post-PDE5-i at baseline, the relatively low shear stress in the brachial and cerebral arteries was insufficient to produce detectable changes in bulk blood flow, suggesting limited hemodynamic impact in young, healthy individuals. In contrast, the shear stress levels that showed enhancements in erythrocyte elongation following PDE5-i administration (7.34 to 50 Pa) are more representative of the microcirculation, where values frequently exceed 12 Pa (Koutsiaris et al., 2013). In this environment, where vessel diameters are comparable to RBC size, deformability plays a more critical role in perfusion. Cellular dynamics such as tank-treading, axial migration, and capillary recruitment are all highly dependent on deformability (Zhang et al., 2009; Basu et al., 2011; Yalcin et al., 2015). Small improvements at these shear stress levels may enhance perfusion and oxygen delivery (Yalcin et al., 2015). Even modest reductions in the proportion of rigid RBCs (e.g., 5%), can significantly affect RBC mechanics and promote inflammatory signaling (Kuck et al., 2022a; Caruso et al., 2024). These findings may be more clinically relevant in populations with impaired microvascular function, such as those with cerebral small vessel disease or peripheral vascular disease, where improved RBC deformability could further support microcirculatory flow (Midttun et al., 1999; Joutel et al., 2010). Chronic PDE5-i administration may further amplify these rheological benefits and improve tissue oxygen delivery (Webb et al., 2024). Additional research is needed to evaluate its therapeutic potential in these settings. Methodological considerations First, it is unclear whether PDE5-i alone affects RBC deformability. We assumed that a one-hour washout period following exercise hyperemia would eliminate any residual effects of exercise; however, this was likely incorrect. Although not statistically significant, EImax appeared slightly elevated post-placebo compared to pre-placebo baseline. It remains unknown whether the observed enhancements in RBC deformability can occur without prior exercise, or how long these effects might persist. Second, venous blood was drawn from the antecubital vein rather than the 99 BA. While minor hemorheological differences between these vessels may exist, prior studies show comparable hematocrit levels between arterial and venous sites during exercise (González-Alonso et al., 2002), and the short arteriovenous transit time suggests that RBC deformability remains stable between circulations (de Winter et al., 2024). Nevertheless, the potential impact of the microcirculation on RBC deformation, should not be overlooked and warrants consideration when interpreting these findings. Third, we assumed that our homogeneous measurement of whole blood viscosity provided a valid estimate of shear stress within the brachial and extracranial cerebral arteries. Although an erythrocyte-poor layer (i.e., cell-free zone) exists near the vessel wall, this zone is negligible relative to conduit artery diameter, supporting the use of whole blood viscosity as a suitable proxy for in vivo conditions (Kim et al., 2007). Finally, testing was conducted during the early follicular phase to control for estradiol-related variability in exercise hyperemia. While this enhances internal validity, it may reduce generalizability, particularly in relation to potential NO/cGMP–estradiol interactions during the late follicular phase. However, any such influence of estradiol on vascular function is likely minimal (D'Urzo et al., 2018). Conclusion Progressive handgrip exercise enhanced RBC deformability, and this effect was prolonged following PDE5-i administration; however, PDE5-i did not further augment deformability. Despite this prolonged enhancement, no changes were observed in CBF or BA Q in healthy individuals. Still, the sustained increase in RBC deformability may have therapeutic potential in conditions with impaired microcirculatory function. Further research is needed to clarify the underlying mechanisms and assess clinical relevance in both healthy and diseased populations. 100 Chapter 5: Study 3 - Hemorheological, cardiorespiratory, and cerebrovascular effects of pentoxifylline following acclimatization to 3,800 m Abstract: Pentoxifylline is a non-selective phosphodiesterase inhibitor used for the treatment of peripheral artery disease. Pentoxifylline acts through cyclic adenosine monophosphate, thereby enhancing red blood cell deformability, causing vasodilation and decreasing inflammation, and potentially stimulating ventilation. A double-blind, placebo-controlled study was conducted to test the hypothesis that pentoxifylline could lower blood viscosity, enhance cerebral blood flow and decrease pulmonary artery pressure in lowlanders following 11-14 days at 3800m. Participants (6M/10F; age=27±4 years) received either a placebo or 400 mg of pentoxifylline orally the night before and again two-hours before testing. Arterial blood gases, venous hemorheology (blood viscosity, red blood cell deformability and aggregation) and inflammation (TNF-α) in room-air (end-tidal oxygen partial pressure ~52 mmHg) was assessed. Global cerebral blood flow (gCBF), ventilation and pulmonary artery systolic pressure (PASP) were measured in room air and again after 8-10 minutes of isocapnic hypoxia (end-tidal oxygen partial pressure: 40 mmHg). Pentoxifylline did not alter arterial blood gases, TNF-α, or hemorheology compared to placebo. Pentoxifylline did not affect gCBF or ventilation during room-air or isocapnic hypoxia compared to placebo. However, in females, PASP was reduced with pentoxifylline during room air (placebo=19±3 and pentoxifylline=16±3 mmHg; p=0.021) and isocapnic hypoxia (placebo=22±5 and pentoxifylline=20±4 mmHg; p=0.029), but not in males. Acute pentoxifylline administration in lowlanders at 3800m had no impact on arterial blood gases, hemorheology, inflammation, gCBF, or ventilation. Unexpectedly, however, pentoxifylline reduced PASP in female participants, indicating a potential effect of sex on the pulmonary vascular responses to pentoxifylline.101 Introduction Exposure to high-altitude prompts a cascade of integrated physiological responses that aim to mitigate the decline in oxygen partial pressure and maintain oxygen delivery (Gill et al., 1962; Lundby et al., 2018; Tymko et al., 2022). However, rapid ascent to high-altitude can result in high-altitude related health problems including acute mountain sickness, and two life threatening diseases: high-altitude pulmonary edema, and high-altitude cerebral edema (Hackett et al., 1998; Maggiorini et al., 2001). These high-altitude illnesses are often associated with heightened whole blood viscosity (Ding et al., 2014), elevated pulmonary vascular pressure (Maggiorini et al., 2001; Swenson et al., 2002), and increased systemic oxidative-inflammatory stress (Bailey et al., 2009; Pham et al., 2022). Hypobaric hypoxia stimulates plasma volume decreases and erythropoiesis leading to a rapid and sustained increases in whole blood viscosity (Tremblay et al., 2020), which can impede microvascular flow (Driessen et al., 1979; Martin et al., 2009). Impaired red blood cell deformability independent of hemoconcentration may also contribute to this detriment in microcirculation flow (Salvi et al., 2023). Red blood cell deformability decreases with increasing hypoxia in a dose-dependent manner (Grau et al., 2016) and with red blood cell oxidative stress (Härtel et al., 2022). Pentoxifylline, a rheologically active drug used in the treatment of microcirculation dysfunction in peripheral artery disease (Bowton et al., 1989), may offer potential therapeutic benefits at high-altitude. For example, pentoxifylline (400 mg – three times daily) prevented an increase in whole blood viscosity and attenuated a decline in red blood cell filtration (a surrogate of red blood cell deformability) during a three week high-altitude expedition at 5150m (Wood & Appenzeller, 1988). Acute pentoxifylline (bolus intravenous 100 mg) lowers pulmonary artery pressure in individuals with chronic cor pulmonale during both rest and exercise and it also attenuates the hypoxic pulmonary vasoconstrictive response in dogs (Hakim & Petrella, 1988). Pentoxifylline's potential to lower pulmonary artery pressure and reduce hypoxic vasoconstrictive responsiveness at high-altitude might be attributed to its capacity to lower blood viscosity (Sonkin et al., 1993), induce pulmonary vasodilation (Sturani et al., 1986b), and decrease inflammation (Liu et al., 1992). 102 Pentoxifylline has also been observed to increase global and regional cerebral blood flow in both healthy participants and patients with cerebrovascular disease (Bowton et al., 1989). Although the exact mechanism is still debated, current evidence suggests that pentoxifylline primarily enhances cerebral blood flow by inducing hemorheological changes (improved red cell deformability), thereby promoting improved cerebral microcirculation flow (Schubotz & Mühlfellner, 1977; Hartmann, 1983; Hartmann & Tsuda, 1988). In addition, pentoxifylline (single dose of oral 400 mg) has demonstrated its ability to prevent decreases in cerebral blood flow during rapid exposure to stimulated 4000 m using a decompression chamber (Koppenhagen, 1984). However, pentoxifylline (400 mg – once daily for five weeks) has also been associated with an increase in arterial oxygen saturation at high-altitude following five weeks at 4600 m (Wood et al., 1992), which would oppose further dilation since arterial oxygen content is tightly linked with cerebral blood flow [as reviewed in (Willie et al., 2014)]. The precise mechanism underlying this augmented arterial oxygen saturation remains unknown, although it is plausible that pentoxifylline could increase ventilation due to its resemblance to other respiratory stimulants (e.g. theophylline) (Tkácová et al., 1989), and even mild hyperventilation is an effective means to increase oxygen saturation in chronic hypoxia. Despite these promising findings, the impact of pentoxifylline on integrated physiology responses and its potential therapeutic use at high-altitude have not been explored. Herein, it was conducted a comprehensive assessment of blood rheology, pulmonary artery pressure, ventilation, inflammation and cerebral blood flow using a double-blind placebo control research study in young healthy male and female volunteers sojourning to high-altitude (3800 meters) following 11-14 days of acclimatization. We hypothesized that pentoxifylline would lower whole blood viscosity, reduce pulmonary artery pressure, and decrease cerebral blood flow by increasing ventilation. Methods Ethical approval 103 All experimental procedures had Institutional Review Board approval from the University of British Columbia (H22-01091) and conformed to the latest revision of the Declaration of Helsinki, except for registration in a database. Before participation, all subjects provided written informed consent. This study was part of a larger research expedition conducted in September 2022 at the Barcroft Field Station (White Mountain, California, USA; 3800 meters). As such, participants took part in a number of studies conducted at the Barcroft Field Station. However, the a priori, primary research questions addressed in the current paper are novel and are exclusively dealt with in this study alone – there was no overlap between this investigation and others completed on the research expedition. Participants Sixteen participants were recruited, including 10 females and 6 males, with an average age of 27 ± 5 years (mean ± standard deviation [SD]). They were normotensive (systolic blood pressure < 140 and diastolic pressure < 90 mmHg), had a body mass index ≤ 30 kg m-2. Participants were non-smokers, who had no previous history of cardiovascular, cerebrovascular or respiratory diseases. All testing occurred at high altitude. However, we additionally made hemorheological measures at low altitude (see Measurements). Low altitude testing occurred at the University of British Columbia-Okanagan Campus in British Columbia (altitude = ~344m), one month before departure to high altitude. All participants arrived at Barcroft Field Station on the same day, and approximately at the same time (i.e. within 1 h). Participants rapidly ascended to 3800 meters by motor vehicle over the course of 2-3 hours. Testing took place between the 11th and 14th day after arrival at 3800 meters. Participants abstained from prophylactic altitude sickness drugs such as acetazolamide and corticosteroids. Participants refrained from using non-steroidal anti-inflammatories for at least 24 hours before the experiments. Before each experiment, all participants abstained from alcohol and caffeine for ≥ 24 h. Participants were sea-level residents and had not traveled to high-altitude within 6 months before the study. None of the participants were experiencing any symptoms of acute mountain sickness, as determined by the Lake Louise questionnaire. 104 Experimental design We used a counter-balanced, cross-over, double-blind, placebo-control design. Participants were tested on two different days with 40 hours between testing conditions. Participants received pentoxifylline or a placebo on any given day, without a predetermined sequence. However, we employed a counterbalanced design to mitigate the impact of day or sequence effects on physiological outcomes. Randomization and drug coordination were conducted by an external member of our research team who was not involved in data collection. This approach guaranteed complete blinding of both participants and researchers to the specific condition being administered. Participants received either a placebo or 400 mg of sustained-release generic pentoxifylline (Apotex Inc, Toronto, Canada) tablets orally on the night before testing and again two hours before the test (total 800 mg). Sustained release tablets are rapidly absorbed and blood concentrations peak within 3 hours and remain elevated for 14 hours (Srinivasu et al., 1999). Testing commenced between 06:00 and 14:00 and was matched for time of day between conditions. Participants received a standardized meal 30 minutes before testing consisting of one Clif bar (Mondelez International Inc, United States). The experiment began with participants laying supine and resting quietly for ~10 min before collections of resting venous blood samples and radial artery blood samples (see Measurements). Following this, carotid body chemoreflex-mediated inhibition of ventilation was assessed. As described previously (Prasad et al., 2020; Gibbons et al., 2022), 100% oxygen via a Douglas bad was delivered for 1-minute followed by ~ 3 minutes of room air breathing or until end-tidal oxygen partial pressure (PETO2) returned to baseline. This process was repeated four times. The 2-3 breath nadir in response to hyperoxia was noted and the average of the four trials was used to provide an index of the carotid body’s contribution to resting room-air ventilation. Subsequently, carotid body-mediated activation of ventilation by progressive isocapnic hypoxia was assessed. Participants were connected to an end-tidal forcing system (see Measurements) to maintain carbon dioxide partial pressure (PETCO2) at baseline values, while being exposed to two progressive 105 hypoxic stages. Isocapnic hypoxia stages were clamped at PETO2 = 45 mmHg and 40 mmHg. Prior to isocapnic hypoxia, participants were initially clamped at their room-air PETCO2 and PETO2 for a 10-minute baseline period to collect baseline echocardiography and cerebral blood flow. Each hypoxic stage consisted of 3-minutes of steady state breathing, with echocardiography and cerebral blood flow scans recollected after this 3-minute period during the last hypoxic stage (40 mmHg). The last minute of each hypoxic stage ventilation was collected and a linear slope between ventilatory variables and peripheral oxygen saturation was used to determine the hypoxic ventilatory response. Figure 5-1. Schematic of the experimental design Pulmonary arterial systolic pressure reactivity was determined by taking the last minute of 40 mmHg isocapnia for PETO2 and pulmonary arterial systolic pressure relative to room-air: PASP reactivity = (∆PASP∆PETO2) = ((PASP)room-air - (PASP)isocapnic hypoxia (40 mmHg)(PETO2)room-air - (PETO2)isocapnic hypoxia (40 mmHg)) Equation 5.1 Measurements 106 Transthoracic echocardiography Echocardiography was conducted using ultrasound (Vivid iq, GE Healthcare, Piscataway, NJ) and a phased array transducer (1.5- to 3.6-MHz M4S-RS, GE Healthcare, Piscataway, NJ) by a trained sonographer (A.M.W.). A three-lead electrocardiograph was connected to the participant to enable cardiac cycle gating during the examination. With the participant in the left lateral decubitus position, images were acquired for assessment of cardiac function according to current guidelines (Lang et al., 2015). The imaging was obtained at end-expiration for five consecutive cardiac cycles, and the data were saved for later offline analysis using EchoPAC software (GE Medical, Horton, Norway). Measurements were taken in triplicate from consecutive cardiac cycles and then averaged to enhance accuracy. To assess the diameter of the inferior vena cava, subcostal images were acquired while the participant was lying supine. Cardiac output was calculated as stroke volume (ml) × heart rate (bpm), and total peripheral resistance was calculated as mean arterial pressure (mmHg) / cardiac output (mL/min). PASP was quantified as the maximum systolic pressure gradient across the tricuspid valve added to right atrial pressure estimated from the collapsibility of the inferior vena cava. To derive pressure, the modified Bernoulli equation (4⋅V2) was applied to the peak systolic regurgitation jet velocity measured via continuous wave Doppler. Cerebral blood flow Diameter and blood velocity of the right vertebral artery and the right internal carotid artery were measured using a 10 MHz mutli-frequency linear array duplex ultrasound (Terason uSmart 3300, Teratech, Burlington, MA, USA). Scans of the vertebral artery and internal carotid artery were performed sequentially. Diameter and peak blood velocity were simultaneously measured using B-mode and pulse-wave mode, respectively. The insonation angle was 60-degrees. For the internal carotid artery, diameter measurements were taken at a location at least 1.5 cm distal to the common carotid bifurcation to avoid capturing turbulent flow. Vertebral artery flow was always measured from the same intervertebral space within participants. Diameter and peak blood flow velocity were captured using screen-capture software and subsequently stored for offline analysis. Peak blood velocity and diameter were analyzed at 30 Hz with a custom wall edge tracking software 107 (Woodman et al., 2001). One-minute averages were collected for internal carotid artery and vertebral artery blood flow at baseline and at the last minute of isocapnic hypoxia. Volumetric blood flow was calculated as: 𝐵𝑙𝑜𝑜𝑑 𝑓𝑙𝑜𝑤 = (0.5 × 𝐵𝑙𝑜𝑜𝑑 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦) × (𝜋(0.5 × 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟)2) × 60 Equation 5.2 Global cerebral blood flow was calculated as twice the sum of the internal carotid artery and the vertebral artery. Cardiorespiratory metrics: Systolic and diastolic blood pressures were measured using an automated cuff sphygmomanometer (Carescape V100, GE Healthcare). Mean arterial pressure was subsequently calculated as follows: (1/3 systolic blood pressure) + (2/3 × diastolic blood pressure). Arterial oxygen saturation (SaO2) was estimated by pulse oximetry (Rad-5, Masimo) using the index finger sensor. PETO2 and PETCO2 were collected continuously using a gas analyzer (model ML206, ADInstruments). PETCO2 and PETO2 were detected using a peak detection analysis. Respiratory flow was continuously measured by a pneumotachometer (model MLT1000L, ADInstruments). The gas analyzer and pneumotachometer were calibrated prior to daily testing and between participants. PETCO2, PETO2 and minute ventilation were recorded at 1000 Hz and integrated with a data-acquisition system (Powerlab 16/30, ADInstruments) and stored for subsequent analysis using associated software (Labchart 8.0 Pro, ADInstruments). A reactivity metric was calculated for hemodynamic variables, respiratory variables, and cerebral blood flow by determining the delta change between room-air conditions and isocapnic hypoxia for each day. Dynamic end-tidal forcing: Both PETCO2 and PETO2 were controlled using a dynamic end-tidal forcing system that has been described in detail previously (Tymko et al., 2015). End-tidal forcing targets breath-by-breath end-tidal PETCO2 and PETO2 thereby controlling arterial gases independent of ventilation. Our end-tidal 108 forcing system controls end-tidal gases through a wide range of PETCO2 and PETO2 and has been used for similar PETO2 manipulations (Tymko et al., 2015). Blood collection Radial artery blood samples (2 mL) were collected using a lithium heparin-coated auto-fill syringe. Immediately following sample collection, any air bubbles were evacuated from the syringe before analysis. Blood analysis, including pH, PaCO2, PaO2, PaSO2, lactate, glucose, sodium and potassium, was performed in duplicate within 2 minutes of sampling using a commercially available gas analyzer (ABL90FLEX PLUS, Radiometer). The gas analyzer was calibrated every eight hours using the manufacturer's standard internal quality checks. Hematocrit was obtained by the micro-method (PowerSpin BX Centrifuge – unico; 10 min at 13,000 rpm). Arterial oxygen content (CaO2) was calculated from [Hb] × 1.34 × (SpO2/100) + (0.003 × PaO2). Venous blood samples were obtained from the antecubital vein using K2 EDTA tubes (12 mL). Following collection, venous samples were promptly centrifuged, aliquoted, and flash-frozen (-196°C). Samples were stored at – 80°C until analysis at our laboratory in Kelowna, British Columbia. Hemorheology and biochemical analysis Whole blood viscosity was measured in duplicate within 15 min of blood sample acquisition at seven shear rates of 47.5, 67.5, 90, 150, 225, 337.5, and 450 s−1 at 37°C with a cone-and-plate viscometer (Model DV2T, CPA-40Z spindle; Brookfield Amtek, Middleboro, MA, USA). These shear rates were selected based on the shear-thinning properties of erythrocytes and the heterogeneity of shear rates within human conduit arteries (Leo et al., 2020). To isolate plasma viscosity from whole blood viscosity, whole blood was centrifuged at 600 g and 4°C for 10 min. The viscosity of the separated plasma was then measured at two shear rates 225 and 450 s−1 at 37°C. The selection of these shear rates was based on their physiological relevance, representing conditions that closely mimic physiological stresses. Red blood cell deformability was analyzed using laser-assisted optical rotational red cell analyzer (LORRCA, RR Mechatronics, Hoorn, The 109 Netherlands). Whole blood (~14 uL) was dissolved in 5 ml of isotonic 0.14 mM polyvinylpyrrolidone (osmolarity 300 mOsmol/L, viscosity 28.38 mPa sec at 37°C). 880 uL of sample was then placed into the couette system (bob) and sheared at nine different rates between 0.3 and 30 Pa. A laser beam is directed through the samples during shearing and deformation of red blood cells was determined based on the diffraction pattern of the laser beam. The LORRCA software used width (W) and length (L) of the diffraction pattern to calculate an elongation index (EI): EI = (L-W)/(L+W) for the nine shear stresses. RBC deformability parameters were derived using the non-linear version of the Lineweaver-Burk model (GraphPad Prism 10.1.0, GraphPad Software Inc., CA, USA) as previously described (Baskurt & Meiselman, 2013). Furthermore, we calculated the ratio between (SS½)/(EImax) as an additional metric for determining alternations of red blood cell deformability. The ratio between (SS½)/(EImax) is a more robust metric for determining alternations in red blood cell deformability compared to SS½ and EImax alone (Baskurt & Meiselman, 2013). A greater SS½ / EImax reflects worse deformability. The red blood cell aggregation was also performed by LORRCA. The computer program analyzed the aggregation parameters of red blood cells, which were based on the syllectogram – the curve of relation between intensity of laser backscattered and time. Red blood cell aggregation was quantified as aggregation index (AI) as previously described (Hardeman et al., 2001). Plasma concentration of tumor necrosis factor alpha was quantified using a sandwich solid-phase enzyme linked immunosorbent assay (R&D: catalogue #: HSTA00E). Statistical analysis Statistical analyses were performed using the statistical language R (R-4.3.1 Foundation for Statistical Computing). Figures were created using GraphPad, Prism 8.3.0 (GraphPad Software Inc.). All data are presented as means ± SD with statistical significance set at p < 0.05. Data were assessed for normality and variance using the Shapiro Wilk and the Bron–Forsythe tests. We employed paired two-tailed Student’s t-tests to compare placebo and pentoxifylline for various parameters, including demographics, blood gases, electrolytes, whole blood and plasma viscosity, TNF-α levels, carotid body chemoreflex-mediated inhibition of ventilation, and carotid body 110 chemoreflex-mediated activation of ventilation. Additionally, we employed a paired two-tailed Student’s t-tests for these comparisons: placebo room-air vs pentoxifylline room-air, placebo isocapnic hypoxia vs pentoxifylline isocapnic hypoxia and between day reactivity for hemodynamic variables, respiratory variables, room-air cerebral blood flow and pulmonary artery systolic pressure. To compare means between placebo and pentoxifylline for global cerebral blood flow and internal carotid artery blood flow during isocapnic hypoxia, we employed a Wilcoxon matched-pairs signed rank test. Although the overall data exhibited a normal distribution, the normality of residual means was not normally distributed. We employed a paired two-tailed Student’s t-tests for these comparisons: placebo room-air vs pentoxifylline room-air, placebo isocapnic hypoxia vs pentoxifylline isocapnic hypoxia and between day reactivity for hemodynamic variables, respiratory variables, room-air cerebral blood flow and pulmonary artery systolic pressure. We also employed an exploratory linear mixed effects model with pulmonary artery systolic pressure as the dependent variable. In this analysis, we included drug (placebo vs. pentoxifylline), and sex as fixed factors, with participant identifiers serving as the random factor. Our aim was to identify any potential interactions among drug and sex that may have existed. When significant interactions were observed, we conducted within-sex comparisons for pulmonary artery systolic pressure using paired two-tailed Student’s t-tests. To gauge effect size, we calculated Cohen’s d using the following formula: Cohen's d = (Mean of Group 1 - Mean of Group 2) / Pooled Standard Deviation Equation 5.3 Pooled standard deviation = √ ([(standard deviation 1^2) + (standard deviation 2^2)]/ 2) Equation 5.4 Results Data are available upon request from the corresponding author. 111 Blood gases and rheology Standard blood gas metrics (pH, PaCO2, PaO2, HCO3- and SaO2) and inflammation (TNF-α) were not different between days (see Table 5-1). TNF-α coefficient of variation was 4.7 %. Whole blood viscosity, plasma viscosity and red blood cell deformability at all shear rates were not different with pentoxifylline compared to placebo. EImax, SS ½, (SS½)/(EImax) and aggregation index, and were also all not different between days (see Table 5-2). Compared with low altitude, whole blood viscosity and hematocrit, but not plasma viscosity were elevated. Additionally, SS ½, and (SS½)/(EImax) were elevated at high altitude, indicating worsened deformability, but there was no change in EImax or aggregation index (see Table 5-3). Table 5-1. Arterial blood gases, electrolytes and venous blood inflammation Placebo Pentoxifylline p-value pH 7.45 ± 0.02 7.46 ± 0.02 0.13 PaCO2 (mmHg) 29 ± 2 29 ± 2 0.83 PaO2 (mmHg) 59 ± 3 58 ± 4 0.65 HCO3- (mmol/L) 22 ± 1 23 ± 1 0.28 SaO2 (%) 91 ± 2 91 ± 2 0.70 Lactate (mmol/L) 0.7 ± 0.2 0.7 ± 0.2 0.91 CaO2 19 ± 2 18 ± 2 0.57 Potassium (mmol/L) 3.9 ± 0.60 3.8 ± 0.60 0.40 Sodium (mmol/L) 138.9 ± 1.5 139.2 ± 1.5 0.26 Glucose (mmol/L) 5.2 ± 0.76 5.0 ± 0.4 0.33 Osmolality (mmol/Kg) 283.2 ± 3.1 283.5 ± 3.1 0.58 TNF-α (pg/mL) 1.2 ± 0.30 1.1 ± 0.28 0.64 List of Abbreviations: PaCO2, carbon dioxide arterial partial pressure; PaO2, oxygen arterial partial pressure; HCO3-, bicarbonate; SaO2 arterial saturation; CaO2 arterial content and TNF-α tumor necrosis factor alpha Values are mean ± standard deviation unless otherwise specified. A paired two-tailed Student’s t tests were used for the statistical comparisons 112 Table 5-2. Whole blood viscosity, plasma viscosity, red blood cell deformability and aggregation. Placebo Pentoxifylline p-value Whole blood viscosity, mPa·s Shear rate of 45 s-1 6.37 ± 0.65 6.54 ± 1.08 0.97 Whole blood viscosity, mPa·s Shear rate of 67.5 s-1 5.84 ± 0.62 5.68 ± 0.69 0.88 Whole blood viscosity, mPa·s Shear rate of 90 s-1 5.51 ± 0.57 5.35 ± 0.64 0.77 Whole blood viscosity, mPa·s Shear rate of 150 s-1 5.11 ± 0.49 4.96 ± 0.59 0.72 Whole blood viscosity, mPa·s Shear rate of 225 s-1 4.84 ± 0.46 4.70 ± 0.57 0.73 Whole blood viscosity, mPa·s Shear rate of 337.5 s-1 4.60 ± 0.50 4.47 ± 0.54 0.72 Whole blood viscosity, mPa·s Shear rate of 450 s-1 4.29 ± 0.34 4.31 ± 0.33 0.74 Plasma viscosity, mPa·s Shear rate of 225 s-1 1.52 ± 0.11 1.47 ± 0.15 0.91 Plasma viscosity, mPa·s Shear rate of 450 s-1 1.43 ± 0.08 1.44 ± 0.09 0.78 Hematocrit (%) 48.2 ± 3.6 47.3 ± 3.5 0.13 Hemoglobin (grams/dL) 15.1 ± 1.5 14.8 ± 1.4 0.89 SS½ 1.88 ± 0.26 1.9 ± 0.32 0.32 EImax 0.64 ± 0.012 0.64 ± 0.012 0.75 (SS½)/(EImax) 2.93 ± 0.46 2.96 ± 0.48 0.64 AI 66.7 ± 9.8 63.8 ± 10.7 0.21 113 List of Abbreviations: SS½, Shear stress at half maximal deformation; EImax, maximal red blood cell deformability and AI, aggregation index. Values are mean ± standard deviation unless otherwise specified. A paired two-tailed Student’s t tests were used for the statistical comparison Cardiorespiratory and pulmonary artery systolic pressure There were no differences between experimental days for both room-air and isocapnic hypoxia minute ventilation, cardiac output, mean arterial pressure (Table 5-4) and global cerebral blood flow (room-air [p = 0.57] and isocapnic hypoxia [p = 0.89]) (Figure 5-2) were not different. Carotid body-mediated activation of ventilation (p = 0.73) and carotid body chemoreflex-mediated inhibition of ventilation (p = 0.56) were also not different between days (Figure 5-3). 114 Table 5-3 Hemrheology parameters measured at low altitude and during the placebo day of testing at high altitude. (n = 14). Low altitude Placebo p-value Whole blood viscosity, mPa·s Shear rate of 45 s-1 5.45 ± 0.59 6.37 ± 0.65 <0.01 Whole blood viscosity, mPa·s Shear rate of 67.5 s-1 5.01 ± 0.43 5.84 ± 0.62 <0.01 Whole blood viscosity, mPa·s Shear rate of 90 s-1 4.70 ± 0.36 5.51 ± 0.57 <0.01 Whole blood viscosity, mPa·s Shear rate of 150 s-1 4.30 ± 0.32 5.11 ± 0.49 <0.01 Whole blood viscosity, mPa·s Shear rate of 225 s-1 4.07 ± 0.33 4.84 ± 0.46 <0.01 Whole blood viscosity, mPa·s Shear rate of 337.5 s-1 3.90 ± 0.31 4.60 ± 0.50 <0.01 Whole blood viscosity, mPa·s Shear rate of 450 s-1 3.81 ± 0.32 4.29 ± 0.34 <0.01 Plasma viscosity, mPa·s Shear rate of 225 s-1 1.45 ± 0.14 1.52 ± 0.11 0.085 Plasma viscosity, mPa·s Shear rate of 450 s-1 1.39 ± 0.11 1.43 ± 0.08 0.87 Hematocrit (%) 46.2 ± 3.0 48.2 ± 3.6 <0.01 SS½ 1.70 ± 0.30 1.88 ± 0.26 0.01 EImax 0.63 ± 0.014 0.64 ± 0.012 0.61 (SS½)/(EImax) 2.68 ± 0.38 2.93 ± 0.46 0.03 AI 60.2 ± 14.2 66.7 ± 9.8 0.34 115 List of Abbreviations: SS½, Shear stress at half maximal deformation; EImax, maximal red blood cell deformability and AI, aggregation index. Values are mean ± standard deviation unless otherwise specified. A paired two-tailed Student’s t tests were used for the statistical comparison 116 Table 5-4 Cardiorespiratory parameters during room-air and isocapnic hypoxia (40 mmHg). Placebo room-air Pentoxifylline room-air p-value Placebo hypoxia Pentoxifylline hypoxia p-value Placebo hypoxia – room-air Pentoxifylline hypoxia – room-air p-value Hemodynamics Heart rate (bpm) 77 ± 11 71 ± 11 0.031 86 ± 11 83 ± 11 0.92 8 ± 5 12 ± 8 0.11 Mean arterial pressure (mmHg) 89 ± 6 88 ± 8 0.70 92 ± 8 93 ± 7 0.98 3 ± 4 6 ± 5 0.13 Systolic arterial pressure (mmHg) 120 ± 9 119 ± 9 0.84 126 ± 9 128 ± 10 0.78 9 ± 6 10 ± 6 0.08 Diastolic arterial pressure (mmHg) 74 ± 7 72 ± 7 0.75 92 ± 7 93 ± 7 0.97 19 ± 5 21 ± 5 0.45 Cardiac output (L/min) 5.9 ± 1.1 5.6 ± 1.2 0.81 6.8 ± 1.3 6.7 ± 1.5 0.99 0.83 ± 0.97 1.2 ± 1.6 0.51 Stroke volume 78 ± 15 79 ± 16 0.94 79 ± 14 78 ± 15 0.98 2 ± 9 3 ± 14 0.93 Respiration PETO2 (Torr) 52 ± 3 53 ± 2 0.67 40 ± 1 39 ± 1 0.56 -13 ± 3 -15 ± 2 0.32 PETCO2 (Torr) 29 ± 3 29 ± 2 0.96 29 ± 3 29 ± 2 0.98 -0.02 ± 0.56 -0.02 ± 0.61 0.59 Peripheral oxygen saturation (%) 90 ± 3 90 ± 2 0.42 76 ± 3 75 ± 3 0.81 -14 ± 3 -16 ± 3 0.43 V̇E (L/min) 14.5 ± 3.8 15.6 ± 3.3 0.84 30.9 ± 16.2 32.9 ± 13.1 0.91 16.3 ± 13.8 17.2 ± 11.3 0.74 Cerebral blood flow ICA flow (mL/min) 247 ± 47 251 ± 34 0.96 289 ± 62 294 ± 50 0.97 42 ± 30 43 ± 42 0.92 VA flow (mL/min) 89 ± 40 83 ± 36 0.49 117 ± 53 113 ± 51 0.79 27 ± 16 29 ± 14 0.59 117 List of Abbreviations: PETO2, end-tidal oxygen partial pressure; PETCO2, end-tidal carbon dioxide partial pressure; V̇E, minute ventilation; ICA, internal carotid artery and VA, vertebral artery. Values are mean ± standard deviation unless otherwise specified. A paired two-tailed Student’s t tests were used for the statistical comparison 118 There was no difference between experimental days for pulmonary artery systolic pressure during room-air (p = 0.94) or isocapnic hypoxia (p = 0.98) (Figure 5-4). Nor was there a change in pulmonary artery systolic pressure reactivity (p = 0.33) (Figure 5-5). However, for the exploratory sex differences, we observed a significant interaction between sex and drug (p = 0.00042), but no other significant sex interactions. Females exhibited lower pulmonary artery systolic pressure during both room-air (p = 0.021) and isocapnic hypoxia (p = 0.029) when given pentoxifylline compared to placebo. There was also a notable effect size for room-air ([pentoxifylline - placebo] effect size = 1.05) and isocapnic hypoxia ([pentoxifylline - placebo] effect size = 0.97) (Figure 5-6). 119 Placeboroom-airPTXroom-airPlacebo hypoxiaPTX hypoxia50010001500gCBF(mL • min-1) Figure 5-2. Pentoxifylline on cerebral blood flow Placebo (blue) and Pentoxifylline [PTX] (red) during room-air and isocapnic hypoxia (40 mmHg). Closed dots are individual responses and bar graphs are group means. A paired two-tailed Student’s t tests were used for the statistical comparisons (n = 14).120 Placebo PTX-80-60-40-200Δ VE (%)Placebo PTX-4-3-2-101Slope (L • min-1 / %)A B Figure 5-3. Pentoxifylline on carotid body tonic activity and tonic control Minute ventilation (V̇E) in response to transient hyperoxia (i.e. carotid body chemoreflex-mediated inhibition of ventilation) reported as a relative reduction from baseline with placebo (blue) and Pentoxifylline [PTX] (red) (Figure 5-3a) (n = 15). Ventilatory sensitivity to isocapnic hypoxia (i.e. carotid body chemoreflex-mediated activation of ventilation) with placebo and PTX. To create a sensitivity slope, the minute ventilation of the last minute of each hypoxic stage was collected and a linear slope between minute ventilation and arterial oxygen saturation was used to determine the acute hypoxic ventilatory response (Figure 5-3b) (n = 14). Closed dots are individual responses and bar graphs are group means. A paired two-tailed Student’s t tests were used for each statistical comparison. 121 Placebo room-airPTXroom-airPTX hypoxiaPlacebo hypoxia1015202530Pulmonary artery sytolicpressure(mmHg) Figure 5-4. Pentoxifylline on pulmonary artery systolic pressure Placebo (blue) and Pentoxifylline [PTX] (red) during room-air (n=14) and isocapnic hypoxia (40 mmHg; n=11). Closed dots are individual responses and bar graphs are group means. A paired two-tailed Student’s t tests were used for the statistical comparisons. 122 Figure 5-5. Pentoxifylline on pulmonary artery systolic pressure reactivity to isocapnia hypoxia PASP sensitivity to isocapnic hypoxia (40 mmHg). The delta increase in PASP divided by the delta decrease in the end-tidal partial pressure of oxygen (PETO2) with placebo (blue) and Pentoxifylline [PTX] (red). Closed dots are individual responses and bar graphs are group means. A paired two-tailed Student’s t tests were used for the statistical comparisons (n = 12). 123 Placebo room-airPTXroom-airPlacebo hypoxiaPTX hypoxia1015202530Pulmonary artery sytolicpressure(mmHg)Placebo room-airPTXroom-airPlacebo hypoxiaPTX hypoxia1015202530Pulmonary artery sytolicpressure(mmHg)✱✱A B Figure 5-6. Sex differences to pentoxifylline and pulmonary artery systolic pressure Placebo (blue) and Pentoxifylline [PTX] (red) during room-air and isocapnic hypoxia (40 mmHg) in females (figure 5-6a) (n = 8) and males (figure 5-6b) (n = 6). Closed dots are individual responses and bar graphs are group means. A paired two-tailed Student’s t tests were used for the statistical comparisons. * = p < 0.05 Placebo vs. PTX. 124 Discussion We characterized the acute impact of oral pentoxifylline on blood rheology and the pulmonary vascular, cerebrovascular and ventilatory responses to hypoxia following 11-14 days of high-altitude (3800 meters) acclimatization. The acute administration of pentoxifylline did not alter arterial blood gases, whole blood viscosity, or red blood cell deformability, nor did it affect hypoxic cerebral or ventilatory responses. Nevertheless, it yielded an intriguing finding: a reduction in pulmonary artery systolic pressure in females, with no such effect observed in males. These results indicate that, in young healthy participants sojourning to 3800 meters, acute administration of pentoxifylline does not influence the acclimatization process but may have sex-specific effects on pulmonary artery pressure. Similar to previous findings (Salvi et al., 2023), high-altitude caused a significant decrease in red blood cell deformability; however, pentoxifylline failed to retifify this decline in red blood cell deformability. Pentoxifylline had no effect on whole blood and plasma viscosity, red blood cell deformability or red blood cell aggregation. Similarly, hikers sojourning to altitudes of 4500 (Wood & Appenzeller, 1988) and 4600 meters (Wood et al., 1992) while taking pentoxifylline (at a dose of 400 mg, once daily) for five weeks reported no significant differences in blood viscosity between the control and pentoxifylline groups. However, pentoxifylline administrated at 5150 meters (400 mg three times daily for three weeks) (Wood & Appenzeller, 1988) and 7350 meters (400 mg once daily for six weeks) (Palareti et al., 1984) attenuated the decline in red blood cell filterability following high-altitude exposure. It is therefore plausible that pentoxifylline at high-altitude does not increase red blood cell deformability per se, but negates the decline in red blood cell deformability. Red blood cell deformability declines with greater altitude (Salvi et al., 2023) and hypoxia (Grau et al., 2016). The absence of any hemorheological benefits of pentoxifylline, despite the elevations in blood viscosity and worsened deformability compared with sea level, may be due to the acute administration. Rheological benefits are often observed following months of usage. For example, blood viscosity in patients with peripheral artery disease is decreased following one month of 125 pentoxifylline administration (400 mg three times daily) and further decreased following continued usage up to two months (Rao et al., 1990a). Pentoxifylline may have greater benefits for blood rheology at higher altitudes used for a longer period of time as previously demonstrated (Palareti et al., 1984; Wood & Appenzeller, 1988). Non-human animal studies have demonstrated that administration of pentoxifylline causes blunted hypoxic pulmonary vasoconstrictive responsiveness (Chick et al., 1988; Hakim, 1988; Hakim & Petrella, 1988; Kaye et al., 1996) both in isolated lung models (Hakim, 1988) and anesthetized dogs (Chick et al., 1988). Humans however have demonstrated both decreased pulmonary circulation tone (Sturani et al., 1986b) and reactivity (Türköz et al., 1996). Notably, we observed no change in reactivity, but a change in pulmonary artery tone in females. This observed difference between males and females could be attributed to variations in plasma concentrations. Although we employed a standardized oral dose (i.e. 400 mg), to ensure clinical applicability (Bowton et al., 1989), this approach may have inadvertently resulted in females receiving a relatively higher dose than males (Dominelli & Molgat-Seon, 2022) (Females 69 ± 11 kg and Males 73 ± 9 kg). Furthermore, females have smaller lungs (McClaran et al., 1998) and pulmonary capillary blood volumes (Bouwsema et al., 2017) compared with age- and height-matched males. Pentoxifylline may therefore have a greater impact on the smaller pulmonary vasculature of females. Interestingly, to our knowledge, no study has specifically investigated the effect of synthetic methylxanthine on pulmonary artery pressure in relation to sex. Pentoxifylline decreases pulmonary artery pressure via nitric oxide. In cats, N-nitro L-arginine methyl ester (L-NAME) has been shown to attenuate pulmonary artery vasodilation with pentoxifylline (David Kaye et al., 1996). Treatment of pulmonary hypertension is more effective in males than females with phosphodiesterase inhibitors (i.e. tadalafil), which is nitric oxide-dependent (Mathai et al., 2015) making it improbable that differences in sex hormones are the sole cause of this sex-specific response. However, sex related hormones cannot be entirely ruled out as a contributing factor. Hence, the possibility arises that pentoxifylline might impact the pulmonary vasculature differently in females compared to males independent of body and pulmonary size. It is also important to recognize the rather small number of males within this cohort and that the aim of this 126 study was not to investigate sex differences. Further studies are needed to confirm this sex-specific interaction; however, these findings are promising. Pentoxifylline has been shown not only to increase ventilation, but also enhance oxygen diffusion in chronic obstructive pulmonary disorder (Haas et al., 1990), and increase p50 in anesthetized dogs (Chick et al., 1988). These effects may provide additional potential avenues for improved arterial saturation at high-altitude beyond ventilation. However, our findings within this cohort do not align with this notion. It is conceivable that a longer administration of pentoxifylline could have resulted in a more pronounced impact on these mechanisms (Wood et al., 1992). However, the exact mechanisms causing this augmented arterial saturation still remains unknown warranting further investigation. We hypothesized that minute ventilation might reduce cerebral blood flow by reducing arterial carbon dioxide levels and increasing arterial content (Willie et al., 2014). However, likely because there were no significant alterations in arterial blood gases or arterial content, we observed no discernible change in cerebral blood flow when pentoxifylline was administered. A previous study demonstrated that pentoxifylline attenuates the decline in cerebral blood flow during acute hypobaric hypoxia (decompression chamber – 4000 meters) in healthy participants (Bowton et al., 1989). However, similar to our findings, Woods and colleagues (1988) found that pentoxifylline compared to control did not change middle cerebral artery velocity in mountaineers at 5150 meters (Wood, 1989). Considering that pentoxifylline enhances cerebral blood flow by improving red blood cell deformability, it is reasonable to propose that the absence of changes in red blood cell deformability led to the lack of subsequent alterations in cerebral blood flow. Similar, the absence of pentoxifylline on inflammation (as indexed via TNF-α) may also provide another examination why cerebral blood flow was unchanged (Berg et al., 2018). Importantly, we only assessed the inflammatory cytokine, TNF-α, because pentoxifylline has been shown to reduce inflammation via sustained cyclic adenosine monophosphate activation of protein kinase A and, in turn, the primary transcription factor for TNF-α, nuclear factor kappa-B (Semmler et al. 1993). Without an observed change in TNF-α, the primary cytokine implicated in pentoxifylline’s anti-inflammatory potential, we did not feel the need to expand the inflammation panel for the present study. However, it should be noted that other inflammatory pathways, particularly concerning certain interleukins (IL; e.g., IL-6, IL-8, IL-1), have been shown to be 127 involved in the pathogenesis of high-altitude illness and, despite the good health of our participants, may be a precursor such high-altitude illness and should be explored in the context of pentoxifylline administration for future studies (Hartmann et al. 2000,, Lundeberg et al. 2018, Brewster et al, 2023). Our cohort of participants were acclimatized lowlanders who were in good health (e.g. low systemic concentrations of TNF-α) without high-altitude illness. It is therefore plausible that pentoxifylline could confer greater advantages to individuals burdened with altitude-related illnesses characterized by hyperviscosity, high levels of inflammation, exaggerated hypoxemia and pulmonary vascular pressures (i.e., chronic mountain sickness and high-altitude pulmonary edema). It therefore seems logical to consider using pentoxifylline at higher altitudes for a longer duration of time. Such an approach could potentially yield more substantial benefits, especially given that pentoxifylline is inexpensive and widely available. Conclusion Acute administration of pentoxifylline in healthy, acclimatized lowlanders did not result in any significant changes in arterial blood gases, whole blood viscosity, red blood cell deformability or aggregation, inflammation, cerebral blood flow, or ventilation at 3800m. However, an unexpected and potentially unique finding emerged, as the treatment resulted in a reduction in pulmonary artery pressure in female participants, while no such effect was observed in males. Further investigation is warranted to confirm and elucidate the mechanism that might determine this sex specific response. 128 Chapter 6: Conclusion 6.1 Main findings: 6.1.1 Study 1 - Effects of plasmapheresis on blood rheology and peripheral vascular function Hemoconcentration was induced via plasma donation and diuresis to examine the effects of increased whole blood viscosity on brachial blood flow and vascular reactivity. • RBC deformability was transiently decreased following plasma donation but normalized within 24 hours; this was not observed with diuresis. • Brachial blood flow and RH-FMD remained unchanged in both conditions; however, total reactive hyperemia increased after plasmapheresis. 6.1.2 Study 2 – Exercise-induced enhancements in RBC deformability are prolonged by oral phosphodiesterase-5 inhibition. RBC deformation was assessed before and after a shear stress inducing-handgrip exercise, with and without a PDE-5 inhibitor (PDE5-i) sildenafil citrate. • RBC deformability increased with exercise intensity; PDE5-i did not enhance this response but prolonged the exercise-induced increases in deformation. • Neither the prolongation of RBC deformability nor the potential vasodilatory effects of PDE5-i influenced exercise hyperemia. 6.1.3 Study 3 - Hemorheological, cardiorespiratory, and cerebrovascular effects of pentoxifylline following acclimatization to 3,800 m The effects of pentoxifylline, a non-specific PDE inhibitor, on blood rheology and blood flow were assessed during 10–14 days of high-altitude exposure. 129 • High altitude increased whole blood viscosity through hemoconcentration and reduced RBC deformability. • Pentoxifylline had no effect on RBC deformability, aggregation, cardiac output, or CBF. 6.2 Contributions to the field Nearly 200 years ago, Jean Léonard Marie Poiseuille demonstrated that fluid flow through a tube is influenced by viscosity. By this principle, increased blood viscosity should reduce flow. However, unlike rigid tubes, blood vessels are compliant and actively adapt to pressure and flow changes. Shear stress, determined by both shear rate and blood viscosity, is a key signal regulating vascular diameter and thus flow. This relationship is further complicated by the shear-thinning behavior of blood (e.g., RBC deformability and aggregation), where blood viscosity decreases at higher shear rates due to erythrocyte elongation and alignment with flow. Consequently, both blood viscosity and vessel diameter vary dynamically in response to physiological demands. This raises a fundamental question: how does blood viscosity and overall blood rheology influence blood flow? With this in mind, this dissertation aimed to isolate the influence of elevated blood viscosity (Study 1) and enhanced RBC deformability (Studies 2 and 3) through a series of targeted investigations, each examining a distinct component of blood rheology to better understand its interaction with blood flow. Study 1 demonstrated that hemoconcentration, whether induced by plasma contraction or donation, increased whole blood and apparent viscosity but did not alter basal shear stress, blood flow, or vascular reactivity in the brachial artery. Although prior studies in animals (Martini et al., 2005) and humans (Connes et al., 2012), suggest that elevated viscosity may reduce vascular resistance, this effect was not evident in the brachial artery. It is possible that the degree of hemoconcentration change was insufficient to activate eNOS, consistent with findings from low-intensity handgrip exercise (Atkinson et al., 2015). Interestingly, only plasma donation, not plasma contraction, increased total reactive hyperemia by ~23 percent, despite a temporary reduction in RBC deformability. This suggests that the observed improvement in microvascular function may 130 not have been driven by blood viscosity or RBC deformability, but instead could reflect the influence of citrate-based anticoagulants used during plasmapheresis. While these results support the idea that acute changes in blood viscosity and RBC deformability may be well-compensated in healthy individuals, particularly at the level of conduit arteries, caution is warranted when drawing broader conclusions about the role of blood rheology in regulating tissue perfusion (Leo et al., 2020). The circulatory system is highly adaptive, and lumped vascular measurements (i.e., reactive hyperemia) may mask meaningful changes at the microvascular level. Reductions in RBC deformability may not affect bulk blood flow but the quality of the microvascular perfusion could be reduced capillary recruitment, axial migration, and alignment with flow, ultimately challenging the efficiency of oxygen delivery at the tissue level (Basu et al., 2011; Yalcin et al., 2015; Caruso et al., 2024). These findings underscore the importance of integrating microcirculatory measures in future research to fully understand the contribution of blood rheology to vascular control. To better understand the influence of blood rheology on blood flow, we aimed to determine whether increasing RBC deformability through related pharmacological agents (e.g., sildenafil citrate and pentoxifylline) has the capacity to (1) enhance RBC deformability and associated blood flow during exercise (Study 2), and (2) enhance blood flow during high-altitude acclimatization (Study 3). Study 2 investigated the in vivo mechanisms regulating RBC deformability. While substantial work has been done using ex vivo shear stress models (Simmonds et al., 2014; Kuck et al., 2019; Ugurel et al., 2022), these models are disconnected from the physiological system (Leo et al., 2020). RBC deformability is essential for shear-thinning behavior during exercise, helping offset hemoconcentration (Leo et al., 2020) and allows RBCs to transverse narrow capillary beds. This study aimed to determine the direct influence of RBC deformation on basal and exercise-induced blood flow. Local handgrip exercise enhanced RBC deformability, but PDE5-i did not further increase RBC deformability, although exercise induced enhancements in RBC deformation were prolonged. These findings suggest that PDE5-i sustains, but does not augment, the rheological benefits of exercise. However, despite sustaining elevated RBC deformability, PDE5-i did not alter 131 resting CBF, brachial artery flow, or exercise hyperemia, suggesting that enhanced deformability alone may not substantially influence basal or active perfusion. These findings led to further investigation of RBC deformability under chronic hypoxia, a condition that reduces RBC flexibility and impairs microvascular function, where enhancing RBC deformability may offer greater physiological benefit. Study 3 explored the effects of pentoxifylline, a non-specific phosphodiesterase inhibitor, under hypoxic conditions at high altitude. Chronic hypoxia is known to increase blood viscosity via hemoconcentration and reduced RBC deformability, which may impair microcirculatory flow (Grau et al., 2016; Salvi et al., 2023). Pentoxifylline has previously been shown to attenuate elevations in whole blood viscosity during a 3-wk high-altitude expedition to 5,150 m (Wood & Appenzeller, 1988). In the present study, high altitude increased blood viscosity and decreased RBC deformation; however, pentoxifylline had no significant effect on rheological properties, CBF, or cardiac output, indicating that this non-specific phosphodiesterase inhibitor does not counteract the hemorheological or vasoconstrictive consequences of chronic hypoxia. 6.3 Integrated summary This series of studies suggests that acute changes in blood viscosity and RBC deformability may not substantially alter bulk blood flow in healthy individuals under the conditions tested (e.g., plasma donation, plasma contraction, exercise and chronic hypoxia). Hemoconcentration, whether induced by diuresis or plasma donation, increased blood viscosity but did not affect basal brachial artery blood flow or vascular reactivity. In contrast, plasmapheresis enhanced microvascular function, possibly reflecting a physiological effect unrelated to blood viscosity or RBC mechanics, potentially hinting at the influence of citrate-based anticoagulants. Pharmacological manipulation of RBC deformability yielded mixed results. PDE5-i prolonged exercise-induced enhancements in RBC deformability but did not increase flow, suggesting an interaction with exercise rather than a direct mechanistic effect. Pentoxifylline, surprisingly, did 132 not alter blood flow or blood rheology metrics at high altitude, where the hypoxic environment is known to reduce RBC deformability. While these findings support the idea that the healthy cardiovascular system is highly adaptive and may compensate for moderate rheological changes, it is important to interpret these outcomes within the limitations of the measurement techniques used. Bulk vascular measurements, (e.g., brachial artery blood flow), does not provide sufficient resolution to assess microvascular changes in perfusion or functional capillarization. It remains possible that local changes in microvascular flow or oxygen extraction occurred without detection at the conduit artery level. Thus, while blood rheology clearly contributes to vascular function and oxygen delivery, its precise role in regulating tissue perfusion requires further studies using more spatially resolved techniques (Yalcin et al., 2015). 6.4 Strengths of the dissertation: Blood viscosity was calculated using a two-phase exponential decay model across seven discrete shear rates to capture the non-linear shear-thinning behavior of blood. This approach improves upon single-point viscosity estimates, which can overestimate or underestimate shear stress depending on the shear rate and physiological stimulus being studied (Tremblay et al., 2019b; Carr et al., 2023). Apparent viscosity and shear stress–specific erythrocyte elongation, were calculated using shear rates derived from duplex ultrasound to better reflect in vivo blood rheology (Leo et al., 2023). Study 1 aimed to isolate the effects of plasmapheresis by comparing plasma donation with diuresis-induced plasma contraction, offering a more physiologically comprehensive understanding of vascular responses to plasma removal. Previous studies have reported reductions in RBC deformability (Bor-Kucukatay et al., 2008) and increases in microcirculatory flow assessed via toenail capillary velocity (Reinhart et al., 1992; Mansouri Taleghani et al., 1997); however, none have directly integrated blood rheology and vascular function to assess their combined response to plasmapheresis. By incorporating a diuresis control arm and measuring both variables over time, 133 Study 1 uniquely isolated the independent contribution of hemoconcentration, allowing for a clearer interpretation of how plasma donation influences blood rheology and flow regulation. Study 2 extends previous research by employing a human in vivo model that combines exercise-induced shear stress with pharmacological modulation to assess RBC deformability and blood flow under physiologically relevant conditions (Grau et al., 2013; Leo et al., 2020). Unlike studies applying ex vivo shear stress (Ugurel et al., 2022) or pharmacological agents to isolated RBCs (Hagley et al., 2011), this integrated model captures both vascular and hemorheological responses to dynamic, systemic stimuli. This approach provides a more physiologically relevant framework for understanding how pharmacological agents interact with exercise to influence blood rheology. Study 3 aimed to extend previous findings by evaluating the efficacy of pentoxifylline under high altitude conditions relevant to clinical syndromes such as excessive erythrocytosis and high-altitude pulmonary edema. While earlier studies reported that pentoxifylline attenuates altitude-induced increases in blood viscosity alongside improved peripheral oxygenation (Wood & Appenzeller, 1988), these findings were limited to narrow outcome measures and lacked detailed physiological context. In contrast, Study 3 employed a double-blind, placebo-controlled design and included a comprehensive assessment of arterial blood gases, blood rheology, cerebral blood flow, and cardiac output. Although pentoxifylline did not yield acute benefits in these variables, the breadth of physiological endpoints assessed provides a more rigorous understanding of its limitations and identifies key areas for future research, particularly regarding treatment duration and its potential role (or lack thereof) in chronic hypoxic acclimatization. 6.5 Weakness Across all three studies, RBC deformability was assessed using ektacytometry (LORRCA), which quantifies cell elongation in response to controlled shear stress. While this technique provides a sensitive and standardized measure of RBC deformability (Hardeman et al., 1994; Bianchi et al., 2015), it does not replicate the geometric constraints of the microcirculation. In contrast, microfluidic filtration devices simulate capillary flow by forcing RBCs through micrometer-scale channels and branching structures, allowing direct visualization of deformation under more 134 physiologically relevant conditions (Guo et al., 2012). Study 2 aimed to assess in vivo RBC deformability by relating it to shear stress in the brachial artery. However, because the brachial artery is a conduit vessel, it may not accurately reflect microvascular dynamics, making the LORRCA a more appropriate tool in this context. While Studies 1 and 3 focused on general systemic RBC deformability, incorporating microfluidic systems could have strengthened this work by better simulating the microvascular environment where deformability is most physiologically relevant (Guo et al., 2012). Study 2 focused on shear stress-specific erythrocyte elongation; however, blood was sampled from the antecubital vein as a surrogate for arterial blood in the brachial artery during handgrip exercise. It was assumed that venous blood reflects arterial under these conditions. Although previous studies have shown minimal differences in hematocrit between arterial and venous blood during exercise (González-Alonso et al., 2002), no research to date has directly compared RBC deformability or aggregation between these vessels. The short arteriovenous transit time likely preserves RBC specific properties between circulations (de Winter et al., 2024), but the potential influence of microvascular transit on RBC deformability should not be overlooked and warrants consideration when interpreting study 2. Duplex ultrasound and vascular reactivity assessments are highly technical procedures that require an experienced sonographer to ensure consistent and repeatable scans. For example, RH-FMD typically results in a diameter change of only ~0.25 mm. Minor factors such as probe movement, suboptimal imaging, or changes in gain or dynamic range can substantially affect the recorded peak dilation (Thijssen et al., 2019). A deviation of just 0.05 mm, half the thickness of a human hair, can alter the peak diameter by ~13.5%, potentially masking true physiological changes. However, the sonographer performing the FMDs in this study demonstrated a coefficient of variation well below reported norms (RH-FMD ~ 9.6%), indicating excellent reproducibility compared to other laboratories (Thijssen et al., 2019). Shear stress is calculated as the product of apparent viscosity and shear rate derived from duplex ultrasound. This calculation assumes homogeneous blood viscosity within conduit arteries; 135 however, a cell-free layer forms near the endothelial wall even in these large vessels (Kim et al., 2007). As a result, calculated shear stress likely overestimates true endothelial shear stress. Nevertheless, the large lumen diameter of conduit arteries likely minimizes the physiological relevance of this overestimation (Leo et al., 2020). Study 1 hypothesized that elevated NO concentrations would promote vasodilation and reduce vascular resistance, as previously shown (Connes et al., 2012). However, brachial artery resistance did not decrease, suggesting that NO levels were unchanged, although NO was not directly measured. It remains possible that NO production increased but was insufficient to elicit vasodilation. Furthermore, a more detailed investigation into the regulatory mechanisms of RBC deformability, including RBC-NO content, eNOS phosphorylation at Ser1177, cGMP signaling, cytoskeletal phosphorylation, and the PI3K/Akt pathway could provide deeper insight into how environmental stressors and pharmacological interventions influence RBC flexibility (Grau et al., 2013; Leo et al., 2023). Blood rheology behaves fundamentally differently in the microcirculation compared to the macrocirculation. Shear rates are much higher, while hematocrit and blood viscosity are lower (Yalcin et al., 2015). Because RBCs are similar in size to microvessels, their orientation and deformability become critical for maintaining flow (Pries et al., 1992). Increased RBC deformability promotes a wider cell-free layer, elevating blood velocity and enhancing tissue oxygenation (Zhang et al., 2009; Yalcin et al., 2015). Blood rheology is therefore critical for oxygen delivery; however, the interaction between blood viscosity, RBC deformability and aggregation, and vessel diameter is complex, making it difficult to isolate their individual effects. In humans, this challenge is further compounded by the limited ability to directly observe the microcirculation, leaving much of it a physiological “black box” (Baez, 1973). In Study 2, the independent effect of sildenafil citrate on RBC deformability could not be determined, as measurements were not taken without prior exercise. While it is possible that sildenafil citrate alone may alter RBC deformability, this remains unknown. In study 3, pentoxifylline was administered twice—once the night before and again prior to testing. Although 136 chronic administration over several weeks has been shown to reduce blood viscosity (Bowton et al., 1989; Rao et al., 1990b), acute dosing typically does not elicit the same effect (Sturani et al., 1986a). Therefore, any influence of pentoxifylline on blood rheology may depend on sustained, long-term use. 6.6 Future directions 6.6.1 Anticoagulants on vascular function and longitudinal influence of plasma donation on cardiovascular health Green et al. (1999) reported that six weeks of EDTA administration, an anticoagulant similar to those used during plasma donation, did not significantly alter forearm blood flow responses to acetylcholine in patients with coronary artery disease (Figure 6-1). However, a trend toward improved endothelium-dependent vasodilation was observed, raising the possibility that calcium chelation may influence vascular function. In that study, vascular reactivity was assessed one week after the final EDTA dose in a small sample of eight participants, which may have limited the ability to detect acute effects. This raises an important question: does acute anticoagulant administration affect vascular reactivity? Future studies should explore the immediate impact of anticoagulant infusion on vascular function using methods such as intrabrachial acetylcholine infusion, RH-FMD, or exercise hyperemia. 137 Figure 6-1. EDTA on endothelium dependent-vasodilation via intrabrachial infusion of acetylcholine. Vasodilation was significantly greater following EDTA combined with vitamin supplementation (▲) than after either EDTA alone (■) or placebo (○), while EDTA alone had no effect. © Green D, O'Driscoll J, Maiorana A, Scrimgeour N, Weerasooriya R, Taylor R. Effects of chelation with EDTA and vitamin B therapy on nitric oxide-related endothelial vasodilator function. Clinical Experimental Pharmacological Physiology. 1999: (11). 853-856. Page 854. Modified with permission from John Wiley & Sons. Study 1 was conducted in young, healthy participants (24 ± 5 years), all of whom had not donated plasma for at least 120 days prior to testing. However, this cohort does not reflect the broader donor population in Canada, where the average plasma donor is 47 years old and donates approximately 21 times per year (Seeley et al.), which may limit the generalizability of these findings. Age-related changes in vascular function and blood rheology may influence responses to plasma donation (Black et al., 2009). Additionally, men and women may respond differently to plasmapheresis due to differences in vascular responsiveness and hormonal regulation (e.g., estradiol) (Liang et al., 1997; Moreau et al., 2003; Black et al., 2009). Sex specific studies are 138 required. Large cohort studies could be used to examine how age, sex, and donation frequency relate to long-term cardiovascular outcomes, including pulse wave velocity, blood pressure, and FMD (Rosa-Bray et al., 2015). 6.6.2 Independent effect of PDE5-i on RBC deformability PDE5-i prolonged exercise-induced enhancements in RBC deformability, suggesting an interaction between pharmacological and mechanical stimuli. However, it remains unclear whether this effect requires prior exercise or if PDE5-i can independently enhance RBC deformability. Future studies should assess the effects of PDE5-i in healthy participants with exercise only performed after drug administration to determine whether prior physical activity is necessary. Sublingual and retinal videomicroscopy could be used to directly visualize microvascular perfusion and evaluate whether changes in RBC deformability lead to functional enhancements in microcirculatory flow (Garvin et al., 2009; Aykut et al., 2015). 6.6.3 Therapeutic potential of PDE5-i in diseases with impaired RBC deformability Prolonging RBC deformability may provide meaningful therapeutic benefits in clinical populations with impaired blood rheology, such as cerebrovascular disease (Bowton et al., 1989) peripheral vascular disease (Reid et al., 1976) and diabetic mellitus (Tan et al., 2020), where reduced RBC flexibility contributes to microvascular dysfunction. In addition to improving RBC mechanics, PDE5 inhibitors (e.g., sildenafil, tadalafil) also promote vasodilation. This combined effect on blood rheology and vascular tone may support enhanced microvascular perfusion. For example, a three-week course of PDE5 inhibitor treatment has been shown to increase CBF in patients with mild to moderate small vessel disease (Webb et al., 2024). However, PDE5 inhibitors are associated with side effects, including headache, hypotension, visual disturbances, and, in rare cases, cardiovascular or auditory complications. Its clinical use should therefore be guided by a clear therapeutic rationale and a careful assessment of risks and benefits. 139 6.6.4 Pentoxifylline in high altitude related disorders Acute administration of pentoxifylline in participants without high altitude related pathologies (e.g., high altitude pulmonary edema, high altitude cerebral edema or chronic mountain sickness) did not result in significant changes in RBC deformability or aggregation, CBF, or cardiac output. Participants were healthy with low systemic TNF-α levels, which may have limited the observable effects of pentoxifylline. It is plausible that individuals with high-altitude maladaptations may benefit more from this intervention. Future studies should consider administering pentoxifylline at higher altitudes and over longer durations to evaluate its potential therapeutic impact in susceptible populations. 6.7 Conclusion Blood rheology has a significant role in attenuating increases in blood viscosity during exercise-induced hemoconcentration (Leo et al., 2020), traversing capillaries bed, facilitating the release of vasodilators from RBCs (Racine & Dinenno, 2019), and transmitting shear stress to the endothelium to promote vasodilation, but its impact on bulk blood flow appears limited in healthy individuals. 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Kinesiology
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Ainslie, Philip N., 1974-
Blood rheology on blood flow during plasmapheresis, plasma contraction, exercise, and chronic hypoxia in humans.
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