Health and Social Development, Faculty of (Okanagan)
Health and Exercise Sciences, School of (Okanagan)
DSpace
UBCO
Shabir, Nadhiyya
Master of Science - MSc
University of British Columbia
Purpose: Variations in shear stimulus has been shown to impact endothelial function. Studies have shown that increases in shear improves endothelial function and overall vascular health while decreases in shear leads to impairments in endothelial function and develops risk of cardiovascular diseases. Although this pattern is noticed in adult studies, there are limited studies that investigate this relationship in children. Therefore, the aim of this study was to examine the relationship between shear rate and flow mediated dilation (FMD) in children and adults. Methods: Nine adults and seven children completed two protocols: passive heat and low-pressure cuff. Superficial femoral artery diameter, flow, shear rate, FMD were measured pre and post condition. Results: Mean shear rate increased significantly post passive heat condition in children (103.1 ± 41.5 s⁻¹ to 230.4 ± 79.1 s⁻¹) and adults (29.9 ± 12.1 s⁻¹ to 140.3 ± 36.0 s⁻¹) with a greater increase in adults than children. Despite the increases in mean shear rate and antegrade shear rate, FMD did not significantly improve post heat. There were no age-related differences in FMD responses post passive heat condition. Mean shear rate decreased significantly following low cuff condition in children (142.4 ± 67.4 s⁻¹ to 101.1 ± 22.1 s⁻¹) and adults (38.7 ± 19.5 s⁻¹ to 23.8 ± 15.4 s⁻¹). The significant increases in retrograde shear rate post low pressure cuff was followed by a significant decline in FMD in children (4.3 ± 1.8% to 2.6 ± 1.4%) and adults (4.1 ± 1.2% to 2.9 ± 0.78%). Conclusions: Passive heat can increase shear rates and flow in both children and adults but may not be enough to stimulate positive endothelial function. Low pressure cuff can impair endothelial function through decreases in shear, flow and FMD in children and adults. These findings have implications for understanding vascular function in different age groups and for developing interventions to enhance vascular health.
EXAMINING THE EFFECTS OF SHEAR RATE ON FLOW MEDIATED DILATION IN CHILDREN AND ADULTS by Nadhiyya Shabir B.S.c, McMaster University, 2021 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Health and Exercise Sciences) THE UNIVERSITY OF BRITISH COLUMBIA Okanagan April 2024 © Nadhiyya Shabir, 2024 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis entitled: EXAMINING THE EFFECTS OF SHEAR RATE ON FLOW MEDIATED DILATION IN CHILDREN AND ADULTS submitted by Nadhiyya Shabir in partial fulfillment of the requirements for the degree of Master of Science in Health and Exercise Sciences Examining Committee: Dr. Alison McManus, Faculty of Health and Social Development Supervisor Dr. Glen Foster, Faculty of Health and Social Development Supervisory Committee Member Dr. Kurt Smith, Faculty of Education Supervisory Committee Member Dr. Brian Timmons, Faculty of Health Sciences University Examiner iii Abstract Purpose: Variations in shear stimulus has been shown to impact endothelial function. Studies have shown that increases in shear improves endothelial function and overall vascular health while decreases in shear leads to impairments in endothelial function and develops risk of cardiovascular diseases. Although this pattern is noticed in adult studies, there are limited studies that investigate this relationship in children. Therefore, the aim of this study was to examine the relationship between shear rate and flow mediated dilation (FMD) in children and adults. Methods: Nine adults and seven children completed two protocols: passive heat and low-pressure cuff. Superficial femoral artery diameter, flow, shear rate, FMD were measured pre and post condition. Results: Mean shear rate increased significantly post passive heat condition in children (103.1 ± 41.5 s-1 to 230.4 ± 79.1 s-1) and adults (29.9 ± 12.1 s-1 to 140.3 ± 36.0 s-1) with a greater increase in adults than children. Despite the increases in mean shear rate and antegrade shear rate, FMD did not significantly improve post heat. There were no age-related differences in FMD responses post passive heat condition. Mean shear rate decreased significantly following low cuff condition in children (142.4 ± 67.4 s-1 to 101.1 ± 22.1 s-1) and adults (38.7 ± 19.5 s-1 to 23.8 ± 15.4 s-1). The significant increases in retrograde shear rate post low pressure cuff was followed by a significant decline in FMD in children (4.3 ± 1.8% to 2.6 ± 1.4%) and adults (4.1 ± 1.2% to 2.9 ± 0.78%). Conclusions: Passive heat can increase shear rates and flow in both children and adults but may not be enough to stimulate positive endothelial function. Low pressure cuff can impair endothelial function through decreases in shear, flow and FMD in children and adults. These iv findings have implications for understanding vascular function in different age groups and for developing interventions to enhance vascular health. v Lay Summary This thesis explores how the force of blood flow against blood vessel walls, known as shear rate, relates to flow-mediated dilation (FMD) in children and adults. FMD measures blood vessel functionality and understanding its connection with shear rate aids in comprehending artery health and potentially preventing cardiovascular diseases early. Given evidence of early artery impairment, studying vascular functions in children becomes crucial. This study investigates this association in children and adults to uncover developmental differences. Results revealed that while enhancements in shear do not consistently improve vascular function in both groups, reductions in shear do impair FMD in children and adults alike. vi Preface Dr. Alison McManus and I designed the rationale and purpose of study as well as planned the research study. Recruitment was done by me with assistance from Dr. Alison McManus. Data collection was completed with assistance from Scott McCall, Jay Carr and Jodie Koep. I completed all the data analysis with assistance and guidance from Dr. Alison McManus. All chapters of the thesis were written by me with feedback from Dr. Alison McManus. This study received ethical approval from The University of British Columbia Clinical Research Ethics Board (H22-0303). vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiii Chapter 1: Introduction ................................................................................................................1 1.1 Structure of Conduit Arteries .......................................................................................... 4 1.1.1 Conduit artery structure during Childhood and Adolescence ................................. 6 1.2 Assessing Maturation in Children ................................................................................... 7 1.3 Importance of the Endothelium to Vasomotor Function .............................................. 11 1.3.1 Mechanisms underlying conduit artery dilation in children. ................................ 15 1.3.2 Shear Rate-FMD Relationships in Adults and Children ....................................... 18 1.4 Clinical Relevance ........................................................................................................ 29 1.5 Summary and Objectives .............................................................................................. 31 Chapter 2: Flow Mediated Dilation Methodology and Passive Heat Feasibility in Prepubertal Children...................................................................................................................32 2.1 Flow Mediated Dilation: Assessing Conduit Artery Function ..................................... 32 2.2 Feasibility of Passive Heat Experimentation in Children ............................................. 37 2.3 Secondary Data Methodology....................................................................................... 38 viii 2.3.1 Passive Heat Experimental Design ....................................................................... 38 2.3.2 Procedures ............................................................................................................. 39 2.3.3 Measures ............................................................................................................... 40 2.4 Findings and Discussion ............................................................................................... 41 2.4.1 Central cardiovascular change .............................................................................. 42 2.4.2 Changes in the Control Leg with Passive Heating................................................ 43 2.4.3 Changes in FMD with passive limb heating ......................................................... 45 2.4.4 Experimental Set up and Sources of Data Loss .................................................... 49 2.5 Main Pilot Study Conclusions ...................................................................................... 50 Chapter 3: Examining the Effects of Shear Rate on Flow Mediated Dilation (FMD) in Children and Adults ....................................................................................................................52 3.1 Introduction ................................................................................................................... 52 3.2 Methods......................................................................................................................... 54 3.2.1 Experimental Design ............................................................................................. 54 3.2.2 Procedures ............................................................................................................. 55 3.2.3 Measures ............................................................................................................... 56 3.2.4 Principal Outcome ................................................................................................ 57 3.2.5 Statistics ................................................................................................................ 60 3.3 Results ........................................................................................................................... 61 3.3.1 Vasodilatory Function with Passive Heat ............................................................. 64 3.3.2 Shear Rates and SRAUC ...................................................................................... 67 3.3.3 Pre, during and post passive heat condition comparisons..................................... 70 3.3.4 Shear rate-FMD relationship................................................................................. 71 ix 3.3.5 Vasodilatory Function with Low Pressure Cuff ................................................... 72 3.3.6 Shear Rates and SRAUC ...................................................................................... 74 3.3.7 Pre, during and post low pressure cuff condition comparisons ............................ 79 3.3.8 Shear rate-FMD relationship................................................................................. 80 3.4 Discussion ..................................................................................................................... 80 3.4.1 Passive Heat Condition ......................................................................................... 81 3.4.2 Low Pressure Cuff Condition ............................................................................... 85 3.5 Limitations .................................................................................................................... 88 Chapter 4: Conclusion .................................................................................................................89 4.1 Strengths and Limitations of the Experimental Design ................................................ 89 4.2 Implications and Future Directions ............................................................................... 92 Bibliography .................................................................................................................................94 x List of Tables Table 2.1 Participant Demographics ............................................................................................. 41 Table 2.2 Cardiovascular responses Pre and Post passive limb heating ....................................... 42 Table 2.3 Shear patterning, and shear rates Pre and Post passive heat condition and in the control leg, in children between the ages of 8-12 years old. ..................................................................... 43 Table 2.4 SRAUC and blood flow Pre and Post passive heat and in the control leg in children between the ages of 8-12 years old. .............................................................................................. 45 Table 2.5 Baseline, peak diameter and FMD changes during passive heat and control conditions, pre and post 1 hour. ....................................................................................................................... 47 Table 3.1 Participant Demographics ............................................................................................. 61 Table 3.2 Cardiovascular data at Baseline, during and Post the passive heat condition in adults and children. .................................................................................................................................. 63 Table 3.3 Superficial femoral artery diameter, blood flow and shear rate responses at baseline (Pre) and Post passive heat in adults and children. ....................................................................... 66 Table 3.4 Hemodynamic responses at baseline prior to the low pressure cuff condition in children and adults ........................................................................................................................ 72 Table 3.5 Superficial femoral artery diameter, blood flow and shear rate responses baseline (Pre) and Post low-pressure cuff in adults and children. ....................................................................... 77 xi List of Figures Figure 1.1 The structure of an arterial wall..................................................................................... 5 Figure 1.2 Diagram of sequence of events at puberty in girls and boys. ...................................... 10 Figure 1.3 NO dependent pathway is shown in the endothelium, affected by shear stress. ......... 14 Figure 1.4 The relationship between sex hormones and the development of secondary sex characteristics regulated by the HPG axis. ................................................................................... 18 Figure 3.1 FMD responses baseline (Pre) and Post passive leg heating in adults (A, n=9) and children (B, n=6). Data are mean ± SD. Lines represent individual data. .................................... 65 Figure 3.2 Mean shear rate and shear patterning pre and post the passive heat condition in adults and children. Data are mean ± SD. Black dots represent adult individual data points and black squares represent child individual data points. ............................................................................. 68 Figure 3.3 Changes in diameter, shear rate and flow are shown from baseline (Pre), to one minute prior to end of condition (60min) and five minutes post passive heat condition (Post) in children and adults. ....................................................................................................................... 69 Figure 3.4 FMD responses Pre and Post the low pressure cuff condition in adults (A, n=9) and children (B, n=6). .......................................................................................................................... 74 Figure 3.5 Shear rate patterning baseline (Pre) and Post passive low pressure cuff condition in adults and children. ....................................................................................................................... 76 Figure 3.6 Changes in diameter, shear rate and flow are shown from baseline low pressure cuff (Pre), to one minute prior to end of condition (30min) and five minutes post low pressure cuff condition (Post) in children and adults (n=7). .............................................................................. 78 xii List of Abbreviations Abbreviation Definition eNOS Endothelium nitric oxide synthase FMD Flow mediated Dilation GTN Glyceral Trinitrate MMPs Matrix metalloproteinases NO Nitric Oxide xiii Acknowledgements This thesis would not be possible without Dr. Alison McManus, my supervisor, whose guidance, and mentorship have been instrumental in the completion of this thesis. Over the past two years at UBC, I have gained invaluable knowledge and improved my skills, because of your support. I also want to thank my lab members for supporting me and making my experience here more enjoyable. To my family and friends, your presence has been my strength. Your unwavering support no matter what path I choose is inspiring. 1 Chapter 1: Introduction Across the globe, the increase in mortality due to cardiovascular diseases is a public health concern for all age groups (Mathers et al., 2009). The alarming annual increase in cardiovascular diseases in both males and females emphasizes the urgency of addressing contributing factors and developing effective prevention strategies (Aggarwal et al., 2023). In order to mitigate the risk of cardiovascular diseases, it is essential to return to the very origin of the problem - the vascular system, and particularly the arteries. The human vascular system undergoes profound changes across the lifespan, with the blueprint for lifelong cardiovascular health being outlined in the formative years during childhood (Sarkola et al., 2012). Arteries can exhibit early signs of impairment well before diseases manifest and consequently, researchers must examine the structure and function of arteries to aid the identification of impairments at an early stage and to develop effective strategies to prevent or slow the development of cardiovascular diseases in adulthood. One pivotal element of the function of the vasculature is the endothelium, the inner cellular lining of arteries. The vascular endothelium is exposed to varying blood flow patterns and changes in pressure during everyday activities, such as standing and posture changes, sitting and exercise. These alterations in blood flow and pressure are meticulously regulated in many ways such as through vascular smooth muscle, and the vascular endothelium via the release of vasoactive substances (Pohl et al., 1986; Dawson et al., 2010; Green et al., 2017). As such the endothelium is paramount to vascular health and is associated with the prevention or development of adverse cardiovascular conditions (Green et al., 2017; Luk et al., 2022). 2 An established non-invasive method for assessing endothelial function is Flow Mediated Dilation (FMD), a technique that quantifies the vasodilation response of the endothelium to hyperemia in research settings where blood flow is experimentally manipulated in conduit arteries (Celermajer et al., 1992). FMD is thought to be primarily endothelial-dependent and reflects the ability of the endothelium to react to changes in blood flow and the corresponding shear stimuli, making it a valuable tool in understanding the etiology of endothelial function. Manipulating shear stimulus and observing FMD responses helps to clarify the mechanisms influencing endothelial function. Shear stimulus can be decreased through manipulations such as prolonged sitting, and this is known to cause a decline in FMD, indicating the endothelium can be impaired by low flow/low shear stress scenarios (Schreuder et al., 2014; Tremblay et al., 2019; Thijssen et al., 2009b). Conversely, increasing the shear stimulus by increasing blood flow to the conduit arteries through various manipulations including exercise, has been shown to increase FMD, indicating that the endothelium can be protected through up-regulation of blood flow/shear stress (Cheng et al., 2021; Restaino et al., 2016; Romero et al., 2017; Mullen et al., 2001). While there have been numerous adult studies manipulating shear stimulus and examining the impact on endothelial function, this area is poorly understood in children. The dominant thought has been that, unlike adults, there is no relationship between shear stress and FMD in children. In support of this line of argument, correlational work concluded that the shear stimulus-FMD relationship is not significant ((Thijssen et al., 2009a; Hopkins et al., 2012). Additionally, 3 experimental work in girls showed a significant decline in FMD following prolonged sitting was not related to any decrease in shear stimulus (McManus et al., 2015). While the evidence to date supports the notion that the relationship between shear rate and FMD is developmental, there have been no studies to date that directly manipulate shear rate in children to fully probe the relationship with FMD and this signifies the need for such investigations. The aim of this thesis therefore was to experimentally increase and decrease shear rate through manipulation of blood flow in boys and men to better understand the developmental course of the shear rate-FMD relationship. The thesis begins with a literature review that provides an overview of conduit arterial structure and development during childhood. An overview of the vasomotor function of arteries follows, including the underlying cellular processes and pathways. Finally, the review concludes by exploring the differences between vascular function in children and adults, specifically focusing on the responses of FMD to different shear stimuli and its clinical significance in the pediatric population. A methodology chapter follows, before the presentation of the experimental work. The thesis concludes with an overall discussion and considerations for future studies. 4 Review of Literature 1.1 Structure of Conduit Arteries Conduit arteries are blood vessels with structural components such as collagen, elastin, and vascular smooth muscle cells designed to ensure they can stretch with each pulse and provide little resistance in blood flow to organs (Greenwald, 2007). This elasticity also provides the Windkessel effect, which means there is a relatively constant pressure in the arteries despite the pulsatile pattern of blood flow. Examples of conduit arteries include the aorta, carotid, brachial and femoral arteries. These arteries vary in size depending on their location. In healthy young adults the carotid artery is about 6-7 mm in diameter, limb arteries such as the superficial femoral artery are around 5-7 mm in diameter (Thijssen et al., 2019), whereas the brachial artery has a diameter of about 3-5 mm (Thijssen et al., 2019). Conduit arteries play a vital role in facilitating blood delivery, particularly during periods of heightened demand when oxygen needs to be efficiently transported to skeletal muscle cells or other organs for maintaining homeostasis. The aorta and carotid arteries, located close to the heart, exhibit a higher content of elastin within their arterial walls which enables these arteries to expand and accommodate the increased blood volume ejected from the heart, acting as a buffer mechanism (Greenwald, 2007). Thus, when blood is ejected from the left ventricle into the aorta during systole, the aortic wall assists in its expansion under pressure. In contrast, conduit arteries situated farther away from the heart, such as limb arteries, face greater resistance to blood flow. To overcome this resistance and regulate blood flow effectively, limb conduit arteries possess a higher concentration of vascular smooth muscle cells. These cells enable the arteries to constrict 5 or dilate in response to varying flow demands, thereby optimizing blood delivery to the extremities (Greenwald, 2007). Figure 1.1 The structure of an arterial wall. The arterial wall comprises of three layers. The outer most layer of the arterial wall, the tunica extrema, mostly consists of connective tissues, elastin, and collagen. The middle layer, the tunica media contains the smooth muscle cells and the inner most layer, the tunica intima is comprised of endothelial cells on the inner wall or endothelium. Obtained from Blausen.com staff (2014) with permission. Blausen.com staff (2014). "Medical gallery of Blausen Medical 2014". WikiJournal of Medicine 1(2). DOI:10.15347/wjm/2014.010. ISSN 2002-4436. The vascular endothelium detects changes in blood flow and is key in the vasomotor control of conduit arteries (Thijssen et al., 2019). In the context of this review, the term "endothelium" will be used to refer to the vascular endothelium. There are physiological advantages to variations in arterial wall organization. For instance, to produce signals from the endothelium to the surrounding cells through the connectivity between these layers (Luk et al., 2022) or to cause the muscle cells to contract through chemical substances that allow the arterial vessel wall to expand 6 (Green et al., 2017) and such alterations in arterial structure can have implications for overall function. However, it should be noted that the organization of the vascular wall layers and the variations between arteries are yet to be fully understood. 1.1.1 Conduit artery structure during Childhood and Adolescence Conduit arteries increase in size with age from infancy to adulthood. Throughout childhood and into adolescence, arteries start to exhibit an increase in wall thickness to adapt to flow and pressure, but some studies suggest that the two innermost layers (i.e., tunica intima and tunica media, see Figure 1) are contributing most to the increase in wall thickness (Sarkola et al., 2012). With increasing age, the smooth muscle cells within the tunica media proliferate (Wenn & Newman 1990). This is more apparent in lower limb arteries compared to upper limb arteries (Sarkola et al., 2012). The increase in vascular smooth muscle cells within the tunica media with increasing age affects length and tortuosity in some arteries. (Wenn & Newman 1990). Wall thickness also influences wall stress (Sugimoto et al., 2003). For example, between 10-14 years of age, femoral artery diameter is about 6.00 mm and arterial circumferential wall stress in the femoral artery is around 62.8  16.9 mm Hg. By 15-18 years of age, when femoral artery diameter is around 7.00 mm, arterial circumferential wall stress almost doubles to 150.2  48 mm Hg (Sarkola et al., 2012). There is not an extensive literature on the development of the peripheral conduit arteries during the prepubertal years (i.e., 6 to 10 years in girls and 6 to 12 years in boys (see section 1.2 for a more detailed description of maturation). Sarkola et al. (2012) noted in 6- to 9-year-olds that brachial artery diameter was about 2.22  0.33 mm, whereas the femoral artery was about 4.63  7 0.57 mm (Sarkola et al., 2012). The most extensive data set on brachial artery diameter comes from data pooled across multiple laboratories (Hopkins et al., 2015). From ages 6-10 years in females, brachial artery diameter increased from 2.38mm to 2.82mm, whereas in males, brachial artery diameter was around 2.81mm at 6 years of age and increased to about 2.99 mm by 10 years of age (Hopkins et al., 2015). During adolescence in males (typically around 13-15 years) brachial artery diameter continues to increase, from about 3.3 to 3.5 mm. In females, there is a small increase in brachial artery diameter across this age range (from about 3.06 mm to 3.1 mm). These increases, and the sex differences, likely align closely with the peak growth rate in height and body mass in early adolescence which is discussed in more detail in section 1.2 (Abbassi, 1998). Adult brachial artery diameter (about 3.22 mm) is attained in females around 16-18 years of age, and in males by about 18 years of age at around 4.00 mm (Hopkins et al., 2015). These data suggest that sex differences in vessel size become more apparent in late adolescence, aligning quite closely with the peak growth rate in lean body mass (Armstrong et al., 2017a). While the data describing changes in conduit artery size are limited, the extant literature stresses the importance of the development in size in conduit arteries and the aligned sex differences in developmental trajectories. These differences in vessel size have implications for measures of vascular function, which will be discussed in section 1.3.1. 1.2 Assessing Maturation in Children Consideration of pubertal status is essential when working with children and adolescents. All individuals undergo a phase of growth characterized by changes in body size, shape, and 8 composition as they advance toward adulthood. However, these changes may not always align or follow identical timelines (Armstrong et al., 2017a). Maturation encompasses the stages of development that individuals progress through from infancy and childhood to adolescence and into adulthood. To determine an individual's maturation status, researchers employ various methods, including assessments of dental development, bone age, hormonal profiles, and more (Armstrong et al., 2017a). These methods collectively help gauge the individual's stage of development. While all children go through the same stages of maturity to reach adulthood, the timing of the stages will differ between individuals (Armstrong et al., 2017b) and is dependent upon genes, environment, diet (mother and child) and hormones (Thomis et al., 2006). This means two individuals can be of the same age but not be in the same maturity status. To assess the maturity status of participants before including them in studies, age at peak height velocity (PHV) is often used to determine somatic maturation. This method looks at growth in stature and particularly differential growth in leg length and torso. The peak growth velocity i.e., the year the maximum growth rate in stature is attained is termed peak height velocity. To measure peak height velocity, serial measures of stature are needed longitudinally e.g., every 6 months throughout childhood and adolescence. Longitudinal data reveals that, on average, girls experience their peak height velocity earlier than boys, typically occurring around 11.8 years of age (with a range of 9 to 15 years old). In contrast, boys tend to reach their PHV at an average age of 13.4 years (with a range of 11.1 to 17.3 years old) (Armstrong et al., 2017b; Iuliano-Burns et al., 2001). 9 Given longitudinal assessment is generally not feasible, equations have been derived to estimate age at peak height velocity (Mirwald et al., 2002). These estimates provide both the predicted age, as well as an offset score i.e., how close the individual is to the predicted age at peak height velocity, with a score of 0 meaning at peak height velocity, a score of -1 meaning one year before and a score of +1 meaning one year after peak height velocity. These provide a crude somatic estimate of maturity status, with scores of -1 being interpreted as prepubertal. However, it is important to note that predicted age at peak height velocity may not serve as an accurate marker of progress across pubertal stages since the measurement relies solely on stature and leg length and specifically on one quantifiable event, the stature growth spurt in males and females. Other methods rely on different markers and therefore predict maturity status at slightly different periods (see Figure 1.2). An alternative marker of maturation is the visual appearance of secondary sexual characteristics or what is more commonly known as Tanner staging (Marshall & Tanner, 1969; Marshall & Tanner, 1970). Marshall and Tanner (1969,1970) charted the changes in secondary sexual characteristics in males and females from childhood to adulthood. The five Tanner stages that emerged use pubic hair, genitalia, and breast development (Marshall & Tanner, 1969; Marshall & Tanner, 1970). Stage 1 is seen as prepubertal where there is no obvious sign of pubic hair, breast, or genitalia development. Stage 2 is the development of each characteristic or early puberty, although the order in which pubic hair, breast, genitalia develop varies depending on the individual. In stages 3 and 4, there is further development of each characteristic until stage 5 when maturity is reached, where secondary sexual characteristics are of adult status (Marshall & Tanner, 1969; Marshall & Tanner, 1970). 10 Figure 1.2 Diagram of sequence of events at puberty in girls and boys. This figure depicts the disparities in determining pubertal status between tanner staging and peak height velocity in males and females. Obtained from Armstrong, N., van Mechelen, W., & Baxter-Jones, A. D. G. (2017). Growth and maturation. In N. Armstrong & W. van Mechelen (Eds.), Oxford Textbook of Children's Sport and Exercise Medicine (3rd ed.). Oxford Academic. ©Oxford University Press. Reproduced with permission of The Licensor through PLSclear. 11 The onset of secondary sexual characteristics is driven by pathways stimulated by hormones such as oestrogen, testosterone, and adrenal hormones. While these hormones become present in the prepubertal Tanner 1 stage, the concentration is not enough to produce morphological changes. As hormone concentrations increase, the related changes noted in the subsequent Tanner stages become apparent. Tanner staging provides a marker of progress across pubertal stages but time in each stage varies greatly between individuals (Armstrong et al., 2017a). Although both predicted age at peak height velocity and Tanner staging have their drawbacks, if prepubertal status is required, then Tanner staging is likely superior. 1.3 Importance of the Endothelium to Vasomotor Function The endothelial wall of an artery is capable of sensing and responding to the stress and strain exerted by the flowing blood within the vessel (Green et al., 2017). This stress is referred to as shear stress and can be described as a frictional force detected by the endothelium. Early research involved applying simulated drag forces on the endothelium, as demonstrated in an animal study by Rodbard et al. (1975). This study revealed that drag forces can elicit an increase in blood flow which then triggers acute vasodilation. If the increased drag persists over an extended period, the vessel undergoes structural remodeling to adapt to the augmented flow and return stimulated drag to normal. This groundbreaking experiment provided two key insights into vascular function: firstly, the endothelium plays a vital role in regulating vasomotor function, and secondly, blood flow itself is a significant determinant of vasomotor function. Further investigations have revealed the endothelium to be a paracrine/endocrine organ, as indicated by recent studies conducted by Nava et al. (2016) and Luk et al. (2022). The 12 endothelium releases various vasoactive substances such as endothelium derived relaxing factor, most commonly known as nitric oxide (NO) and prostaglandins. Notably, a seminal study by Furchgott and Zawadzki (1980) identified the crucial role of endothelial cells in the release of NO, which subsequently induces smooth muscle relaxation and promotes vasodilation. Prostaglandins released by endothelial cells, are also involved in endothelial repair and vasodilation (Moncada et al., 1976). The release of these vasoactive substances is stimulated by shear stress on the endothelium, as demonstrated by Rubanyi et al. (1986) and removal of the endothelium abolishes the ability to dilate in response to changes in shear stress (Pohl et al., 1986). Collectively these studies provide evidence of endothelial dependent vasodilation, and the key roles played by NO and prostaglandins. Conduit arteries are exposed to various stressors, including circumferential strain, shear stress, and fluctuations in blood pressure. Endothelial cells possess receptors that detect stress signals and other mechanical forces acting on the artery. This enables the activation of cellular pathways that facilitate the vessel's adaptation to these dynamic changes. The endothelium has a significant role in modulating vascular tone, as highlighted in a study by Thijssen et al. (2007). Vascular tone refers to the degree of dilation and constriction of a blood vessel. Endothelin-1 (ET-1), a vasoconstrictor, is released by the endothelium and contributes to the regulation of vascular tone in peripheral arteries. This effect is mediated through the interaction of ET-1 with receptors located in both the endothelial cells and smooth muscle cells (Thijssen et al., 2007). Blood pressure affects the growth rate of endothelial cells, such that increases in pressure increases concentration of endothelial cells (Laughlin et al., 2008). Pressure distends arteries 13 where changes in pressure which is also pulsatile produces cyclic strain on the vessel wall. This strain exposed to endothelial cells, allows it to trigger the activation of G-coupled proteins, key players in cell signaling responsible for translating external signals into cellular responses. Furthermore, blood pressure can stimulate kinase receptors, which, in turn, activate transcription factors. This can cause changes to both endothelial cells and vascular smooth muscle cells as stimulating these sensors can lead to cell proliferation or cell death. This cascade of intracellular signaling ultimately leads to modification of matrix proteins such as matrix metalloproteinases (MMPs) and nonmatrix substrates (Cummins et al., 2007; Green et al., 2017). Matrix proteins allow for increases in extracellular matrix components such as collagen, essential for vessel structure remodeling and function (Cummins et al., 2007). Inhibition of MMPs hinders arterial remodeling in response to shear stress, as MMPs facilitate arterial enlargement. Shear stress, on the other hand, triggers the activation of ion channels (see Figure 1.3) that initiate specific cellular pathways responsible for generating proteins involved in structural and functional changes within the vessel (Lehoux & Castier, 2006). Shear stress-induced activation of nuclear factor kB (NF-kB) results in the production of proteins that prevent cell apoptosis, which is particularly important for vascular smooth muscle cells (VSMCs) that are exposed to stress and susceptible to cell death (Lehoux & Castier, 2006). Similar to blood pressure, shear stress is also detected by cell membrane receptors like kinase and G-coupled proteins (Green et al., 2017). Most pathways triggered by shear stress or blood pressure culminate in the activation of genes responsible for the production of chemical agents, including NO. These chemicals play a pivotal 14 role in supporting vascular remodeling, enabling the adaptation to heightened flow or pressure conditions, and protect against development of atherosclerosis. Figure 1.3 NO dependent pathway is shown in the endothelium, affected by shear stress. Ion channels such as Ca2+ that are present in the endothelium allow ions to enter, allowing eNOS and other eNOS transcription factors to be activated to release NO from the cell and cause structural changes in the vessel. This is one simple and specific pathway that leads to structural changes; however, pathways are active and simultaneously activate other chemical substances and proteins in response to shear stress. Obtained from Moens et al., 2005 with permission. Manipulation of shear stress has been shown to have significant effects on various pathways, resulting in changes in gene expression (Cheng et al., 2005). Specifically, altering shear stress patterns leads to an increase in the production of endothelial nitric oxide synthase (eNOS) proteins and gene expression (Cheng et al., 2005). It is worth noting that for proper activation, the eNOS enzyme must be localized to the Golgi complex within the cell, where it interacts with 15 protein kinase B (Akt) or other proteins (Cheng et al., 2005). In cases where eNOS fails to distribute to the Golgi complex under unusual shear stress patterns, its activation is hindered, leading to a decrease in NO availability. To adapt to such circumstances, the Golgi complex undergoes morphological changes, increasing in size, in response to alterations in shear stress (Cheng et al., 2005). Consequently, both low and high shear stress can exert an impact on the structural and functional aspects of the endothelium, influencing processes such as eNOS activation, NO production, and the overall response of the endothelium to shear stress. In vitro studies show that oscillatory shear stress (where there is flow reversal) leads to increases in inflammation, oxidative stress, and abnormal alignment of cells within the endothelium, whereas laminar shear stress (unidirectional flow) reduces inflammation and leads to overall healthy endothelial function (Green et al., 2017). Thus, understanding the role of shear stress, shear stress patterning and its effects on vasomotor function is relevant for vascular health across the lifespan. 1.3.1 Mechanisms underlying conduit artery dilation in children. Although the pathways mentioned above are derived largely from work in adults, the development of these pathways in children, particularly during the prepubertal stage, remains inadequately understood. Age-related differences in vasomotor function have been speculated to stem from two main factors (Thijssen et al., 2008; Seals et al., 2008; Thijssen et al., 2007). Firstly, changes in the structure of the vessel wall occur with development, such as elasticity, wall thickness, and diameter, as discussed in section 1.1.2 (Thijssen et al., 2008). Secondly, differences in the sensitivity to vasoactive pathways (i.e., NO pathway) have been suggested 16 between young adults and older adults (Seals et al., 2008; Thijssen et al., 2008), which may result in differing vasomotor responses in children compared to adults. Thijssen et al. (2008) also suggest the presence of an age-related disparity in vascular adaptations to blood flow. Nonetheless, deeper investigation within the younger population, which remains currently underexplored, is imperative. To understand these age-related differences, it is imperative to explore the development of the vascular tree. Vasculogenesis, the formation of new blood vessels, occurs during the gestation period, while angiogenesis, the growth of vessels from pre-existing vessels, takes place post-birth. The development of endothelial cells occurs through a series of pathways following vasculogenesis (Hislop & Pierce, 2000). Post birth, the increase in blood flow increases shear stress which then stimulates the release of NO and prostacyclin (Hislop & Pierce, 2000). In addition to NO, the endothelial cells also release endothelin-1 and it appears that these pathways are established from birth (Hislop & Pierce, 2000). Prostacyclin has been observed to play a vital role in the vascular structure during fetal development, with high concentrations present during this stage (Hislop & Pierce, 2000). However, in lambs, prostacyclin levels decrease a few hours after birth (Dukarm et al., 1996). This suggests that prostacyclin may be crucial for vessel development during gestation. Animal studies have shown that prostacyclin causes relaxation of vascular smooth muscle cells, leading to dilation through the activation of adenylate cyclase, another enzyme that also decreases after birth (Hislop & Pierce, 2000), at least in animal models. 17 In prepubertal boys and girls, growth hormones, thyroid hormones, and insulin-like growth factor 1 (IGF-1) play key roles in regulating growth. Growth hormones, secreted from the anterior pituitary gland, are regulated by IGF-1 through the Growth Hormone/IGF-1 axis. IGF-1 has multiple functions, including promoting muscle tissue growth, bone growth, and collagen formation (Rogol et al., 2002). It is important to note that the IGF-1 axis plays a crucial role in endothelial function and regulates the function of vasoactive and growth factor endothelin-1 (Hasdai et al., 1998). Animal studies show that insulin stimulates the release of endothelin-1 from the endothelium (Hasdai et al., 1998). Endothelin-1 is of relevance here because fetal lamb and pig studies have demonstrated its high concentration at birth, followed by a decrease due to increased pulmonary pressure (Dukarm et al., 1996; Abman et al., 1999). Endothelin-1 has two receptors, ETa and ETb, activation of which on endothelial cells leads to vasoconstriction. However, ETb activation has also been shown to stimulate the production of NO and prostacyclin (Hislop & Pierce, 2000). A study conducted on piglets revealed the presence of ETb receptors on endothelial cells 1-3 days after birth (Naguchi et al., 1997), exhibiting developmental vasomotor pathways. In the pubertal stages of a child, sex steroid hormones are the primary hormones that drive maturational processes in boys and girls. At this age, the hypothalamus, pituitary gland, and gonads form a pathway called the HPG axis (Figure 1.4). Gonadotropin hormone-releasing hormone (GnRH) stimulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the pituitary gland by binding to the GnRH receptor and causing activation of signal transduction pathway in the pituitary gland (Lee et al., 2002). LH is released in a pulsatile manner and in the pre-pubertal stage, it is released during the night and less in the day 18 in stable concentrations. LH slowly starts to increase prior to puberty and causes the first change in pubertal status biologically with increasing LH concentrations in girls first (8-9 years) and then boys, (10-11years) explaining the earlier pubertal onset observed in girls. This is important as the HPG axis (Figure 1.4) is active in adults and can produce apparent sex differences in vascular measures such as artery diameter and flow. Figure 1.4 The relationship between sex hormones and the development of secondary sex characteristics regulated by the HPG axis. Ellison PT. Puberty. In: Cameron (ed.) Human growth and development. San Deigo: Academic Press; 2002. p. 65–84. © Oxford University Press. Reproduced with permission of The Licensor through PLSclear. 1.3.2 Shear Rate-FMD Relationships in Adults and Children As established in section 1.3.1, the influence of blood flow patterns on endothelial functions underscores the need to explore flow manipulation and its repercussions on vasomotor function. If reduced blood flow indeed impacts the extent of vasodilation, this carries significant implications for cardiovascular health on a broader scale. Consequently, in-depth investigations 19 into the study of flow manipulation and its effects on vascular mechanisms are paramount. This is especially pertinent for individuals who are unable to engage in physical exercise due to health constraints. It is imperative therefore to explore alternative methods that enhance blood flow and stimulate the endothelium, all to mitigate the risk of cardiovascular disease. Furthermore, variations in blood flow patterns and the consequential endothelial response have been observed across diverse age groups (Thijssen et al., 2008). These disparities necessitate a closer examination of developmental differences to determine the underlying mechanisms of the vasodilatory response and the degree to which shear stimulus alone influences this. This section will delve into studies that scrutinize variations in shear stimulus – both increases and decreases – and its impact on the key measure of the vasodilatory response – the FMD response. Decreases in Shear Stimulus The utilization of a low-pressure cuff intervention to reduce blood flow and decrease shear stimulus in research studies is a valuable approach to assess the impact of altered blood flow on vasomotor function. This technique has been applied to the lower limb and involves placing a cuff on the upper part of the leg which, inflated to varying pressures, can effectively reduce flow to the superficial femoral artery. By examining FMD post-intervention, researchers have gained insight into how the reduction of flow results in a reduction in the shear stimulus and subsequently how this influences FMD. Schreuder et al. (2014) conducted a comprehensive investigation of the effects of a low-pressure cuff intervention on shear patterning, blood flow, and FMD in healthy adult males. Their 20 methodology involved employing a thigh cuff inflated to pressure levels ranging from 30 to 75 mm Hg. The pre-and post-intervention measurements in the superficial femoral artery were then compared to assess the impact of the varying cuff pressures on shear rate and FMD. One of the key findings was the emergence of a dose-dependent relationship between retrograde shear rate and FMD. As the cuff pressure increased, there was a significant greater decrease in the mean shear rate and an increase in retrograde shear rate at 60mm Hg compared to 30mm Hg. When examining FMD, the study revealed a significant decline in FMD at 60 mm Hg, while there was no FMD change at 30 mm Hg. These findings parallel an earlier study by Thijssen et al. (2009) that also demonstrated that at 25mm Hg, there were no significant changes in retrograde shear rate and FMD post intervention. The consensus among these studies (Johnson et al., 2012; Gurovich & Braith 2013) seems to be that lower cuff pressures, such as 25 to 30 mm Hg, do not have a substantial negative impact on FMD responses, suggesting that a certain threshold of retrograde shear rate must be reached before FMD begins to decline. It is in the higher cuff pressures, specifically ranging from 50 to 75 mm Hg, that the decline in FMD was consistently observed due to significant increases in retrograde shear rate and decreases in mean shear rate (Schreuder et al., 2014; Thijssen et al., 2009b). In line with previous studies, Tremblay et al. (2019) used a low pressure cuff intervention to understand the effects of low mean shear rate and increased retrograde shear rate on FMD. This study showed a significant increase in retrograde shear rate and a decrease in antegrade and mean shear rate following a 30-minute cuff intervention inflated to 70 mm Hg, accompanied by a decline in FMD. Tremblay et al. (2019) observed that alterations in retrograde shear rate were not the sole factor responsible for changes in FMD. In their investigation, they found that low 21 mean shear rate, even without changes in retrograde shear rate also had an impact on the FMD response. This modification in mean shear rate alone was associated with a potential 30% decline in brachial artery FMD. This pivotal finding challenges the long-held belief that retrograde shear rate is the primary driver of reduced FMD. Instead, it highlights the importance of considering mean shear rate as a crucial, if not the predominant factor affecting vasomotor responses. In contrast, Totosy et al. (2014) applied higher cuff pressures within the range of 75-100 mm Hg and noted augmentation of antegrade shear rate and increases in retrograde shear rate, alongside declines in FMD. In the 100 mm Hg cuff condition, the augmentation of mean shear rate was significant whereas in 75 mm Hg, it was not. This is noteworthy because the decline in FMD was present whether or not concurrent declines in mean shear rate were observed. These findings highlight the complex relationship between shear rate and FMD, emphasizing that both retrograde and antegrade, as well as mean shear rates should be thoroughly investigated to comprehend the intricate mechanisms influencing FMD.This study also showed a possible influence of the oscillatory shear index (OSI) on FMD responses, since with increased cuff pressures, OSI increased and FMD decreased. OSI quantifies the changes in shear stress direction within the vessel and a review by Peiffer et al. (2013) shows various studies report an association between high OSI and the development of atherosclerosis. A different approach to decreasing shear stimulus which may affect FMD may be achieved through prolonged sitting. Studies in adults have shown that prolonged sitting can cause changes in shear stimulus and reductions in FMD (Patterson et al., 2020; Taylor et al., 2022; Thosar et al., 2015). The impact of prolonged sitting on FMD and shear rate has been investigated in pre-adolescent girls (McManus et al., 2015). Results revealed a significant decline in FMD after 22 three hours of prolonged uninterrupted sitting, however, this decline was not due to decreases in mean shear rate or blood flow. Despite the three-hours of sitting, mean shear rate and flow remained unaffected. This leads to a compelling conclusion that shear stimulus may not be the primary driver behind the observed decrease in FMD responses in children with prolonged sitting. Instead, it is plausible that other mechanisms, such as NO-independent dilation, play a more substantial role in the observed reduction in FMD. It is also possible that other physiological factors such as blood pooling (Kitano et al., 2005) and muscle sympathetic nerve activity (Ray et al., 1993) also play a role in the reduced FMD response with prolonged sitting. It would seem therefore that, at least in children, prolonged sitting is not be the ideal manipulation for the interplay between reductions in blood flow, shear stimuli and FMD. To date, there have been no experiments in children that have used the low-pressure cuff technique, but this would be valuable to better understand the relationship between changes in shear stimuli and FMD. Increases in Shear Stimulus Cheng and colleagues (2021) explored the effects of lower limb passive heating on shear rate and FMD in a group of young adults. This study included two visits, each involving participants’ lower limbs immersed in a water bath heated to 42ºC for 45 minutes. In one visit, the heating extended up to the knees, while in the subsequent visit, it was extended only to the ankles. The aim was to assess how the level of heating might influence shear rate and, subsequently, FMD in the superficial femoral artery. A notable finding was the rise in both antegrade and mean shear rates during both knee and ankle-level heating in the superficial femoral artery. Shear rate area under the curve (SRAUC) expressed in arbitrary units (au), (see section 2.1 for further 23 information) in the knee-level heating condition exhibited a statistically significant effect post-condition, increasing from 45.8 ± 40.6 au to 55.1 ± 39.2 au. This outcome was in line with the general expectation that increased blood flow with passive heating and shear rates typically contribute to vasodilation. However, the study also unveiled a noteworthy aspect of FMD responses in the participants. When examining FMD expressed as a percentage, there was considerable variability in the superficial femoral artery response compared to the brachial artery. Surprisingly, some participants experienced a decrease in superficial femoral artery FMD 20 minutes after the passive heat intervention. This variation in FMD responses is consistent with earlier research findings, such as those by Romero et al. (2017) and Thomas et al. (2016), who also noted that passive heating could lead to mixed or even negative FMD responses. The reasons behind this variability in FMD responses are believed to be multifaceted. Individual responses to heat stimulus are influenced by various factors, including genetic predisposition, baseline vascular health, and fitness level that impact the ability of the endothelium to adapt to changes in blood flow. Romero and colleagues (2017) share findings that parallel the research conducted by Cheng et al. (2021) in the exploration of the effects of acute lower limb heating on superficial femoral artery function in both young and older adult participants. In this study, the researchers similarly employed a hot water immersion technique, immersing the participants' legs in a 42°C heated water bath up to top of thigh for a duration of 45 minutes. The results from Romero et al. (2017) revealed a compelling age-dependent pattern. Specifically, older adult participants experienced a significant increase in blood flow and mean shear rate following the passive heat intervention and demonstrated a significant increase in FMD after passive heating (Romero et al., 2017). In 24 contrast, young adults’ average FMD data did not exhibit significant differences between pre- and post-heating conditions, even though mean shear rate and flow increased significantly post passive heat condition (Romero et al., 2017). This disparity in FMD responses suggest an age-dependent alteration on macrovascular function. These authors propose passive heating as a therapeutic approach that could hold promise for older adults, potentially contributing to the enhancement of vascular function and overall cardiovascular health (Romero et al., 2017). However, it is important to note that a critical element in the comparison with the work of Cheng et al. (2021) is the timing of FMD measurements. While Romero et al. measured FMD 30 minutes after the end of the passive heating condition, Cheng et al. recorded FMD measurements 20 minutes post-heating intervention. This variance in timing may influence the dynamic nature of FMD responses and the potential for distinct patterns of recovery between age groups. Understanding these nuances is important in developing tailored interventions and therapeutic strategies for diverse populations that use passive heat. Further work combining passive heating with prolonged sitting has also provided valuable insight into the impact of shear stress on FMD and the potential benefits of passive heating, particularly in the lower limb. Restaino et al. (2016) investigated the effects of passive heat on mitigating reductions in FMD during prolonged sitting. They achieved this by immersing one leg of participants in a water bath heated to 42°C, while the contralateral limb remained unheated, allowing for a direct comparison between heated and non-heated conditions over three hours of prolonged sitting.Throughout the course of the study, FMD and shear rate were assessed in the popliteal artery, including baseline pre-heating, assessment after each hour of passive heat, as well as post-heating measurements. The findings revealed a contrast in responses between the 25 heated and non-heated limbs. In the contralateral non-heated limb, there was a significant reduction in shear rate following three hours of sitting, reflecting the potential negative impact of prolonged sitting on shear rate. This decrease in shear rate was also accompanied by a substantial decline in FMD, from an initial 7.1 ± 1.4% to 2.8 ± 0.9%. Conversely, in the heated leg, the mean shear rate remained consistently elevated throughout the protocol, exhibiting an increase from 38.9 ± 3.4 s−1 to 63.9 ± 16.9 s−1. Importantly, this increase in mean shear rate corresponded to improvements in FMD, which increased from an initial 7.3 ± 1.5% to 10.9 ± 1.8%. These findings suggest that passive heating in the lower limb can effectively counteract the negative impact of prolonged sitting on shear rate and FMD. Of note, despite a significant increase in shear rate and FMD with passive heating, there was considerable intra-individual differences in the response with some participants experiencing a slight decline or no significant difference in mean shear rate following the passive heating intervention. Thomas et al. (2016) also conducted a study that explored the effects of hot water immersion on shear rate and FMD in a cohort of young adults. This study involved immersing participants in a 42°C heated water bath up to their waist for a duration of 30 minutes. One of the key observations from this study was a significant increase in mean and antegrade shear rate following the heating intervention. Contrary to expectations, there was no significant difference in the average FMD observed post intervention and no significant decrease in retrograde shear rate. In this study, there was remarkable degree of variability in the post-intervention FMD responses among the participants. This could have been due to the lack of reductions in retrograde shear rate and might mean oscillatory shear was not altered enough to induce significant changes in FMD. Another explanation might be that perhaps a threshold exists for 26 antegrade shear rate and any increases in antegrade shear rate below this threshold will not stimulate the endothelium enough for adaptation. Findings from Tinken et al. (2009) support the possibility of antegrade shear rate threshold. In this study, participants completed a thirty-minute bilateral forearm heating (40°C) intervention. In one forearm where only passive heating was applied, they observed an increase in antegrade and mean shear rate, and a significant increase in FMD responses in the brachial artery post intervention. In the contralateral arm, in addition to passive heating, a cuff was placed which decreased antegrade shear rate and they noted FMD did not increase post intervention. Difference in the magnitude of antegrade shear rate was believed to be the primary reason that led to differences in FMD seen in this study. Similarly, Cheng et al. (2019) conducted a study exploring the influence of shear stimulus on FMD. In their research, they implemented a ten-minute passive heating intervention of the arm in a group of young and healthy participants, maintaining a temperature of 42°C using heat pads. The results of this intervention were striking: a notable increase in antegrade shear rate within the brachial artery correlated with a significant improvement in FMD. Additionally, participants engaged in a ten-minute hand grip exercise during a separate visit, which induced even higher antegrade shear rates in the brachial artery compared to the passive heating intervention and this also led to a substantial increase in FMD. The findings from this study would also suggest that to elicit acute enhancements in FMD, it is imperative to achieve increased antegrade shear rates (Cheng et al., 2019). 27 The studies mentioned above primarily focused on the impact of steady increases in shear stimulus on FMD responses. Holder et al. (2019) took a different approach by delving into the effects of fluctuating shear rates while keeping the mean shear rate constant, shedding light on the impact of oscillating shear rate, as well as mean shear rate on FMD. This study explored the impact of intermittent negative pressure applied to the brachial artery. The technique involved a pattern of pressure fluctuations, with ten seconds of negative pressure at -40 mm Hg, followed by seven seconds of no pressure (0 mm Hg), and this cycle was repeated for a total of 30 minutes. These fluctuations in pressure had a profound effect on shear patterning within the vessels. During periods of negative pressure, mean and antegrade shear rate increased, while retrograde shear rate decreased, however the opposite occurred when no pressure was applied. What was particularly intriguing about this study is that a significant increase in FMD followed the 30 minute negative pressure intervention, even though average shear rate and patterning was not significantly different from baseline. The overall large increases in antegrade shear rate compared to retrograde shear rate could have led to improvements in FMD. The findings challenge the notion that only steady increases in mean shear rate i.e., from passive heating, can lead to acute improvements in FMD. Additionally, Morishima et al. (2016) demonstrated that endothelial impairments resulting from prolonged sitting can be effectively countered through a fidgeting intervention, an innovative approach to fluctuating shear rate. In this study, participants engaged in one minute of fidgeting followed by four minutes of rest during a three-hour sitting period in the condition leg, while the contralateral leg served as a ‘sitting’ control. The condition leg exhibited a significant increase in FMD post-intervention whereas the control leg had a significantly decreased FMD. Another 28 study has also shown that increases in FMD were greater with intermittent exercise than continuous exercise (Hanson & Casey, 2023). These studies offer compelling examples of how manipulating shear stress intermittently can confer protective benefits to endothelial function. Fluctuations in shear rate, even when the mean shear rate remains unaltered, can still positively influence FMD in healthy adults (Holder et al., 2019; Hanson & Casey, 2023). Such adaptability may have implications for the development of interventions aimed at improving endothelial function. There are limited studies looking at the effects of increased shear stimulus in children. A study conducted by McManus and colleagues (2019) explored how varying exercise intensities affected FMD responses in children aged 7 to 12 years. Following both moderate continuous and high-intensity interval exercise, there was a noticeable increase in superficial femoral artery diameter. This increase suggested that both types and intensities of exercise stimulated vasodilation in the superficial femoral artery, likely due to increased blood flow and shear rates. Indeed, shear rate increased immediately following both moderate and high-intensity exercise. It is worth noting that the increase in shear rate was more pronounced after high-intensity interval exercise, indicating a more substantial vascular response to intermittent exercise at a higher intensity (McManus et al., 2019), but further work is needed to understand if it is the intensity, or intermittent nature of the exercise that causes these differences. To date, there remains a noticeable absence of experimental data examining the interplay between shear rate and FMD in children, particularly in terms of deliberate manipulation of shear stimulus and closely observing the corresponding flow and dilatation responses. This lack of 29 empirical research in this population represents a critical knowledge gap. The importance of addressing this knowledge gap becomes even more evident when looking at the findings presented in Thijssen et al. (2009). This work examined the association between brachial FMD data and shear rate in children and adults (Thijssen et al., 2009b). No significant relationship between shear rate and FMD was apparent in children, whereas young adults exhibited a significant correlation between shear rate and FMD. The authors suggest that vasodilatory mechanisms underlying FMD may vary with age. While this study provided correlational evidence, experimental research is still needed to really probe the shear rate-FMD relationship in children. 1.4 Clinical Relevance While the preceding section reviewed various studies examining the relationship between shear rate, shear patterning and FMD, this section shifts focus to the importance of investigating endothelial function within the broader context of cardiovascular health. According to the American Heart Association, there is evidence that places emphasize on the importance of early strategies that help improve cardiovascular health in the younger population (Benjamin et al., 2017). Conditions like atherosclerosis and hypertension have their roots in childhood (Charakida et al., 2010; Deanfield et al., 2007; Urbina et al., 2009). Furthermore, obesity and insulin resistance, increasingly prevalent in children (Benjamin et al., 2017), are associated with impaired endothelial function (Benjamin et al., 2017; Jarvislo et al., 2004). 30 Juonala et al. (2004) found that those with increased risk factors in childhood had impaired FMD. Therefore, by protecting endothelial function, it is speculated that the risk of developing atherosclerosis will decrease (Juonala et al., 2004). Therefore, experimental studies that help in our understanding of optimizing vascular health in children, such as manipulations of shear stimulus using passive heat or low pressure cuff occlusions may help us understand the subtle changes that the endothelium may be subjected to that in turn may prevent future cardiovascular disease. Passive heating provides a unique window into the upregulation of blood flow, the accompanying alterations in shear patterns and the impact this may have on FMD. As seen in section 1.3, passive heating can increase flow without causing a central cardiovascular response or activating muscle sympathetic nerve activity, unlike conventional exercise, (Cheng et al., 2015; Cheng et al., 2018; Padilla et al., 2010). Additionally, research into therapies designed to augment blood flow to lower limbs, exemplified in a study on the expedited healing of foot ulcers (Sundby et al., 2016), shows the potential for accelerated healing processes through increased lower limb blood flow. The use of passive heat as a non-pharmacological means of upregulating vascular function in children at risk or in those unable to exercise (Charkida et al., 2010) can potentially serve as a preventative strategy for cardiovascular risk in children and adolescents, contributing to long-term cardiovascular health (Juonala et al., 2004; Benjamin et al., 2017; Urbina et al., 2009). 31 1.5 Summary and Objectives This literature review concludes by stating that to understand and prevent the risk of cardiovascular diseases in adulthood, it is vital that vascular health particularly of the conduit arteries is a priority. The conduit arteries vary in composition, structure, and function. Variations in conduit artery size exist based on location and function, while age-related changes involve growth, thickening, and diameter alterations. These age related changes may also affect cellular mechanisms regulating vasomotor functions within the artery. For instance, the impact of shear stress on the release of vasoactive substances like NO and prostaglandins, causing vasodilation is not established in children. Other mechanisms such as the role of hormones in growth regulation, including growth hormones, insulin, and sex steroid hormones in pubertal stages, and the impact on endothelial function are studied using animal models but are challenging to investigate in children. Therefore, experimentally manipulating flow and shear stress in children, is a non-invasive way to examine the impact of shear stress on endothelial function in children. At least in adults, reduced shear stimulus reduces FMD, while increased shear stimulus can lead to improved FMD. Age-dependent responses to shear stimulus underlines the need for further research in children. Studies that experimentally explore the shear rate-FMD relationship in childhood have the potential to assist in our understanding of the development and prevention of cardiovascular diseases, as well as provide additional treatment options for clinical populations. Therefore, the primary aim of this thesis was to investigate the relationship between shear rate and FMD in children and adults by manipulating shear stimulus and analyzing FMD responses. 32 Chapter 2: Flow Mediated Dilation Methodology and Passive Heat Feasibility in Prepubertal Children 2.1 Flow Mediated Dilation: Assessing Conduit Artery Function Since flow affects the dilation of conduit arteries and this is dependent on the endothelium as noted by studies reviewed in Section 1.3, the non-invasive FMD technique was developed to manipulate flow and observe arterial responses. Short-term changes in FMD have subsequently been used in studies to evaluate improvements in endothelial function and assess the risk of atherosclerosis and cardiovascular disease (Celermajer et al., 1992; Inaba et al., 2010). The FMD procedure involves cuffing the limb of interest (arm or leg) for a specific duration (typically 5 minutes) to induce arterial occlusion and ischemia. The cuff is then deflated, leading to a hyperemic response, in other words an increase in blood flow, which results in vasodilation (see Figure 3) (Anderson & Mark, 1989). Doppler ultrasound is used for FMD measurements and detailed guidelines for measurement have been provided (Thijssen et al., 2019). These guidelines highlight how it is crucial to obtain clear images of the artery with well-defined vascular walls throughout the procedure. The position of the probe must remain consistent during the procedure for determination of artery diameter, blood velocity and subsequent calculation of blood flow, shear rate and FMD. One common mistake to avoid is changing the probe angle during the ultrasound scan, as this can lead to underestimation of the arterial diameter (Thijssen et al., 2019). 33 It is important to note that certain limitations exist in the FMD technique. Changing the settings on the ultrasound machine can affect the obtained image, and it is recommended to maintain consistent settings throughout the experiment to ensure reliable measurements of arterial diameter (Thijssen et al., 2019). Additionally, in studies involving repeated measurements, it is essential to scan the same segment of the artery consistently, which can be challenging and requires appropriate training and guidance (Thijssen et al., 2019), as well as the use of pre-determined landmarks and marking up of the probe position. Figure 2.1 The process in which flow mediated dilation (FMD) occurs from baseline assessment, occlusion and post occlusion and how artery diameter and shear rate are affected by the procedure. Changes in an artery’s diameter and shear stress post occlusion and the sequence of these changes can be seen in this figure. 34 When considering the technical aspects of the FMD protocol, it is important to place the cuff distal to the artery. This positioning allows for vasodilation mediated by nitric oxide (NO), which is necessary for studying endothelium-dependent dilation (Thijssen et al., 2019). Placing the cuff proximal to the artery does not stimulate NO release to the same extent and is therefore not recommended (Thijssen et al., 2019). Accurate calculation of FMD requires careful measurement of the baseline diameter, which should be obtained prior to occlusion of the artery (Thijssen et al., 2019). Baseline diameter serves as a reference point and enables comparisons within and between groups, especially when different conditions are involved that can affect the baseline diameter (e.g., exercise). During the occlusion period, the cuff should be inflated to approximately 200-300 mm Hg in adults, and approximately 50 mm Hg over systolic in children. It is crucial to ensure that the cuff pressure is 50 mm Hg above systolic pressure to prevent arterial inflow (Thijssen et al., 2019). Although the high pressure cuff may cause some discomfort for participants, using a smaller cuff can help minimize discomfort during the occlusion period. The post-deflation phase of the FMD protocol should be recorded for approximately three minutes to capture the peak diameter, as it may not occur within the first 60 seconds for all individuals. In larger arteries such as the superficial femoral artery, the concentration of NO synthase may differ from the brachial artery, leading to delayed peak dilation (Thijssen et al., 2008). FMD is usually expressed as a percentage and calculated as follows (Thijssen et al., 2011): %FMD = [(Peak diameter - Baseline diameter) / Baseline diameter] × 100 35 Outside factors such as diet, lifestyle, and mental health (e.g., stress) can influence FMD values. To ensure accurate measurements, it is recommended that the FMD guidelines by Thijssen et al. (2019) are strictly adhered to. This means participants should avoid engaging in exercise or strenuous physical activity for at least 24 hours before obtaining FMD measurements. Participants should refrain from consuming foods or beverages high in caffeine and vitamin C, as well as any supplements that may impact the cardiovascular system, for approximately 12 hours before undergoing FMD testing. Participants are advised to rest in a supine position for 10-15 minutes before the FMD measurements. This allows the body to reach a state of physiological equilibrium and minimizes the potential influence of mental stress on FMD results. Smokers should abstain from smoking for approximately 6 hours prior to the FMD assessment. For premenopausal women, it is recommended to conduct FMD measurements during the same phase of the menstrual cycle to standardize the potential effects of hormonal fluctuations on vascular function. Consideration of these factors and implementing the recommended guidelines contribute to obtaining reliable and standardized FMD measurements (Thijssen et al., 2019). Shear rate Shear rate data is derived from the pulse wave velocity mode as this displays blood velocity. However, a significant limitation arises from the competition for transducer resources in the ultrasound device, as the ultrasound beam needs to be at a 90° angle for diameter measurements and the Doppler signal for blood flow needs to be at a 0° angle. Therefore, to accommodate both requirements, the insonation angle should always be set to 60-70° throughout the study. Shear rate can be calculated using the following formula (Thijssen et al., 2019): 36 Shear rate = 4 𝑥 𝑉𝑒𝑙𝑜𝑐𝑖𝑡𝑦𝐷𝑖𝑎𝑚𝑒𝑡𝑒𝑟 , for smaller arteries such as brachial Shear rate = 8 𝑥 𝑉𝑒𝑙𝑜𝑐𝑖𝑡𝑦𝐷𝑖𝑎𝑚𝑒𝑡𝑒𝑟, for bigger arteries such as superficial femoral Figure 2.1 depicts the alteration in shear stress from baseline to post-deflation during the FMD procedure. It is noteworthy that after cuff deflation the shear rate response precedes the change in diameter of the vessel (Thijssen et al., 2019). This pattern of response has therefore led to the understanding that the post-occlusion hyperaemia results in a shear mediated dilation (Pyke & Tschavosky, 2007). Shear rate area under the curve (SRAUC) SRAUC is a measure of the area under the curve when shear rate is plotted against time, as seen in Figure 2.1, particularly during the reactive hyperemia phase following a temporary occlusion of blood flow. This curve reflects the changes in shear rate, and the area under this curve represents the cumulative effect of shear rate on the endothelium during this phase. When investigating the association between FMD and hyperemic shear rate during FMD protocol (see Figure 2.1), Thijssen et al. (2009) pointed out that previous studies have attempted to normalize FMD for shear rate. However, it is important to acknowledge that the normalization of FMD using shear rate may not be applicable across all age groups. As demonstrated by Thijssen et al. (2009), the correlation between SRAUC and FMD was significant in young adults, but not in children or older adults. In a healthy population, SRAUC was found to account for only 9% of the changes in FMD (Thijssen et al., 2009a) and therefore FMD normalization using shear rate is not recommended. 37 Baseline diameter is inversely related to FMD, and therefore different baseline diameters could influence FMD. For instance, studies that compare age related differences, would have groups with different baseline diameters and this also applies to interventions that alter diameter, which can lead to changes in baseline diameter pre and post intervention. Therefore, Atkinson et al., (2013) developed a method to allometrically scale for baseline diameter to account for these differences in baseline diameters, either between or within groups. This approach standardizes FMD for any differences in baseline diameter and provides group specific adjusted means. 2.2 Feasibility of Passive Heat Experimentation in Children As discussed in section 1.4, prior adult studies have used passive heat to manipulate shear rate and FMD, but with mixed results. Some passive heat studies show an increase in FMD, and shear rate post passive heat intervention (Bailey et al., 2016; Brunt et al., 2016; Romero et al., 2017; Cheng et al., 2021), whereas others only show an increase in shear rate, while FMD remained unchanged (Alali et al., 2020; Thomas et al., 2016; Engelland et al., 2020). It is likely that methodological differences account for the inconsistency in findings between studies and the available literature highlights several key issues related to the use of passive heat as an experimental condition to alter lower limb vascular function that are pertinent to conducting this type of experimental manipulation with children: 1. Does passive heating of one leg lead to a central cardiovascular response? 2. Does passive heating of one leg cause a systemic change in the contralateral leg? 3. Is the between limb baseline shear rate and FMD acceptable to allow the non-heated leg to be used as a control condition? 38 4. What is the ideal experimental set-up for a passive heat manipulation of shear rate in children and what are the potential sources of data loss? As preparatory work for my primary study, I undertook secondary analyses of pilot data that had been collected in the laboratory in 2017-18, with the goal of understanding the feasibility of running a shear manipulation experiment in children. The aim was to utilize the findings from the pilot work to design an experimental condition using passive lower limb heating to up-regulate shear rate and potentially FMD. 2.3 Secondary Data Methodology 10 healthy children (6 females, 4 males) between the ages of 7-11 years completed the pilot testing. Participants were all pre-pubertal based on parental assessment of Tanner stage (all participants were Tanner 1). Written and verbal consent and assent were obtained from the parents and children prior to testing. This study was approved by the UBC Clinical Review Board (H16-00997). 2.3.1 Passive Heat Experimental Design This study involved one visit to our pediatric research laboratory where the participants underwent the Heat condition on one leg, with the contralateral leg acting as the control condition. In the Heat condition, one of the upper legs (randomly selected as either left or right) underwent passive heating for a duration of one hour. To achieve this, a heat pad set at 42°C was wrapped around the thigh above the knee. The choice of a 42°C heat pad was deliberate, as it was intended to enhance blood flow without invoking any systemic changes in the nervous 39 system, consistent with prior research involving adult subjects (Padilla et al., 2011). Conversely, the contralateral leg had no heat stimulus administered to the thigh. The preference for a heat pad over a water bath in this study was primarily influenced by practical considerations. In a laboratory setting involving children, the use of a water bath was expected to be potentially distracting and messy. Therefore, the heat pad was considered a more pragmatic choice, with an optimistic expectation that it would induce a passive heat response similar to that achieved with warm water baths in previous studies involving adult participants. The children were advised to avoid rigorous physical activity, caffeine, foods high in fat (Nicholls et al., 2006) and vitamin C (Thosar et al., 2015) prior to their first visit, consistent with the FMD guidelines (Thijssen et al., 2019). 2.3.2 Procedures The study began with a familiarization session, during which participants were introduced to the laboratory environment, equipment, and an overview of the protocol. Prior to start of experiment, anthropometric measurements were taken. Participants were seated with their legs positioned horizontally, and baseline measurements of blood pressure and heart rate were recorded. Simultaneously, ultrasound assessments of superficial femoral artery function were conducted on both legs. In the Heat condition leg, a heat pad was applied and heated to 42°C and participants were instructed to remain seated and limit leg movements for one hour. Throughout this time, the children were given the option to engage in leisure activities, such as playing games on their phones or watching movies on an iPad provided to them prior to the start of the condition. At the conclusion of the hour, blood pressure and heart rate were reassessed, and ultrasound scans of the superficial femoral artery were performed on both legs. FMD scans were conducted 40 simultaneously in both legs, Pre and Post the experiment, by two sonographers. Each sonographer was assigned to one leg for each participant; however, the choice of the leg (Heat or Control) was randomized to ensure unbiased assessments. 2.3.3 Measures Body mass, measured in kg, was obtained using Tanita TBF-410 scales, with participants wearing light clothing and no shoes during measurement. Stature, or height, was measured in cm using a Seca 217 stadiometer while participants stood barefoot. Blood pressure measurements were taken using a manual sphygmomanometer (Prestige Medical 79-BLK standard aneroid, Northridge CA). Systolic and diastolic blood pressures were measured twice in the right arm, with each reading rounded to the nearest 2 mm Hg. If the two systolic and diastolic measurements differed by more than 10 mm Hg, a third measurement was taken for accuracy. Heart rate was monitored using heart rate telemetry (Polar Vantage NV, Polar Electro Oy) before the start of the experimental condition and at the conclusion of the condition. To assess the impact of movements of the lower limbs and posture changes, an ActivPal accelerometer (ActivPal3 micro; PALtechnologies, Glasgow, UK) was employed. This tracking provided insights into participants' adherence to the study protocol, particularly in terms of minimizing major posture changes and leg movements, as these could significantly affect artery function, as suggested by Morishima et al. (2016). 41 2.4 Findings and Discussion All 10 children recruited completed the study. Demographics of participants are shown in Table 2.1. ActivPAL accelerometer data showed that the participants in average sat for 59 minutes of the intervention and spent less than one minute moving and/or standing. Due to data loss during scanning, a total of five cases were excluded from FMD analysis. The main contributing factor to these exclusions was the occurrence of angle changes during the scans, which are likely to be attributed to movements made by the participants during the scanning process. It was observed that most children experienced difficulty remaining still during the FMD procedure, leading to leg movements. These movements were reported to have occurred due to a sensation of itchiness in their lower limbs and discomfort. Table 2.1 Participant Demographics Variables N=6 Sex (M/F) 1/5 Age (years) 10.3 (1.6) Height (cm) 144.5 (9.2) Body Mass (kg) 34.0 (7.2) Data are means (SD) Similarly, six cases were excluded from the shear rate analyses. The exclusion of these cases was predominantly due to specific settings on the Doppler ultrasound, which picked up noise and generated a reflection trace in the pulse wave velocity mode. Given that these issues deviate from the guidelines outlined by Thijssen et al. (2019), I made the decision to either exclude the cases or exclude the data from the analyses. 42 2.4.1 Central cardiovascular change In this study, the systolic and diastolic blood pressures, as well as heart rate pre to post heating are shown in Table 2.2. These findings indicate that the passive heating applied to one leg did not induce systemic changes in blood pressure or heart rate, aligning with previous research using passive limb heating (Cheng et al., 2021; Chiesa et al., 2016; Chaseling et al., 2022; Esteves et al., 2021). Table 2.2 Cardiovascular responses Pre and Post passive limb heating Abbreviations: HR, heart rate, SBP, Systolic blood pressure, DBP, Diastolic blood pressure However, since core temperature was not measured, the exact increases in core temperature caused by heat pad remains unknown. A review of heat stress suggests that due to thermoregulatory responses, any exposure to a heat stimulus, even a minor increase of 0.3°C in internal core temperature, can instigate heat dissipation reactions, including vasodilation and sweating (Crandall & Wilson, 2015). These responses can lead to elevated cardiac output, HR SBP DBP Cases PRE POST PRE POST PRE POST 2 74 70 116 100 70 60 5 79 78 110 100 80 70 6 74 74 100 100 68 70 7 76 81 99 100 63 73 8 89 88 107 102 75 79 9 80 79 104 100 80 72 Mean (SD) 79 (6) 78(6) 106 (6) 100 (1) 73 (7) 71 (6) 43 regulation of blood pressure, and improved perfusion to the skin, especially within the cutaneous region (Crandall & Wilson, 2015; Cheng et al., 2018). However, we did not see changes in blood pressure in this experiment. 2.4.2 Changes in the Control Leg with Passive Heating Table 2.3 provides details on shear patterning and mean shear rates for each participant before and after the passive heat condition as well as mean and standard deviations values for those parameters. Table 2.3 Shear patterning, and shear rates Pre and Post passive heat condition and in the control leg, in children between the ages of 8-12 years old. Data are presented as individual cases, as well as mean (SD). Pre Post Heat Cases Antegrade Shear Rate (1/s) Retrograde Shear Rate (1/s) Mean Shear Rate (1/s) Antegrade Shear Rate (1/s) Retrograde Shear Rate (1/s) Mean Shear Rate (1/s) 1 144.30 -21.63 122.73 191.50 -14.29 177.30 2 126.44 -15.11 111.37 159.45 -9.45 150.04 3 115.36 -17.37 98.03 167.74 -4.22 163.64 4 172.28 -1.23 171.12 304.54 163.64 304.68 Mean (SD) 139.59 (24.83) -13.84 (8.83) 125.81 (31.85) 205.81 (67.21) -6.99 (6.22) 198.91 (71.38) Controls 2 166.84 -18.86 148.05 140.22 -15.25 125.03 3 142.08 -28.86 113.29 147.40 -22.79 124.68 4 143.02 -7.28 135.79 115.13 -10.62 104.55 Mean (SD) 150.65(14.03) -18.33(10.80) 132.38(17.63) 134.25(16.94) -16.22(6.14) 118.09(11.73) 44 The results in Table 2.3 indicate an increase in both antegrade shear rate and mean shear rate in the Heat condition leg for all participants. In the Control leg, two out of the three participants exhibited a decline in antegrade and mean shear rate. After one hour of passive heating in the Heat condition leg, the Control leg remained similar to start of condition. A similar finding can be observed in Table 2.4 for blood flow and SRAUC measurements. While SRAUC and blood flow show an increase post one hour of heat in the condition leg, the values in the control leg were not different from pre one hour. In summary, the overall pattern suggests that passive heating leads to an increase in shear patterning and blood flow in the condition leg but not in the contralateral control leg. This is similar to the work of Chiesa et al. (2016) who conducted a study using isolated leg passive heating and found that heat stress applied to one leg resulted in significant changes in blood flow. Similarly, they did not observe any changes in the contralateral control limb during the heat protocol. Other studies conducted on adults have also reported similar results, where localized interventions targeting one limb did not influence the flow, shear patterns, or FMD of the contralateral limb (Totosy et al., 2014; Cheng et al., 2021). These data demonstrate that with localized limb heating, there is an increase in flow and mean shear rate in children. Antegrade shear rate increases post intervention and retrograde shear rate decreases. This is also consistent with previous literature that show retrograde shear is associated with endothelial dysfunction by decreasing shear stimulus (Tremblay et al., 2019; Schreuder et al., 2014). 45 Table 2.4 SRAUC and blood flow Pre and Post passive heat and in the control leg in children between the ages of 8-12 years old. Data are presented as individual cases, as well as mean (SD). Abbreviations: SRAUC, shear rate area under the curve. 2.4.3 Changes in FMD with passive limb heating Table 2.5 presents individual and mean data for the baseline diameter, peak diameter, and FMD before and after passive heat condition. Additionally, it includes measurements of the Control limb baseline diameter, peak diameter, and FMD. Pre Post Heat Cases SRAUC Blood Flow (ml/min) SRAUC Blood Flow (ml/min) 1 37388 60.03 35055 71.34 2 31199 46.83 37535 70.41 3 7218 63.51 10005 112.53 4 56098 57.72 64939 81.99 Mean (SD) 32976 (20171) 57.02 (7.20) 36884 (22459) 84.07 (19.69) Controls 2 29725 82.20 25887 66.93 3 30600 52.74 23098 66.03 4 14119 77.40 20228 87.06 Mean (SD) 24815 (9273) 70.78(15.81) 23071(2829) 73.34(11.89) 46 Only three out of the five participants had an increase in baseline diameter post heating, which is what we would expect to occur post heat due to the increase in flow from the heat stimulus. Two of the participants had a decrease in baseline diameter post intervention, however, because peak diameter post heat was still higher than pre heat peak diameter, the overall FMD were consistently higher post intervention for each of the five participants in the Heat condition. The reason could be physiological, where the differences in baseline diameter seen are caused by vasodilatory mechanisms or it is a technical error from the scan itself. However, the intratester coefficient of variation for FMD was 10% for FMD. 47 Table 2.5 Baseline, peak diameter and FMD changes during passive heat and control conditions, pre and post 1 hour. Pre Heat Post Heat (1hr) Cases Baseline Diameter (mm) Peak Diameter (mm) Delta Diameter (mm) FMD (%) Baseline Diameter (mm) Peak Diameter (mm) Delta Diameter (mm) FMD (%) 1 4.28 4.56 0.28 6.54 4.1 4.53 0.43 10.49 2 4.12 4.41 0.29 7.04 4.28 4.83 0.55 12.85 3 3.86 4.28 0.42 10.88 3.58 4.00 0.42 11.73 4 3.70 4.01 0.31 8.38 3.58 4.09 0.51 14.25 5 4.05 4.29 0.24 5.93 4.17 4.52 0.35 8.39 Mean (SD) 4.00 (0.23) 4.31 (0.20) 0.31 (0.07) 7.75 (1.97) 3.94(0.34) 4.39 (0.34) 0.45 (0.08) 11.54 (2.24) Control 2 4.51 4.96 0.45 9.98 4.47 4.89 0.42 9.40 3 4.60 4.89 0.29 6.30 5.16 5.68 0.52 10.08 Mean (SD) 4.56(0.06) 4.93(0.05) 0.37(0.11) 8.14(2.60) 4.82(0.49) 5.29(0.56) 0.47(0.07) 9.74(0.48) Data are presented as individual cases, as well as mean (SD). 48 Is the between limb variance in shear rate and FMD acceptable to allow the non-heated leg to be used as a control? Differences in shear rate and FMD were observed between the Heat leg and Control leg Pre passive heating. Some participants exhibited different baseline diameters in their two legs prior to heating and variations in shear patterning, blood flow, and shear rates were also observed (see table 2.3 and 2.5). For instance, participant 1 displayed an antegrade shear rate of 126.44 1/s in the Heat condition leg Pre heat, while the Control leg had a shear rate of 166.84 1/s. Similarly, the blood flow measurements in this participant were 46.83 ml/min for the Heat condition leg and 82.2 ml/min for the Control leg, Pre heat. These findings suggest that the structural characteristics of the superficial femoral arteries may differ leg to leg within the same participant. Therefore, examining the effects of localized limb heating, using the contralateral limb as the Control may not be as useful as it first appears. Indeed, other methodological problems can emerge such as dyssynchronous scanning. Totosy et al. (2014) acknowledged this as a limitation in their study, namely the absence of simultaneous post-FMD measurements in both the control and condition legs. They reported a 15-minute delay in obtaining FMD measurements for the contralateral leg after the end of the intervention. Additionally, it is noteworthy that both the condition and control legs displayed distinct baseline shear rates, including antegrade and retrograde shear rates, prior to the initiation of the intervention in this study. Variability in FMD responses with passive heating may relate to the timing of the FMD after heat condition. This is highly variable across studies, with the FMD measurement ranging from 30 minutes (Cheng et al., 2021; Romero et al., 2017) to 60 minutes (Brunt et al., 2016) after 49 passive heating. The acute effects of heating may recover during this time, potentially leading to no significant changes in FMD response (Cheng et al., 2018). A time course study of passive heating indicates that FMD measurements taken within 30 minutes of the end of heating condition can represent the acute effects of heating on the vessel of interest (Chaseling et al., 2022). 2.4.4 Experimental Set up and Sources of Data Loss The wall tracking software utilized in this pilot work was at times unable to accurately detect the vascular wall due to probe or limb movements, leading to artificial changes in arterial diameter that were not physiological. In simpler terms, the alterations in diameter could have stemmed from technical errors caused by movements of the probe (or child), resulting in an artificial peak in diameter measurement. During the heating condition, the heat pad was wrapped around the participants' leg for the entire duration of one hour. However, it is important to note that the consistency of the wrapping technique varied between participants because of limb size, and thus, it is uncertain whether the heat pad provided a uniform heating stimulus to each participant. Table 2.5 provides some insight into the observed changes in blood flow (ml/min) post heat condition and supports the notion that the heat pad tightness may have contributed to these variations. For instance, in one participant, the baseline flow prior to applying the heat pad was measured at 60.03 ml/min, and post passive heat condition, it increased to 71.34 ml/min. Similarly, in another participant, the baseline flow was 63.51 ml/min, and post passive heat condition, it surged to 112.53 ml/min, representing a nearly twofold increase in flow in the superficial femoral artery. While it is 50 important to consider other potential factors that might have influenced the differences in flow increase, the heat pad itself could be considered a contributing factor to these observed variations as mentioned in detail in section 2.4.2. Although the heat pad was manufactured to reach a temperature of 42°C, there was no temperature controller incorporated directly into the pad itself. Consequently, it was not possible to determine whether this temperature was indeed reached or remained constant throughout the hour. These limitations introduce uncertainty regarding the consistency and reliability of the heating stimulus during the intervention. To address these limitations in future studies, a heated water bath likely provides a more consistent heating environment. The heated water bath allows for better control of the heat stimulus given the measurement of temperature is easier with the water bath and can be kept consistent regardless of limb size. By utilizing a heated water bath in my primary study, I hope to provide a more controlled and standardized heat stimulus for each participant. 2.5 Main Pilot Study Conclusions Exposure to heat stimulus in humans triggers a cascade of thermoregulatory mechanisms aimed at heat dissipation, which in turn directs blood flow to the cutaneous region. As part of this process, vasodilation occurs, activating mechanisms related to vasomotor function in response to heat stimulus. However, it is evident that several factors, including the temperature of the heat stimulus, the chosen passive heating methodology, whether the stimulus is localized to a single limb, and core temperature, play crucial roles. Additionally, adherence to the FMD methodology 51 outlined by Thijssen et al. (2019) and the timing of post-heating measurements also contribute to the observed differences. In the upcoming study, we will employ a water bath heating method, which allows for better control of temperature, taking into consideration the significance of skin temperature, and limiting potential sources of data loss. Moreover, the study will be conducted with more meticulous adherence to FMD guidelines, including the careful acquisition of artery images to prevent overt angle changes, addressing a major issue encountered in this current study. Lastly, timing of the ultrasound measures will be standardized post heating in the primary study. 52 Chapter 3: Examining the Effects of Shear Rate on Flow Mediated Dilation (FMD) in Children and Adults 3.1 Introduction Adult studies have highlighted the relevance of mean shear and shear patterning influencing endothelial function (Green et al., 2017; Thijssen et al., 2009; Green et al, 2014). From the literature, it is shown that antegrade shear stress fosters positive effects on the endothelium and overall vascular health (Chappell et al., 1998; Hsiai et al., 2003; Wang et al., 2013), while retrograde shear rate has negative implications on the endothelium (Thijssen et al., 2009b; Schreuder et al., 2014). These studies inform strategies for preserving cardiovascular health, particularly before onset of arterial diseases. However, the complexity of the interplay between shear stress and endothelial function is such that the magnitude and patterning of shear stress needed to impact FMD still requires further investigation. Passive heating intervention on specific limbs or the entire body is shown to enhance blood flow and shear stress without inducing central changes, unlike exercise (Cheng et al., 2021; Restaino et al., 2016; Romero et al., 2017). While exercise can be used to examine the shear rate-FMD relationship, the stimulus from exercise induces neurohormonal and metabolic factors. Nevertheless, despite the common increase in mean and antegrade shear rate in passive heating studies, the effects on FMD are diverse and inconsistent. For instance, Cheng et al. (2021) found no significant FMD improvement in the superficial femoral artery following passive heating in 53 the lower limb despite significant increases in antegrade shear rate and flow. Romero et al. (2017) found improvements in superficial femoral FMD in older adults but not young adults. Effects of decreased shear stimulus have been linked to reductions in endothelial function via the use of low pressure cuff interventions (Tremblay et al., 2019; Thijssen et al., 2009b; Schreuder et al., 2014). These investigations have consistently demonstrated decreased retrograde shear rate and mean shear rate, resulting in a significant decline in FMD responses, at least in adults. While the relationship between shear rate and FMD has been extensively explored in adults, no study to date has compared the effects of increasing or decreasing shear rate on superficial femoral artery function between children and adults. In contrast to young adults, no association between shear rate and FMD was found in children (Thijssen et al., 2009a). Notably, in adults, a decline in FMD after prolonged sitting is accompanied by corresponding decreases in shear rate and alterations in shear patterns (Restaino et al., 2015; Thosar et al., 2015), but this is not observed in children (McManus et al., 2015). Collectively, these studies suggest age-dependent vasomotor mechanisms. No studies to date have investigated the shear rate-FMD relationship in children using low pressure cuff or passive heat interventions to isolate the precise effects of these flow-related changes on the function of the endothelium, as well as explore developmental differences. Therefore, the aim of this study was to manipulate shear stress in both children and adults and analyze the FMD responses that follow. Our hypotheses are that; (i) passive lower leg heating will increase antegrade shear rate, mean shear rate and shear rate under the curve (SRAUC) to a similar magnitude in children and adults; (ii) increases in antegrade shear rate and mean shear 54 rate with passive limb heating will lead to an increase in superficial femoral artery FMD in adults but not children; (iii) low pressure cuff inflation will increase retrograde shear rate, decrease mean shear rate to a similar magnitude in children and adults; (iv) increases in retrograde shear rate and decreases in mean shear rate with a low pressure cuff intervention will lead to decreases in FMD in adults but not children. 3.2 Methods Ten healthy adult males aged 19 to 29 years, and seven healthy young males, aged 7 to 12 years, participated in this study. None of the participants had any preexisting medical conditions. Written consent was obtained from parents and written and verbal assent from children before testing. This study received approval by the institutional Clinical Review Ethics Board (H22-03037). 3.2.1 Experimental Design This experiment involved all participants completing two conditions designed to either up-regulate or down-regulate blood flow and shear rate to the superficial femoral artery. The up-regulation condition consisted of passive heating of the lower leg, while the down-regulation condition consisted of applying a low-pressure cuff to the upper thigh, both of which are described below. Passive Heat: In the passive heat condition, the left leg was subjected to passive heating for a duration of one hour. To achieve this, a hot water bath was heated through a sous vide machine to ensure the temperature stayed consistent throughout the condition. The temperature of the 55 water bath was maintained at 42°C and specifically selected from the prior adult literature that demonstrates an increase in blood flow to the leg, without any central cardiovascular changes (Padilla et al., 2011). Low-pressure Cuff: In the low-pressure cuff condition, the left leg was exposed to a low pressure (70mm Hg in adults and 40mm Hg in children) occlusion of the superficial femoral artery using a cuff placed on the upper thigh for thirty minutes. The respective pressures chosen for the adults and children were based on prior adult studies where a cuff pressure of 70 mm Hg was found to reduce blood flow and shear rate (Tremblay et al., 2019; Thijssen et al., 2009b; Schreuder et al., 2014). Since 70 mm Hg in adults is approximately 50 below systolic, the same formula was used in children as there are no prior child studies in literature for reference. Therefore, we obtained 40 mm Hg using the lower limit of average systolic blood pressure value in children (Gebara et al., 2005). 3.2.2 Procedures Participants completed each experimental condition on a separate day, with at least 24 hours between laboratory visits. The order of completion of the two conditions was counterbalanced with half of the participants starting with passive heat, while half started with low-pressure cuffing. Participants were advised to avoid rigorous physical activity, caffeine, foods high in fat (Nicholls et al., 2006) and vitamin C (Thosar et al., 2015) prior to both laboratory visits as this has been shown to influence the assessment of endothelium function (Thijssen et al., 2019). During the first visit, anthropometric measures were taken upon arrival. Participants were asked to sit with their upper thigh horizontal, with a 90-degree bend at the knees during both laboratory 56 visits. During both visits, participants were instructed to sit still, avoid standing and limit leg movements. To track leg movements during the experimental time, an ActivPAL accelerometer, (ActivPAL3TM micro; PALtechnologies, Glasgow, UK) was attached to the right upper thigh. After sitting for 10 minutes, baseline measurements of blood pressure and heart rate were taken, followed by ultrasound assessment of superficial femoral artery diameter, blood velocity and flow mediated dilation of the left leg. While sitting, participants could play on their phones or watch shows / play games on an iPad. During the last minute of both conditions, an ultrasound scan of the superficial femoral artery diameter and blood velocity was taken. Five minutes after the end of each condition, a repeated ultrasound scan of the left leg was completed for diameter and blood velocity of the superficial femoral artery. To improve ultrasound reliability pre and post condition, a probe markup was done on participants leg, to ensure the same artery site was scanned post condition. 3.2.3 Measures Body mass (kg) was measured wearing light-weight clothing and no shoes using electronic scales (Tanita TBF-410) to the nearest 0.1kg. Stature (cm) was measured barefoot using a stadiometer (Seca 217) to the nearest 0.1 cm. Body mass index (BM) was calculated from body mass (kg) divided by stature (m2). Body fat (%) was calculated using electronic scales (Tanita TBF-410). The pubertal status of the children was classified using parental assessment of Tanner stage (Marshall & Tanner, 1969; Marshall & Tanner, 1970) for pubic hair and genital development (Rasmussen et al., 2015). As noted in section 1.2, Tanner stage uses physical appearance to 57 identify changes in sexual maturation. Tanner 1 denotes pre-puberty, with the subsequent stages representing ensuing maturation. Movements of lower limb and posture changes were tracked using ActivPAL accelerometer (ActivPAL3 micro; PALtechnologies, Glasgow, UK). These accelerometers are more commonly used in studies to track movement, posture, and sedentary behavior, (Kim et al., 2015). This study used accelerometers to monitor the average time spent standing, moving during condition in each participant. This can be useful in determining whether the participants adhered to the protocol as movements during sitting is shown to have an impact on shear stress (Ray et al., 1993; Morishima et al., 2016). Systolic and diastolic blood pressures were taken using an automated blood pressure machine with three lead electrocardiogram for heart rate (SunTech Medical Tango M2, NC, USA) twice in the right arm to the nearest 2 mm Hg after resting for five minutes, pre, 30 minutes into passive heat condition and post. If both measurements were more than 10 mm Hg apart, a third measurement was taken. During the passive heat condition, heart rate measurements were also assessed at 15-minute intervals to monitor any potential central cardiovascular changes caused by the passive heating of the leg using the same automated machine. For the low pressure cuff condition, heart rate and blood pressure measurements were only reported at baseline. 3.2.4 Principal Outcome Superficial femoral artery images were acquired using a duplex ultrasound system (Terason USMART3300TM; Teratech, Burlington, MA, USA) equipped with a 15 MHz multi-frequency 58 array vascular ultrasound probe (Terason T3200; Teratech, Burlington, MA, USA). B-mode ultrasound was utilized to obtain artery diameter measurements, while pulse wave mode was activated simultaneously to record blood velocity data. A baseline recording of at least one minute was initially captured. Subsequently, the artery was occluded by rapidly inflating a small cuff, placed 2-3 cm from the superficial femoral artery bifurcation to a pressure of 50 mm Hg over systolic blood pressure for five minutes (Thijssen et al. 2019). Thirty seconds before cuff deflation, diameter and pulse wave velocity recordings resumed and continued post deflation for three minutes. Video capture software, Camtasia (TechSmith, Okemos, MI), was employed to record both baseline and post-deflation artery measurements for later analysis. These recordings were securely stored as video files, prepared for offline analysis. Throughout the process, technical recommendations outlined by Thijssen (2019) were followed. Visual inspection of the scans was conducted before analysis to ensure data integrity. Recordings were excluded from the analysis based on three specific criteria: 1. Overt Angle Change: Recordings showing a significant change in the angle of imaging were excluded. 2. Excessive Vessel Movement: Recordings in which there was excessive movement of the vessel, potentially impacting diameter measurement accuracy, were also excluded. 3. Poor Image Quality: Recordings with general issues related to image quality, such as unclear vessel walls, were not included in the analysis. 59 To extract precise measurements of diameter, velocity, blood flow, and shear rate, custom-designed software equipped with advanced edge detection and wall tracking capabilities was used in this study (Woodman et al., 2001). This ensured that the data processing approach is consistent, reducing the potential for observer bias and enhancing the reliability of the data. Synchronized diameter and velocity recordings allowed the calculation of superficial femoral artery blood flow and shear rate. Shear rate was determined using the formula: Shear rate = 4 × (Velocity / Diameter). Both antegrade and retrograde flow were calculated using the positive and negative shear rate area under the curve (SRauc) during baseline. Shear rate area under curve is recognized as the more accurate method for understanding flow mediated dilatatory responses than peak or mean shear rate and hence has also been reported in this study (Thijssen et al., 2009a). All data processing of the ultrasound data were conducted by a single researcher to minimize the potential for bias in the results. FMD was calculated following the guidelines set forth by Thijssen et al. (2019) where FMD is the difference between peak diameter (Dpeak) and baseline diameter (Dbase), expressed as a percentage: % FMD = ((Dpeak - Dbase) / Dbase) × 100. To assess the reliability of the sonographer an intra-individual Technical Error of Measurement (TEM) was calculated for diameter and FMD. The relative technical error of measurement for FMD is 16% for this study and the coefficient of variation is 36.2%. 60 3.2.5 Statistics Normality of data was assessed for all primary outcome variables using the Shapiro-wilk test and q-q plots. All main outcome variables were logarithmically transformed before conducting the statistical analyses since the data were not normally distributed. All data are presented as group means (±SD) unless otherwise stated. Repeated Measures Analysis of Variance (RM ANOVA) was used to investigate both within and between group main effects and interactions for both the passive heat and low pressure cuff conditions, for superficial femoral artery diameter, blood flow, FMD, shear rate, shear patterning and SRauc. T-tests were used to deconstruct main effects and interactions, with Bonferroni adjustments. RM ANOVA was used to compare shear rate, flow and diameter changes in baseline, prior to end of condition and post condition within and between groups. To test the relationship between changes in shear rate and FMD, Pearson’s correlational analysis was performed. FMD was adjusted for baseline diameter by log transforming baseline (logdbase) and peak diameter (logdpeak). A log-linear mixed model was used with the difference between logdbase and logdpeak as dependent variable, and logdbase as a covariate, with time and group as fixed factors. The estimated means from time and group specific output was back transformed to obtain allometrically scaled FMD. Heart rate and blood pressure measurements taken baseline, during and post the passive heat condition were also investigated using a between and within group RM ANOVA. A α-level of 0.05 was established a priori to determine statistical significance. All statistical procedures and analyses were executed using SPSS (Version 28, SPSS; Chicago IL). 61 3.3 Results Anthropometric data are presented in Table 3.1 for the nine adults and seven children included in the analyses. Of the seven children, five were Tanner stage 1, one was Tanner stage 2, and one was Tanner stage 3. Table 3.1 Participant Demographics Adults (n=9) Children (n=7) Mean ± SD Minimum and Maximum Mean ± SD Minimum and Maximum Age (years) 22.8 ± 3.0 20-29 10.0 ± 1.7 7-12 Stature (cm) 177.9 ± 8.2 163-187 149.2 ± 12.3 134-172 Body mass (kg) 78.3 ± 9.3 70-93 42.9 ± 12.2 28-56 %Body fat 16.4 ± 3.1 11.1-19.6 17.7 ± 8.5 9.1-30.4 BMI (kg/m2) 24.8 ± 2.6 20-28 19.2 ± 4.4 14-26 Passive Heat Condition One adult participant was excluded from the analyses due to an inability to obtain a clear image of the artery during any of the visits. Data from one child participant was excluded from the passive heat condition due to poor scan quality. Table 3.2 presents the cardiovascular responses at various time points, including baseline, during the passive heat condition, and post-condition, for both the adults and children. There were significant main effects for time (F(3.221, 38.6648)= 12.24, p=.000) and group (F(1,12)=11.093, p=.006) for heart rate, but no interaction. Heart rate was higher in children compared to adults at all time points. Pairwise comparisons with 62 Bonferroni corrections showed that heart rate was significantly different (p<0.001) from baseline at all time points. Heart rate significantly decreased post condition compared to during condition at 60 minutes in children and adults (p=.003). Systolic and diastolic blood pressures remained constant baseline to post heat. Systolic blood pressure was lower in the children (F(1,11)=10.09, p=.009), while diastolic was similar in children and adults. 63 Table 3.2 Cardiovascular data at Baseline, during and Post the passive heat condition in adults (n=9) and children (n=6). Data are presented as mean ± SD. Abbreviations: HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure. Baseline 15min 30min 45min 60min Post Time Group TimexGroup Adult HR (bpm) 64 ± 13 67 ± 13 68 ± 12 68 ± 14 69 ± 12 64 ± 12 p<0.001 0.006 0.116 SBP (mm Hg) 125 ± 7 120 ± 12 123 ± 6 0.082 0.009 0.12 DBP (mm Hg) 73 ± 12 73 ± 10 72 ± 11 0.334 0.415 0.804 Child HR (bpm) 77 ± 9 83 ± 9 88 ± 14 86 ± 15 86 ± 13 85 ± 12 p<0.001 0.006 0.116 SBP (mm Hg) 109 ± 7 110 ± 8 116 ±12 0.082 0.009 0.12 DBP (mm Hg) 71 ± 6 75 ± 9 74 ± 7 0.334 0.415 0.804 64 3.3.1 Vasodilatory Function with Passive Heat Table 3.3 displays the superficial femoral artery diameter (mm) and blood flow (ml/min) at baseline and peak hyperemia before and after the passive heat condition. There was a main effect for time for baseline diameter (F(1,13)=4.943; p=.045), a main effect for group (F(1,13)=29.304; p=.000), but no interaction. A larger baseline diameter was apparent in adults compared to children, with baseline diameter declining with passive heat in both children and adults. Only a main effect for group (F(1,13)=30.492; p=.000) was apparent for peak diameter, with a larger arterial diameter in adults. Blood flow at baseline was similar between the children and adults. A main effect for time was present (F(1,13)=121.423; p=.000), as well as an interaction (F(1,13)=14.456; =.002). Both children and adults experienced a significant increase in flow post passive heat, however, the magnitude of this increase was greater in adults compared to children (p<0.001). There was no significant main effect for group for baseline blood flow. There was an increase in hyperaemic blood flow from baseline to post passive heat in both children and adults (F(1,13)=39.685; p=.000), with an interaction (F(1,13)=6.18; p=0.027). The increase in hyperemic blood flow with passive heat was greater in the adults compared to the children (p<0.001). There was no main effect for group. Figure 3.1 illustrates the mean and the within subject superficial femoral artery FMD at baseline and post passive heat in both children and adults. Baseline FMD and post passive heat was 3.23 65 % and 4.10 % respectively in the adults and 3.44 % and 4.28 % respectively in children. There were no main effects for time or group and no interaction. Allometrically scaling FMD for baseline diameter also showed no main effects for time and group. Scaled FMD in adults increased from 3.87 ± 1.51% to 4.60 ± 1.82% and 2.74 ± 1.73% to 3.36 ± 1.73% post condition in children. Figure 3.1 FMD responses baseline (Pre) and Post passive leg heating in adults (A, n=9) and children (B, n=6). Data are mean ± SD. Lines represent individual data. 66 Table 3.3 Superficial femoral artery diameter, blood flow and shear rate responses at baseline (Pre) and Post passive heat in adults and children. Passive Heat ANOVA Main Effects and Interaction Adult (n=9) Child (n=6) Pre Post Pre Post Time (p-value) Group (p-value) Group x Time (p-value) Baseline Diameter (mm) 6.6 ± 0.5 6.5 ± 0.5 5.0 ± 0.7 4.8 ± 0.6 0.045 p<0.001 0.731 Mean blood flow (ml/min) 38.9 ± 13.3 161.6 ± 46.2* 52.4 ± 5.9 113.1 ± 54.6 p<0.001 0.017 0.002 Mean SR (1/s) 29.9 ± 12.1 140.3 ± 36.0* 103.1 ± 41.5 230.4 ± 79.1 p<0.001 p<0.001 0.002 Peak Diameter (mm) 6.8 ± 0.5 6.7 ± 0.5 5.14 ± 0.66 5.0 ± 0.6 0.086 p<0.001 0.664 Mean blood flow (ml/min) 62.0 ± 18.6 104.4 ± 31.8* 80.9 ± 15.3 103.8 ± 30.1 p<0.001 0.313 0.027 Mean SR (1/s) 50.3 ± 19.0 82.5 ± 27.4 146.6 ± 57.7 207.1 ± 109.2 p<0.001 p<0.001 0.103 SRAUC 2940.0 ± 1013.0 4950.0 ± 1595.0 8656.0 ± 3276.0 12155.0 ± 6446.0 p<0.001 p<0.001 0.068 Data represented as mean ± SD. Abbreviations: FMD, Flow mediated dilation; SR, Shear rate; SRAUC, Shear rate area under the curve. Bold text indicates P < 0.05. * denotes significant post hoc differences (p<0.001). 67 3.3.2 Shear Rates and SRAUC SRauc increased significantly with passive heat in both children and adults (see Table 3.3) (F(1,13)=44.634; p=.000). There was a main effect for group (F(1,13)=25.218; p=.000), but no interaction, with SRauc values higher in the children compared to the adults. Mean shear rate at baseline and peak hyperemia are presented in Table 3.3. At baseline mean shear rate increased with passive heat in both children and adults (F(1,13)=146.177; p=.000), and there was a significant main effect for group (F(1,13)=33.315; p=.000), as well as interaction (F(1,13)=15.339; p=.002). Follow-up tests show mean shear rate was higher at baseline in the children (p< .001) and increased more so in adults with passive heat (see also Figure 3.2A). Peak mean shear rate increased with passive heat in children and adults (F(1,13)=43.219; p=.00) with no interaction. There were significant differences between groups (F(1,13)=23.91; p=.00) with higher values in the children. Antegrade shear rate increased with passive heat in both children and adults (F(1,13)=66.686; p=.000), and there was a significant main effect for group (F(1,13)=21.86; p=.000) but no interaction (see Figure 3.2B). Antegrade shear rate was higher at baseline in children (132.08 1/s compared to 60.7 1/s in adults). Retrograde shear rate decreased significantly with passive heat (F(1,11)=13.184; p=.004) in both children and adults but with no interaction and no main group effects (see Figure 3.2C). 68 Figure 3.2 Mean shear rate and shear patterning pre and post the passive heat condition in adults (n=9) and children (n=6). Data are mean ± SD. Black dots represent adult individual data points and black squares represent child individual data points. * denotes significance (p<0.001). * 69 Figure 3.3 Changes in diameter, shear rate and flow are shown from baseline (Pre), to one minute prior to end of condition (60min) and five minutes post passive heat condition (Post) in adults (n=9) and children (n=6). Data are mean ± SD. * denotes significance from baseline to 60min (p<0.001). ** denotes significance from 60min to post (p<0.001). a denotes significance from baseline to post (p<0.001). * ****de* * ** a ** * a 70 3.3.3 Pre, during and post passive heat condition comparisons A separate RM anova analysis was done where data from baseline, one minute before end of condition (60 minutes) and post were compared for main and group effects. In both children and adults, there were main effects for time (F(1.79, 23.3)=166.727; p=.000) and group (F(1, 13)=32.83; p=.000) in mean shear rate with interaction (F(1.79, 23.3)=11.44, p=.000). Pairwise comparisons with bonferonni corrections showed mean shear rate significantly increased from baseline to 60 minutes (p<0.001). There were significant decreases in mean shear rate (p<0.001) from 60 minutes to post condition in children and adults (See Figure 3.3A). Blood flow showed main effects for time (F(1.85, 24.02)=163.579; p=.000) and interaction (F(1.85, 24.02)=10.053; p=.000) but no group effects (Figure 3.3C). There were significant differences between each time points (p<0.001). Blood flow increased throughout condition but significantly decreased from 60 minutes to post condition. Baseline diameter had main effects for time (F(1.72, 23.3)=6.936; p=.006) and group (F(1,13)=30.955; p=.000) with no interaction. Baseline diameter significantly decreased from 60 minutes to post condition (p=0.001) as seen in Figure 3.3D. SRauc changed significantly throughout (F(1.66, 21.57)=93.866; p=.000) and had group effects (F(1,13)= 27.808; p=.000) with interaction (F(1.66, 21.57)=93.866; p=.021). There were significant differences between each time points (p<0.001) with increases from baseline to all time points but significantly decreased from 60 minutes to post condition (See Figure 3.3E). 71 Antegrade shear rate observed main effects for time (F(1.67, 21.75)=81.141; p=.000) and group effect (F(1,13)=18.711; p=.000) with no interaction (Figure 3.3B). Like SRauc, anterograde shear rate also experienced the same pattern and observed significant differences from baseline to each time point (p<0.001) and a significant decrease from 60 minutes to post condition (p=.046) (Figure 3.3B). Retrograde shear rate had main effects for time (F(1.981, 21.793)=10.063; p=.000) but no group effects and no interaction (Figure 3.3F). There were significant decreases from baseline to 60 minutes (p<0.001) and from baseline to post condition (p=.003) but no significant differences from 60 minutes to post condition. 3.3.4 Shear rate-FMD relationship Changes in mean shear rate andSRauc were not significantly correlated with changes in FMD in adults. Changes in antegrade shear were not significantly correlated with FMD, however, the positive correlation was close to significance (r=.653, p=.056) in adults. Retrograde shear rate was significantly correlated with FMD (r=.759, p=.048). In children, changes in antegrade and retrograde shear rate were not significantly correlated with changes in FMD. However, there were almost significant correlations between SRauc and FMD (r=.808, p=.052) and peak mean shear rate and FMD (r=.809, p=.051). Low Pressure Cuff Condition Resting heart rate, systolic and diastolic blood pressure were measured prior to the onset of the low pressure cuff condition for both children and adults, and is reported in Table 3.5. 72 Table 3.4 Hemodynamic responses at baseline prior to the low pressure cuff condition in children and adults Baseline Adult HR (bpm) 64 ± 14 SBP (mm Hg) 125 ± 8 DBP (mm Hg) 69 ± 10 Child HR (bpm) 80 ± 11 SBP (mm Hg) 108 ± 10 DBP (mm Hg) 70 ± 7 Data are presented as Mean ± SD 3.3.5 Vasodilatory Function with Low Pressure Cuff Superficial femoral artery diameter (mm) and blood flow (ml/min) at baseline and during hyperemia are presented in Table 3.6 for the adults and children. There was a main effect for time (F(1,14)=4.977; p=.043) and a main effect for group (F(1,14)=32.438; p=.000), but no interaction for baseline diameter. Adults had a significantly larger baseline arterial diameter compared to children and baseline diameter increased with the lower pressure cuff in both children and adults. Similarly, a main effect for group (F(1,14)=31.059; p=.000) was apparent for peak diameter, with no main effects for time or interaction. Peak diameter was significantly larger in adults than children. There were main effects for time (F(1,14)=8.308; p=.012), and group (F(1,14)=8.545; p=.011), for mean blood flow, with no interaction. Mean blood flow decreased significantly following the low pressure cuff condition in children and adults. There were no main effects for group and time for peak blood flow in the low pressure cuff condition and no interaction (see Table 3.6). 73 Figure 3.4 illustrates mean as well as individual data for FMD before and after the low pressure cuff condition in the adults and children. There was a main effect for time for FMD (F(1,14)=21.339; p=.00), but no main effect for group and no interaction. FMD decreased significantly in children (4.3% to 2.58%) and adults (4.13% to 2.86%). Allometrically scaling FMD for baseline diameter showed the main effects for time (F(1,14.67)=19.27; p=.000) persisted but no main effect for group and no interaction. Scaled FMD values were 3.98 ± 1.51% to 2.74 ± 1.82 post condition in adults and 4.50 ± 1.73% to 2.74 ± 1.48% in children. 74 Figure 3.4 FMD responses Pre and Post the low pressure cuff condition in adults (A, n=9) and children (B, n=6). Data are mean ± SD. Lines represent individual data. * denotes significance p<0.05 3.3.6 Shear Rates and SRAUC There was a main effect for time for mean shear rate at baseline (F(1,14)=8.73; p=.010) and a main effect for group (F(1,14)=53.21; p=.000), but no interaction. Mean shear rate was greater in the children and decreased significantly with the low pressure cuff condition in both children and adults (Figure 3.5A). Peak mean shear rate decreased significantly with low pressure cuff * * 75 condition (F(1,14)=7.604; p=.015), with children exhibiting a greater decline post low pressure cuff condition (see Table 3.6). There was a main effect for time for SRauc (F(1,14)=6.565; p=0.023) and a main effect for group (F(1,14)=18.577; p=.000), but no interaction. SRauc was higher in children and decreased significantly following the low pressure cuff condition in children and adults (see Table 3.5). There was a main effect for time (F(1.14)=6.421; p=.024) for antegrade shear rate and a main effect for group (F(1,14)=16.037; p=.001), but no interaction (see Figure 3.5B). Antegrade shear rate was greater in children and declined significantly in both children and adults. There was a main effect for time for retrograde shear rate (F(1,14)=14.9; p=.002), a main effect for group (F(1,14)= 29.551; p=.00), and an interaction (F(1,14)=10.709; p=.006). Retrograde shear rate increased significantly with the low pressure cuff condition in both children and adults, however, there was a significantly greater increase in children (p=0.008) than adults (see Figure 3.5C). 76 Figure 3.5 Shear rate patterning baseline (Pre) and Post passive low pressure cuff condition in adults (n=9) and children (n=7). Data are mean ± SD. Black dots represent adult individual data points and black squares represent child individual data points. * denotes significance (p<0.05).* 77 Table 3.5 Superficial femoral artery diameter, blood flow and shear rate responses baseline (Pre) and Post low-pressure cuff in adults and children. Data represented as mean ± SD. Abbreviations: FMD, Flow mediated dilation; SR, Shear rate; SRAUC, Shear rate area under the curve. Bold text indicates P<0.05. Low pressure Cuff ANOVA Main Effects and Interaction Adult (n=9) Child (n=7) Pre Post Pre Post Time (p-value) Group (p-value) Group x Time (p-value) Baseline Diameter (mm) 6.39 ± 0.53 6.51 ± 0.48 4.80 ± 0.56 4.92 ± 0.58 0.043 p<0.001 0.827 Mean blood flow (ml/min) 44.2 ± 13.9 31.0 ± 14.2 64.6 ± 24.9 51.2 ± 17.3 0.012 0.011 0.468 Mean SR (1/s) 38.7 ± 19.5 23.8 ± 15.4 142.4 ± 67.4 101.1 ± 22.1 0.01 p<0.001 0.393 Peak Diameter (mm) 6.65 ± 0.54 6.69 ± 0.47 5.01 ± 0.63 5.05 ± 0.62 0.468 p<0.001 0.990 Mean blood flow (ml/min) 67.0 ± 25.6 54.7 ± 14.9 70.8 ± 27.0 61.0 ± 17.0 0.074 0.581 0.716 Mean SR (1/s) 57.1 ± 26.6 43.4 ± 14.0 141.8 ± 76.6 112.9 ± 35.7 0.015 p<0.001 0.636 SRAUC 3465 ± 1742 2609 ± 819 8478 ± 4577 6693 ± 2180 0.023 p<0.001 0.751 78 Figure 3.6 Changes in diameter, shear rate and flow are shown from baseline low pressure cuff (Pre), to one minute prior to end of condition (30min) and five minutes post low pressure cuff condition (Post) in adults (n=9) and children (n=7). Data are mean ± SD. * denotes significance from baseline to 60min (p<0.05). ** denotes significance from 60min to post (p<0.05). a denotes significance from baseline to post (p<0.05). * ** * a 79 3.3.7 Pre, during and post low pressure cuff condition comparisons A separate RM Anova analysis was done where data from baseline, one minute before end of condition (30 minutes) and post were compared for main and group effects. In both children and adults, there were main effects for time (F(1.99, 23.888)=4.971; p=.016) and group (F(1, 12)=56.236; p=.000) in mean shear rate with no interaction. There were significant decreases in mean shear rate (p=.047) from baseline to post condition with no other significant differences between time points (See Figure 3.6A). SRauc changed significantly throughout (F(1.79,21.50)=16.203; p=.000) and had group effects (F(1,12)=32.228; p=.000) with interaction (F(1.79, 21.50)=4.057; p=.036). There were significant differences from baseline to 30 minutes (p=.001) and also from 30 minutes to post condition where SRauc increased significantly post condition compared to during condition (p=.007) (See Figure 3.6C). Baseline diameter had main effects for time (F(1.84, 22.05)=4.344; p=.028) and group (F(1,12)=33.714 ; p=.000) with no interaction. There were also no significant differences between time points (Figure 3.6D). Antegrade shear rate did not observe significant main effects for time but did exhibit a group effect (F(1,12)=26.774; p=.000) with no interaction (Figure 3.6B). There were no significant differences between time points (Figure 3.6B). Retrograde shear rate had main effects for time (F(1.42, 17.00)=11.717; p=.000), group (F(1,12)=16.669; p=.002) and interaction (F(1.42, 17.00)=15.286; p=.000) (Figure 3.6F). There were significant differences from baseline to 30 80 minutes (p=.001) and from baseline to post condition (p=.023) but no significant differences between 30 min and post condition time points. Blood flow showed main effects for time (F(1.95, 23.26=3.584; p=.045) and group (F(1,12)=8.098; p=.015) but no interaction. There were no significant differences between each time points (See 3.6E). 3.3.8 Shear rate-FMD relationship Changes in mean peak shear rate, SRauc and antegrade rate were not significantly correlated with changes in FMD in adults and children. Changes in retrograde shear rate was significantly correlated with FMD (r=.710; p=.032) in adults and children (r=.834; p=.020). 3.4 Discussion In this study, the impact of shear rate manipulation on FMD responses was examined in both children and adults. We hypothesized that both low pressure cuff and passive heat condition would alter shear rate and shear patterns similarly, however, our study revealed contrasting results. Passive heat increased mean shear rate in both children and adults, with a greater increase seen in adults. Following low pressure cuff, retrograde shear rate increased in both groups with greater increases in children. However, while it was hypothesized that FMD would be altered in adults and not children, our findings showed that the increases in mean SR, SRauc and antegrade shear, alongside declines in retrograde shear with passive heat in children and adults resulted in no changes in FMD in both children and adults. In contrast, the low pressure cuff intervention resulted in declines in mean SR, SRauc, and antegrade shear, alongside 81 increases in retrograde shear and was accompanied by reductions in FMD in both children and adults. This study is the first to show that reductions in shear rate results in reductions in FMD in both children and adults, and that although passive heat did not result in an increase in FMD, there were associations between shear rate and FMD in children. 3.4.1 Passive Heat Condition The passive heat condition successfully increased mean shear rate, antegrade shear rate and blood flow and decreases in retrograde shear rate in both children and adults, as predicted. However, mean shear rate increased to a greater extent in adults, from 29.9 s-1 to 140 s-1 post passive heat whereas in children mean shear rate increased from 103.1 s-1 to 230.4 s-1. Similarly, blood flow was also increased to a greater extent in adults from 38.9 ml/min to 161.6 ml/min compared to 52.4 ml/min to 113.1 ml/min in children. This also contradicts previous passive heat studies that also show elevated shear in smaller vessels (Larson et al., 2021; Chaseling et al., 2022). However, it could be that the exposure to heat stress in children differs from that of adults and this could impact shear rate. For instance, it is shown that during passive heating, children experience greater blood flow in the back and chest but not in thigh compared to young adults (Shibasaki et al., 2001; Notley et al., 2020). While the studies on heat exposure and vascular responses are limited in children, the differences in body mass, lean muscle and adipose tissue concentration can cause differences in sweat rate, core temperature and skin blood flow experienced by children. Indeed, our findings showed that there were significant increases in heart rate during and post passive heat condition in children, with a more substantial increase noted 30 minutes into passive heat compared to post. This pattern of heart rate elevation, although relatively modest, raises concerns as it may signify the activation of thermoregulatory 82 pathways and sympathetic nervous activity that could potentially impact vascular function. Given that passive heat intervention acts as a thermal stimulus (Crandall & Wilson, 2015), the activation of the central nervous system due to heat stress could potentially explain variations in FMD post passive heat exposure. The autonomic nervous system is recognized for its role in elevating heart rate in response to heat stress, primarily aimed at redistributing blood flow to the skin while reducing flow to non-cutaneous areas (Crandall & Wilson, 2015; Cheng et al., 2018). Consequently, heart rate and blood pressure measurements were integrated into the protocol to monitor any significant changes in cardiac output resulting from heat stress. Overall, this could mean that the variability in FMD as well as the lack of change in children and adults post passive heat could be due to the role of central cardiovascular response seen from the significant changes in heart rate, a reflection of changes in core temperature. Studies have associated core temperature changes in the range of 1-1.5°C with the maximum release of NO (Kellogg, 1985), while other research suggests the release of vasoactive substances with core temperature increases of approximately 0.6°C (Low et al., 1985). The heating stimulus applied to all participants was limited to the mid-calf, and this might have caused a modest increase in core temperature, causing lack of FMD improvement. This is also observed in Cheng et al. (2021) where knee-level heating resulted in higher mean shear rate and antegrade shear rate compared to ankle-level heating in the superficial femoral artery but neither level heating led to changes in core temperature of over 0.5°C, which might be why they did not see a significant improvement in lower limb FMD. 83 Despite the significant increases in shear rate, patterning and flow, superficial femoral artery FMD response did not change significantly to the passive heat condition, even when scaled for baseline diameter. This observation challenges the prior literature that shows that increases in mean shear rate and/or antegrade shear rate leads to an improvement in FMD. The increased antegrade shear and decreased retrograde shear potentially enhancing nitric oxide bioavailability, reducing oxidative stress, exhibiting anti-inflammatory effects, and minimizing vasoconstriction (Brunt & Minson, 2021). The findings reported in this thesis do however support the variability in the FMD response to passive heat as demonstrated in various studies (Chiesa et al., 2016; Esteves et al., 2021; Crandall & Wilson, 2015) and in our findings (refer to figure 3.1). Our findings showed that increases in mean shear rate and antegrade shear rate were not significantly correlated with changes in FMD in adults and children, but the values were close to significance. This could mean that our analysis was underpowered and/or thermoregulatory mechanisms underlying heat exposure plays a role in the FMD responses seen. It is plausible that there exist thresholds for both core temperature and shear rate that need to be met to observe significant changes in vascular measures such as FMD. From our findings and previous passive heat studies (Chaseling et al., 2022; Alali et al., 2020), it appears that the previously observed dose-dependent relationship between retrograde shear rate and FMD (Thijssen et al., 2009b; Schreuder et al., 2014) does not have the same effect for antegrade shear rate and FMD, rather a threshold exists for antegrade shear rate, perhaps a threshold of over leads to vasodilation in adults (Cheng et al., 2019) which did not occur in our findings. Antegrade shear rate in our study only increased to 154 s-1 in adults. Although our data show the possibility 84 of an association between shear rate and FMD in children, defining specific shear rate thresholds while controlling for core temperature, required to elicit responses in FMD, remains a challenge. Timing of passive heat and FMD We also found that passive heating led to significantly greater increases in shear rate and blood flow during the condition than post the condition. This is in line with previous literature, where one hour into heating, blood flow was observed to be greater compared to post-condition observations in upper and lower leg heating protocols (Esteves et al., 2021). We also found that baseline diameter did not change during condition but did significantly decrease post condition in adults and children (p<0.001). This change post condition was also seen with mean shear rate, SRauc, shear patterning and flow. The decline in diameter could be the effect of thermoregulatory mechanisms regulating skin temperature post condition when limb is out of water bath. Furthermore, the findings from Chaseling et al. (2022) suggest that the improvement in FMD following passive heating might not be solely governed by mean shear rate or SRauc. This is indicated by a significant association between FMD and time. Studies assessing FMD responses within 30 minutes after the end of heating have consistently shown marked improvements in FMD (Chaseling et al., 2022; Cheng et al., 2021; Romero et al., 2017). In Chaseling et al. (2022), although mean shear rate and antegrade shear rate began to recover 5 minutes after the end of heating, the increase in FMD was only significant 30 minutes post-condition. Perhaps our FMD findings might improve if taken thirty minutes post end of condition. 85 3.4.2 Low Pressure Cuff Condition This intervention aimed to investigate whether reductions in shear stimulus and altered flow would impair endothelial function. In contrast to the passive heat condition, the low-pressure cuff intervention, deliberately increasing retrograde shear rate, resulted in a notable decrease in FMD in both children and adults, as anticipated. All flow and shear-related variables significantly decreased post-condition and retrograde shear rate increased, which aligned with a consequential decline in FMD, affirming that increases in retrograde shear rate can indeed impede the superficial femoral artery FMD in both adults and children. These observations substantiate previous research where the adverse effects of reduced mean shear rate and increased retrograde shear rate has been shown on FMD following low pressure cuff interventions (Thijssen et al., 2009b; Schreuder et al., 2014; Tremblay et al., 2019; Totosky et al., 2014). Furthermore, this study validates applying a 60mm Hg pressure cuff in adults and a 40mm Hg cuff in children results in significant declines in flow, shear rates, increases in retrograde shear, and a decline in FMD. Therefore, our success in using a low-pressure cuff to manipulate shear rate in the superficial femoral artery confirms its efficacy in eliciting the necessary alterations in shear patterning to induce impairments in FMD. Our findings showed a significant increase in retrograde shear rate following 30 minutes of a low pressure cuff in both children and adults. This elevation in retrograde shear rate was more pronounced in children. It was expected that retrograde shear rate would increase, but we predicted that it would increase similarly in adults and children. It is also important to note that the increase in retrograde shear rate was also accompanied by decreases in mean and anterograde shear rate in both adults and children. This is unlike a previous child study that showed 86 prolonged sitting for three hours caused no significant differences in mean shear rate and retrograde shear rate (McManus et al., 2015). This could mean that prolonged sitting does not lead to shear mediated declines in FMD, and is most likely due to changes in blood viscosity, blood pooling or increases in muscle sympathetic activity that leads to decline in FMD. We also found a significant correlation between changes in retrograde shear rate and changes in FMD in both children and adults. While such relationships are often documented in adults in existing literature (Thijssen et al., 2009; Totosy et al., 2014; Schreuder et al., 2014), this association remains unexplored in the pediatric literature. Our study shows that even in children, an association exists between increased retrograde shear rate and a decline in FMD, implying similar vasomotor mechanisms between children and adults when exposed to reduced shear stimulus. The low NO bioavailability associated with reduced shear stress is a possible reason for the decline in FMD in children and adults. Perhaps with higher cuff pressures, it could also be possible to see a similar dose dependent relationship with retrograde shear rate and FMD, as seen in adults (Thijssen et al., 2009). However, an unresolved aspect of this intervention is whether increased retrograde shear rate alone is adequate to cause the decline in FMD, or if decreases in mean shear rate can also lead to decline in FMD. These variations in shear patterning hold significance as they can influence critical mechanisms for vessel adaptation, often discussed in similar low-pressure cuff studies. The intervention, causing reduced blood flow to the leg and subsequent FMD impairment, presumably leads to increased endothelin-1 expression, decreased eNOS expression, and activation of cellular pathways stimulating reactive oxygen species (ROS) production, inducing 87 oxidative stress (Tinken et al., 2009). The substantial declines observed in SRauc, mean shear rate, and antegrade shear rate in our study diverge from findings in literature concerning low-pressure cuff interventions. For instance, Totosy et al. (2014) demonstrated a significant decrease in FMD following a low-pressure cuff condition in the superficial femoral artery. However, their study did not show a significant decline in mean shear rate after a 75mm Hg cuff condition; instead, they observed a significant increase in antegrade shear rate. Nonetheless, the mechanisms behind differences in shear patterning leading to impaired endothelial function should be investigated further in adults but especially in children. Our findings might have been influenced by smooth muscle cells, as they can also be impacted by decreased shear stress within the artery, stimulating mechanisms that cause structural alterations in the artery (Green et al., 2017). This study did not incorporate the use of glyceryl trinitrate (GTN) to evaluate endothelium-independent dilation, as discussed by Thijssen et al. (2017). Therefore, we cannot rule out that the observed alterations in FMD could also be attributed to the involvement of vascular smooth muscle cells. It is important to note that the intervention involving the low-pressure cuff to investigate peripheral artery changes, conducted by Anderson et al. (1989), laid the foundation for the FMD approach utilized today, established by Celermajer et al. (1992). Hence, it is plausible to speculate that the consistent observations of reduced FMD in studies employing the low-pressure cuff, irrespective of varying mean and antegrade shear patterns, might be attributed to bias within the methodology itself. 88 3.5 Limitations Some limitations of this study include the relatively small sample size in both child and adult cohorts, limiting the generalizability of the results. Further studies with larger populations are necessary to confirm these findings. Our study only includes male participants. An initial desire was to recruit an equal number of male and female participants. We had difficulties recruiting girl participants and with time constraints ended up only testing males. Future studies should recuit females too and examine sex differences in the FMD response to shear rate manipulation. The choice of the superficial femoral artery for assessment might not represent the overall vascular response. Studying multiple vascular sites, and comparing results would provide a more comprehensive understanding of the relationship between shear rate and FMD. This is observed in studies like Cheng et al., (2021) where brachial and superficial femoral arteries showed different FMD responses to passive heating. Furthermore, this study did not measure endothelium-independent dilation, which means that the observed FMD responses might also be influenced by smooth muscle mechanisms. 89 Chapter 4: Conclusion This study provides valuable insights into the complex relationship between shear rate and FMD in both children and adults. The results suggest that increasing shear rate alone, through passive heating, may not be sufficient to induce significant improvements in FMD. On the other hand, the reduction of shear rate through a low pressure cuff intervention led to a decline in FMD. These findings suggest that a careful balance of shear rate, including both antegrade and retrograde components, may be necessary for optimal endothelial function. The study did not find age-dependent differences in FMD responses, suggesting that children and adults may exhibit similar endothelial sensitivities to manipulations in flow and shear rate in the superficial femoral artery. 4.1 Strengths and Limitations of the Experimental Design The strengths of this thesis originate from insights gained in the pilot study, while certain limitations identified in Chapter Two contributed to the refinement of the main study design. An important limitation in the pilot study pertained to uncontrolled posture movements among participants, potentially influencing the collected data. In response, a researcher was stationed in the lab during the intervention to ensure adherence to the protocol, particularly during low-pressure cuff application and passive heating. Although the heat pad used in the pilot study might not have consistently produced the same stimulus and posed difficulties in wrapping around the upper thigh, employing a water bath in the main study ensured standardized stimuli for both children and adults, enhancing the homogeneity of the intervention. 90 Initially aiming to investigate sex-based differences within the pediatric group, recruiting prepubertal girls for the study proved challenging, necessitating a shift to a same-sex study design due to time constraints. Although this precluded exploring sex-related disparities in the shear-FMD relationship, earlier research findings did not reveal significant differences between boys and girls post passive heating (Tochihara et al., 1995). Additionally, vascular developmental data suggested no sex-based differences in vessels before 16 years of age (Sarkola et al., 2012). Similarly, research in adults by Chaseling et al. (2022) did not highlight statistically significant sex-based differences in the acute effects of heating on brachial artery FMD. Nonetheless, it remains pertinent to conduct dedicated sex differences studies to corroborate these earlier findings in children. Despite efforts to address scanning issues and data loss encountered in the pilot study by implementing stricter guidelines for FMD scans, marking where ultrasound probe was on participants after baseline measurements to measure the same site post condition, etc., challenges persisted in obtaining clear scans and analyzing the data in the main study. It seems that children and some adults find the FMD procedure uncomfortable and are unable to keep their leg relaxed during the FMD recordings, especially during the occlusion period. This causes movements in the probe, and changes in angle which in turn leads to more issues during analysis. While it would be nice to have shear and FMD measured in the contralateral limb, it was not feasible for the study and research design as this would mean that an additional scanner would have to simultaneously scan with condition leg scanner. While it is already difficult to scan one limb, we felt including another limb in the protocol could lead to more errors, either with 91 adherence to protocol itself or the scanning. Furthermore, a brief literature review and the pilot study showed that contralateral control limb for low pressure cuff or passive heat condition might not be ideal, since there are variances in both limbs and the main objective of this study is to localize the shear stimulus to one limb and observe its effects pre and post condition. For this objective, we felt that data from contralateral limb was not needed. There is evidence to show that the contralateral limb does not affect the condition limb and vice versa. For instance, one study showed that inducing hyperthermia in one limb can lead to notable alterations in flow and arterial diameter, while leaving the non-heated limb unaffected (Esteves et al., 2021). Similarly, Restaino et al., (2016) also showed that the passive heating of one leg for three hours had no positive effects on the contralateral limb. During this time, the condition leg experienced substantial increases in shear rate and flow, along with improvements in FMD. In contrast, the control leg exhibited significant decreases in shear rate, primarily attributed to the effects of prolonged sitting on the popliteal artery (Restaino et al., 2016). These findings underscore the clear and localized nature of the effects induced by passive heating interventions, with minimal impact on the contralateral limb. One notable aspect of passive heat studies is the variation in core temperature measurements. Some studies define heat stimulus based on the increase in the core temperature of participants whether rectal or esophageal (Chiesa et al., 2016; Larson et al., 2021), while others determine heat stimulus according to the temperature of the water bath or skin temperature (Cheng et al., 2021). This difference in approach does mean that these studies are exposing participants to either higher or lower heat stress which introduces the involvement of the sympathetic nervous system in participants, and this may potentially contribute to the inconsistencies in FMD 92 responses to passive heating (Chiesa et al., 2016). Notably, approximately 60% of the increase in heart rate in response to heat stress is attributed to sympathetic nervous activity (Crandall & Wilson, 2015). Our findings did show an increase in heart rate 30 minutes into the passive heat condition, especially in children. This means we were not successful in preventing a central response elicited by the heat stimulus and this is a limitation that should be addressed in future studies looking at passive heating in children. 4.2 Implications and Future Directions This study contributes significantly to the limited data available in children with regards to conduit artery function. The investigation into the shear-FMD relationship across age groups, albeit with a small sample size, has shown responses were similar in children and adults. Prior literature suggested developmentally different vasomotor control, which no relationship between shear rate and FMD (Thijssen et al., 2009) . Given the heightened retrograde shear rate observed in children during low-pressure cuff intervention and the lack of FMD change to passive heat, it would be intriguing to combine these interventions in children to observe endothelial responses to varying shear patterns while maintaining a constant mean shear rate. This experimental approach has been undertaken in studies involving adults, such as the work by Alali et al. (2020). Replicating this investigation in children could elucidate whether the increased shear seen in children, regulated by the cuff, results in improved FMD. This study could perhaps provide more of an insight into shear-FMD relationship than just looking at passive heating alone. 93 Additionally, this study focused on acute interventions to manipulate shear rate. Examining the long-term effects of repeated passive heating or low-pressure cuff interventions on vascular health and FMD would be beneficial. However, scheduling repeated visits in children can be challenging, necessitating both child and parent availability. 94 Bibliography Abbassi, V. (1998). Growth and normal puberty. Pediatrics, 102(2 Pt 3), 507-1 Aggarwal, R., Yeh, R. W., Joynt Maddox, K. E., & Wadhera, R. K. (2023). 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Thesis/Dissertation
eng
Health and Exercise Sciences
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
McManus, A. M. (Alison M.)
Examining the effects of shear rate on flow mediated dilation in children and adults
Text