The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

The present series of in vivo experiments in healthy subjects, were performed to investigate wall stiffness in peripheral vessels and how this modality adapts to iterative increments or sustained reductions in local intravascular pressures. Vascular stiffness was measured as changes in arterial and venous diameters, and in arterial flow, during graded increments in distending pressures in the vasculature of an arm or a lower leg. In addition, effects of intravascular pressure elevation on flow characteristics in veins, and on limb pain were elucidated. Arteries and veins were stiffer (i.e. pressure distension was less) in the lower leg than in the arm. The pressure-induced increase in arterial flow was substantially greater in the arm than in the lower leg, indicating a greater stiffness in the arterioles of the lower leg. Prolonged reduction of intravascular pressures in the lower body, induced by 5 wks of sustained horizontal bedrest (BR), decreased stiffness in the leg vasculature. BR increased pressure distension in the tibial artery threefold and in the tibial vein by 86 %. The pressure-induced increase in tibial artery flow was greater post bedrest, indicating reduced stiffness in the arterioles of the lower leg. Intermittent increases of intravascular pressures in one arm (pressure training; PT) during a 5-wk period decreased vascular stiffness. Pressure distension and pressure-induced flow in the brachial artery were reduced by about 50 % by PT. PT reduced pressure distension in arm veins by 30 to 50 %. High intravascular pressures changed venous flow to arterial-like pulsatile patterns, reflecting propagation of pulse waves from the arteries to the veins either via the capillary network or through arteriovenous anastomoses. High vascular pressures induced pain, which was aggravated by BR and attenuated by PT; the results suggest that the pain was predominantly caused by vascular overdistension. In conclusion, vascular wall stiffness constitutes a plastic modality that adapts to meet demands imposed by a change in the prevailing local intravascular pressure. That increased intravascular pressure leads to increased arteriolar wall stiffness supports the notion that local pressure load may serve as a “prime mover” in the development of vascular changes in hypertension. / medicine doktorsexamen QC 20101109

Smoking and environmental tobacco smoke (ETS) exposure are major risk factors for cardiovascular disease (CVD), although the exact components and pathophysiological mechanisms responsible for this association remain unclear. Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental contaminants that form during organic material combustion and are thus found in cigarette smoke, vehicle exhaust particles, and air pollution. We hypothesize that PAHs are key agents responsible for mediating the cigarette smoke effects in the cardiovascular system, including increased oxidative stress, inflammation, and arterial stiffness.<p> Arterial stiffness is a powerful, independent predictor of cardiovascular risk and is regulated, in part, by vasoactive mediators derived from the endothelium. The first objective of this project was to determine whether pulse wave dP/dt collected from radiotelemetry-implanted rats is a reliable indicator of changes in arterial stiffness following administration of vasoactive drugs or acute ETS exposure. Anaesthetized rats were administered a single dose of saline (vehicle control), acetylcholine, norepinephrine, and N(G)-nitro-L-arginine methyl ester (L-NAME) via the tail vein, allowing a washout period between injections. Acetylcholine decreased and norepinephrine increased dP/dt compared to saline vehicle. Injection of the nitric oxide (NO) synthase inhibitor L-NAME decreased plasma nitrate/nitrite (NOx), but transiently increased dP/dt. For the ETS experiment, rats were exposed for one hour to sham, low dose ETS, or high dose ETS. Exposure to ETS did not significantly alter dP/dt or plasma endothelin-1 (ET-1) levels, but increased plasma NOx levels at the high ETS exposure and increased plasma nitrotyrosine levels in both ETS groups. In conclusion, acute changes in NO production via acetylcholine or L-NAME alter the arterial pulse wave dP/dt consistently with the predicted changes in arterial stiffness. Although acute ETS appears to biologically inactivate NO, a concomitant increase in NO production at the high ETS exposure may explain why ETS did not acutely alter dP/dt.<p> The second objective of this project was to compare the effects of subchronic ETS and BaP exposure on circadian blood pressure patterns, arterial stiffness, and possible sources of oxidative stress in radiotelemetry-implanted rats. Pulse wave dP/dt was used as an indicator of arterial stiffness, and was compared to both structural (wall thickness) and functional (NO production and bioactivity, ET-1 levels) features of the arterial wall. In addition, histology of lung, heart, and liver were examined as well as pulmonary and hepatic detoxifying enzyme activity (cytochrome P450 specifically CYP1A1). Daily ETS exposure for 28 days altered the circadian pattern of heart rate and blood pressure in rats, with a loss in the normal dipping pattern of blood pressure during sleep. Subchronic ETS exposure also increased dP/dt in the absence of any structural modifications in the arterial wall. Although NO production and ET-1 levels were not altered by ETS, there was increased biological inactivation of NO via peroxynitrite production (as indicated by increased plasma nitrotyrosine levels). Thus, vascular stiffness and failure of blood pressure to dip precede structural changes in rats exposed to ETS for 28 days. Exposure to ETS also caused increased number of lung neutrophils as well as increased CYP1A1 activity in lung microsomes.<p> Since ETS-induced increases in arterial stiffness occurred as early as day 7, radiotelemetry-implanted rats were exposed daily to intranasal BaP for 7 days. Similar to ETS, BaP exposure altered circadian blood pressure patterns and reduced blood pressure dipping during sleep. Thus, in support of part of our hypothesis, the PAH component of cigarette smoke may be responsible for the ETS-induced increase in blood pressure and the loss of dipping pattern during sleep. Increased neutrophil recruitment was observed in the lungs of both ETS- and BaP-exposed rats, suggesting that lung inflammatory reactions may be involved in the disruption of circadian blood pressure rhythms. Unlike ETS however, BaP exposure did not significantly alter pulse wave dP/dt, endothelial function, or lung CYP1A1 activity. Thus, contrary to our hypothesis, the reduction in NO bioactivity and increased arterial stiffness caused by ETS cannot be explained by BaP at the dose and length of the exposure in the current study. Production of reactive metabolites in the lung following ETS exposure may be responsible, at least in part, for the increases in oxidative stress in the vasculature, leading to reduced NO bioactivity and increased arterial stiffness. Oxidative stress caused by BaP exposure may have been insufficient to reduce NO bioactivity in the peripheral vasculature. Therefore arterial stiffness was not increased and factors other than NO may be responsible for the increase in blood pressure observed with ETS and BaP exposure.

An age-related decline in cerebral blood flow (CBF) is widely acknowledged. However, uncertainty exists as to whether this reduction is the result of a reduced metabolic demand (cerebral atrophy) or an impaired delivery system (cerebrovascular disease). The purpose of these experiments was to examine the relationship of CBF and dynamic cerebrovascular regulation with changes in common carotid intima-media thickness (cIMT), brachial-ankle pulse wave velocity (baPWV) and common carotid distensibility. Additionally, we took an exploratory view into the effect of vascular aging and CBF reduction on brain function, as expressed through the performance of motor and cognitive tasks. An important finding in elderly participants was that seated anterior CBF declined as a function of arterial stiffness, independently of age. Linear regression analysis developed a model that predicts CBF drops 22 ml/min (95% confidence interval (CI): 6, 38) for each 100 cm/s increase in baPWV and 8 ml/min (95% CI: 1, 15) for each additional year in age. The effect of baPWV appears to be mediated through an increase in cerebrovascular resistance (r2 = 0.84, p < 0.0001). Additionally, CBF showed postural dependency and the volume of the drop in CBF between supine and seated positions was greatest in elderly participants (YOUNG: 65 ± 81 ml/min; ELDERLY: 155 ± 119 ml/min; p = 0.001). Despite this negative impact of vascular aging on steady state flow, dynamic regulation does not appear to be affected. Cerebrovascular responses to an acute drop in blood pressure or to activation of the motor cortex were not attenuated in the elderly participants. Finally, seated CBF had modest directionally relevant relationships with perceptuo-motor and complex sequencing processes; while cIMT appeared to influence performance on initiation and inhibition tasks.

An age-related decline in cerebral blood flow (CBF) is widely acknowledged. However, uncertainty exists as to whether this reduction is the result of a reduced metabolic demand (cerebral atrophy) or an impaired delivery system (cerebrovascular disease). The purpose of these experiments was to examine the relationship of CBF and dynamic cerebrovascular regulation with changes in common carotid intima-media thickness (cIMT), brachial-ankle pulse wave velocity (baPWV) and common carotid distensibility. Additionally, we took an exploratory view into the effect of vascular aging and CBF reduction on brain function, as expressed through the performance of motor and cognitive tasks. An important finding in elderly participants was that seated anterior CBF declined as a function of arterial stiffness, independently of age. Linear regression analysis developed a model that predicts CBF drops 22 ml/min (95% confidence interval (CI): 6, 38) for each 100 cm/s increase in baPWV and 8 ml/min (95% CI: 1, 15) for each additional year in age. The effect of baPWV appears to be mediated through an increase in cerebrovascular resistance (r2 = 0.84, p < 0.0001). Additionally, CBF showed postural dependency and the volume of the drop in CBF between supine and seated positions was greatest in elderly participants (YOUNG: 65 ± 81 ml/min; ELDERLY: 155 ± 119 ml/min; p = 0.001). Despite this negative impact of vascular aging on steady state flow, dynamic regulation does not appear to be affected. Cerebrovascular responses to an acute drop in blood pressure or to activation of the motor cortex were not attenuated in the elderly participants. Finally, seated CBF had modest directionally relevant relationships with perceptuo-motor and complex sequencing processes; while cIMT appeared to influence performance on initiation and inhibition tasks.

Smoking and environmental tobacco smoke (ETS) exposure are major risk factors for cardiovascular disease (CVD), although the exact components and pathophysiological mechanisms responsible for this association remain unclear. Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental contaminants that form during organic material combustion and are thus found in cigarette smoke, vehicle exhaust particles, and air pollution. We hypothesize that PAHs are key agents responsible for mediating the cigarette smoke effects in the cardiovascular system, including increased oxidative stress, inflammation, and arterial stiffness.<p> Arterial stiffness is a powerful, independent predictor of cardiovascular risk and is regulated, in part, by vasoactive mediators derived from the endothelium. The first objective of this project was to determine whether pulse wave dP/dt collected from radiotelemetry-implanted rats is a reliable indicator of changes in arterial stiffness following administration of vasoactive drugs or acute ETS exposure. Anaesthetized rats were administered a single dose of saline (vehicle control), acetylcholine, norepinephrine, and N(G)-nitro-L-arginine methyl ester (L-NAME) via the tail vein, allowing a washout period between injections. Acetylcholine decreased and norepinephrine increased dP/dt compared to saline vehicle. Injection of the nitric oxide (NO) synthase inhibitor L-NAME decreased plasma nitrate/nitrite (NOx), but transiently increased dP/dt. For the ETS experiment, rats were exposed for one hour to sham, low dose ETS, or high dose ETS. Exposure to ETS did not significantly alter dP/dt or plasma endothelin-1 (ET-1) levels, but increased plasma NOx levels at the high ETS exposure and increased plasma nitrotyrosine levels in both ETS groups. In conclusion, acute changes in NO production via acetylcholine or L-NAME alter the arterial pulse wave dP/dt consistently with the predicted changes in arterial stiffness. Although acute ETS appears to biologically inactivate NO, a concomitant increase in NO production at the high ETS exposure may explain why ETS did not acutely alter dP/dt.<p> The second objective of this project was to compare the effects of subchronic ETS and BaP exposure on circadian blood pressure patterns, arterial stiffness, and possible sources of oxidative stress in radiotelemetry-implanted rats. Pulse wave dP/dt was used as an indicator of arterial stiffness, and was compared to both structural (wall thickness) and functional (NO production and bioactivity, ET-1 levels) features of the arterial wall. In addition, histology of lung, heart, and liver were examined as well as pulmonary and hepatic detoxifying enzyme activity (cytochrome P450 specifically CYP1A1). Daily ETS exposure for 28 days altered the circadian pattern of heart rate and blood pressure in rats, with a loss in the normal dipping pattern of blood pressure during sleep. Subchronic ETS exposure also increased dP/dt in the absence of any structural modifications in the arterial wall. Although NO production and ET-1 levels were not altered by ETS, there was increased biological inactivation of NO via peroxynitrite production (as indicated by increased plasma nitrotyrosine levels). Thus, vascular stiffness and failure of blood pressure to dip precede structural changes in rats exposed to ETS for 28 days. Exposure to ETS also caused increased number of lung neutrophils as well as increased CYP1A1 activity in lung microsomes.<p> Since ETS-induced increases in arterial stiffness occurred as early as day 7, radiotelemetry-implanted rats were exposed daily to intranasal BaP for 7 days. Similar to ETS, BaP exposure altered circadian blood pressure patterns and reduced blood pressure dipping during sleep. Thus, in support of part of our hypothesis, the PAH component of cigarette smoke may be responsible for the ETS-induced increase in blood pressure and the loss of dipping pattern during sleep. Increased neutrophil recruitment was observed in the lungs of both ETS- and BaP-exposed rats, suggesting that lung inflammatory reactions may be involved in the disruption of circadian blood pressure rhythms. Unlike ETS however, BaP exposure did not significantly alter pulse wave dP/dt, endothelial function, or lung CYP1A1 activity. Thus, contrary to our hypothesis, the reduction in NO bioactivity and increased arterial stiffness caused by ETS cannot be explained by BaP at the dose and length of the exposure in the current study. Production of reactive metabolites in the lung following ETS exposure may be responsible, at least in part, for the increases in oxidative stress in the vasculature, leading to reduced NO bioactivity and increased arterial stiffness. Oxidative stress caused by BaP exposure may have been insufficient to reduce NO bioactivity in the peripheral vasculature. Therefore arterial stiffness was not increased and factors other than NO may be responsible for the increase in blood pressure observed with ETS and BaP exposure.

Background Anthropogenic air pollution is a global health problem estimated to contribute to millions of premature deaths. Exposure to biomass smoke is common due to varying sources, such as wildfires, indoor cooking over open fires, and residential heating from wood stoves. In urban environments transportation and industry rely heavily on the combustion of fossil fuels yet environmental policies increasingly support a shift to renewable fuels such as biodiesel. It has not been investigated how either wood smoke or biodiesel exhaust affect human health in general or the cardiovascular system in particular. We hypothesized that wood smoke exposure would induce acute cardiovascular impairment via similar underlying mechanisms as have been established for petrodiesel exhaust exposure. We also hypothesized that replacing petrodiesel with biodiesel, as a blend or pure biodiesel, would generate an exhaust profile with a less harmful effect on the cardiovascular system than petrodiesel exhaust. Methods In four separate studies healthy non-smoking subjects were exposed to different air pollutants in controlled exposure chambers followed by clinical investigations of the cardiovascular system. All studies were performed as randomized controlled trials in a crossover fashion with each individual acting as her own control. In study I healthy volunteers were exposed to wood smoke at a target concentration of particulate matter (PM) 300 µg/m3 for three hours followed by measures of blood pressure, heart rate variability and central arterial stiffness. In study II subjects were exposed to wood smoke at a target concentration of PM 1000 µg/m3 for one hour followed by measures of thrombus formation using the Badimon technique and vasomotor function using forearm venous occlusion plethysmography. In study III subjects were exposed to petrodiesel exhaust and a 30% rapeseed methyl ester (RME30) biodiesel blend for one hour at a target concentration of PM 300 µg/m3. Following exposure, thrombus formation and vasomotor function were assessed as in study II. In study IV subjects were exposed to petrodiesel exhaust at a target concentration of PM 300 μg/m3for one hour and pure rapeseed methyl ester (RME100) exhaust generated at identical running conditions of the engine. Following exposure, thrombus formation and vasomotor function were assessed as in study II and III. Results In study I fourteen subjects (8 males) were exposed to wood smoke at P M 294±36 μg/m3. Compared to filtered air exposure, measures of central arterial stiffness were increased and heart rate variability was decreased following wood smoke exposure. No effect was seen on blood pressure. In study II sixteen males were exposed to wood smoke at PM 899±100 μg/m3. We found no evidence of increased thrombus formation or impaired vasomotor function following wood smoke exposure. In study III sixteen subjects (14 males) were exposed to petrodiesel exhaust (PM 314±27 µg/m3) and RME30 exhaust (PM 309±30 µg/m3). Thrombus formation and vasomotor function were equal following either exposure. In study IV nineteen males were exposed to petrodiesel exhaust (PM 310±34 µg/m3, 1.7±0.3 x105 particles/cm3) and RME100 exhaust (PM 165±16 µg/m3, 2.2±0.1 x105 particles/cm3). As in study III, thrombus formation and vasomotor function were identical following both exposures. Conclusions We have for the first time demonstrated that wood smoke exposure can increase central arterial stiffness and decrease heart rate variability in healthy subjects. We did not, however find evidence of increased thrombus formation and impaired vasomotor function following wood smoke exposure at a higher concentration for a shorter time period. We have, for the first time, demonstrated that exhaust from RME biodiesel induced acute adverse cardiovascular effects of increased thrombus formation and impaired vasomotor function in man. These effects are on par with those seen following exposure to petrodiesel exhaust, despite marked physicochemical differences of the exhaust characteristics.

In today’s aging population, cerebrovascular health plays a pivotal role in maintaining independence. The identification of early markers of change might help to plan more appropriate preventative and/or therapeutic measures. Recent focus has been placed on the relationship between peripheral vascular characteristics and cerebral hemodynamics. Given the compliant nature of the cerebral circulation, examination of passive properties, including critical closing pressure (CrCP) and resistance area product (RAP), might provide sensitive information about early functional changes. The purpose of this thesis was to provide a comprehensive view of peripheral vascular and cerebrovascular regulation in community-living older adults. In doing so, the thesis covered a spectrum, ranging from an examination of lifestyle factors, including habitual physical activity and sleep quality, to the impact of cerebrovascular health on functional status, characterized by gait speed. Key findings included the observation that while participants showed the ability to regulate cerebral blood flow (CBF) appropriately in most circumstances, the underlying mechanisms used to achieve this regulation was dependent on baseline vascular tone. During sit-to-stand transitions, individuals with lower baseline resistance relied primarily on fluctuations in RAP, which have been suggested to more closely reflect myogenic pathways. In contrast, individuals with elevated resistance had lower baseline CBF and relied relatively more on fluctuations in CrCP during the dynamic transition. The greater reliance on CrCP might indicate that these individuals were required to tap further into reserve pools to avoid hypoperfusion during the transition. Notably, those who exhibited a smaller dynamic RAP response during the posture change also had slower gait speed and higher occurrence of falls over the past year. These results provide evidence that passive cerebrovascular dynamics are sensitive markers linking peripheral and cerebrovascular properties with functional consequences for brain health in the elderly.

In today’s aging population, cerebrovascular health plays a pivotal role in maintaining independence. The identification of early markers of change might help to plan more appropriate preventative and/or therapeutic measures. Recent focus has been placed on the relationship between peripheral vascular characteristics and cerebral hemodynamics. Given the compliant nature of the cerebral circulation, examination of passive properties, including critical closing pressure (CrCP) and resistance area product (RAP), might provide sensitive information about early functional changes. The purpose of this thesis was to provide a comprehensive view of peripheral vascular and cerebrovascular regulation in community-living older adults. In doing so, the thesis covered a spectrum, ranging from an examination of lifestyle factors, including habitual physical activity and sleep quality, to the impact of cerebrovascular health on functional status, characterized by gait speed. Key findings included the observation that while participants showed the ability to regulate cerebral blood flow (CBF) appropriately in most circumstances, the underlying mechanisms used to achieve this regulation was dependent on baseline vascular tone. During sit-to-stand transitions, individuals with lower baseline resistance relied primarily on fluctuations in RAP, which have been suggested to more closely reflect myogenic pathways. In contrast, individuals with elevated resistance had lower baseline CBF and relied relatively more on fluctuations in CrCP during the dynamic transition. The greater reliance on CrCP might indicate that these individuals were required to tap further into reserve pools to avoid hypoperfusion during the transition. Notably, those who exhibited a smaller dynamic RAP response during the posture change also had slower gait speed and higher occurrence of falls over the past year. These results provide evidence that passive cerebrovascular dynamics are sensitive markers linking peripheral and cerebrovascular properties with functional consequences for brain health in the elderly.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.