The Structural and Functional Changes of Blood Vessels during Aging

The vascular adventitia is recognized as a dynamic mediator of vascular structure and function, yet its role in aging is not understood. The purpose of this thesis was to examine the age-related changes of the vascular adventitia and determine the underlying mediators responsible. Male Sprague-Dawley rats were aged to 15, 30, 50 and 80 weeks before being anesthetised and euthanized by exsanguination. Thoracic aortas, mesenteric and pudental arteries were isolated, formalin fixed, and embedded in paraffin then sectioned at 5μm. Vessels were examined by microscopy and protein expression was determined by indirect immunofluorescence. The thickness of the adventitia increased dramatically with age. Immunofluorescence revealed a robust expression of endothelin system proteins in the adventitia. Additionally, extracellular matrix proteins collagen and fibronectin, and the proliferation marker Ki67 showed strong adventitial origin. The changes observed in the vascular adventitia with aging clearly demonstrate an important role in the process of vascular aging.

The structural and functional changes of blood vessels during aging

The vascular adventitia is recognized as a dynamic mediator of vascular structure and function, yet its role in aging is not understood. The purpose of this thesis was to examine the age-related changes of the vascular adventitia and determine the underlying mediators responsible. Male Sprague-Dawley rats were aged to 15,30,50 and 80 weeks before being anesthetised and euthanized by exsanguination. Thoracic aortas, mesenteric and pudental arteries were isolated, formalin fixed, and embedded in paraffin then sectioned at 51lm. Vessels were examined by microscopy and protein expression was determined by indirect immunofluorescence. The thickness of the adventitia increased dramatically with age. Immunofluorescence revealed a robust expression of endothelin system proteins in the adventitia. Additionally, extracellular matrix proteins collagen and fibronectin, and the proliferation marker Ki67 showed strong adventitial origin. The changes observed in the vascular adventitia with aging clearly demonstrate an important role in the process of vascular aging.

Expression and function of endothelin and its receptors in vascular adventitial fibroblasts

Objective: The adventitia has been recognized to play important roles in vascular oxidative stress, remodelling and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin-1 (ET-1) in response to angiotensin II (ANG II). However, the mechanisms by which ANG II induces ET-1 expression are unknown. It is also unclear whether the ET-1 receptors are expressed in the adventitia. We therefore examined the role of oxidative stress in the regulation of ET-1. We also investigated the expression of both the ETA and ETB receptors and the roles of these two types of receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Methods and Results: Adventitial fibroblasts were isolated and cultured from the thoracic mouse aorta. Cells were treated with ANG II (lOOnM), ET-1 (lOpM), NADPH oxidase inhibitor apocynin (lOOfiM), the superoxide anion scavenger tempol (lOOfiM), the ANG II receptor antagonists (100[aM), losartan (AT| receptor) and PD 1233 19 (AT2 receptor), the ET-1 receptor antagonists (lOOuM), BQ123 (ETA receptor) and BQ788 (ETB receptor), and the ETB receptor agonist (lOOnM) Sarafotoxin 6C. ET-1 peptide levels were determined by ELISA, while ETA ,ETB and collagen levels were determined by Western blot. ANG II increased ET-1 peptide levels in a time-dependent manner reaching significance when incubated for 24 hours. NAD(P)H oxidase inhibitor, apocynin, as well as the superoxide scanverger, tempol, significantly reduced ANG Il-induced ET-1 peptide levels while over-expression of SOD1 (endogenous antioxidant enzyme) significantly decreased ANG Il-induced collagen I expression, therefore implicating reactive oxygen species in the mediation of ET-1. ANG II increased ETA receptor protein as well as collagen in a similar fashion, reaching significance after 4, 6, and 24 hours treatment. ANG II induced collagen was reduced while in the presence of the ETA receptor antagonist suggesting the role of the ETa receptor in the regulation of the extracellular matrix. ANG II treatment also increased ETB receptor protein levels in a time-dependent manner. ANG II treatment in the presence of the ETB receptor antagonist significantly increased ET-1 peptide levels. On another hand, the ETB receptor agonist, Sarafotoxin 6C, significantly decreased ET-1 peptide levels. These data implicate the role of the ETb receptor in the clearance of the ET-1 peptide. Conclusion: ANG II-induced increases of ET-1 peptide appears to be mediated by reactive oxygen species derived from NAD(P)H oxidase. Both the ETA and ETB receptors are expressed in adventitial fibroblasts. The ETA receptor subtype mediates collagen I expression, while the ETB receptor may play a protective role through increasing the clearance of the ET- 1 peptide.

Mechanisms of endothelin-1 induced reactive oxygen species production in vascular adventitial fibroblasts

With the relationship between endothelin-1 (ET-1) stimulation and reactive oxygen species (ROS) production unknown in adventitial fibroblasts, I examined the ROS response to ET-1 and angiotensin (Ang II). ET-1 -induced ROS peaked following 4 hrs of ET-1 stimulation and was inhibited by an ETA receptor antagonist (BQ 123, 1 uM) an extracellular signal-regulated kinase (ERK) 1/2 inhibitor (PD98059, 10 uM), and by both a specific, apocynin (10 uM), and non-specific, diphenyleneiodonium (10 uM), NAD(P)H oxidase inhibitor. NOX2 knockout fibroblasts did not produce an ET-1 induced change in ROS levels. Ang II treatment increased ROS levels in a biphasic manner, with the second peak occurring 6 hrs following stimulation. The secondary phase of Ang II induced ROS was inhibited by an ATi receptor antagonist, Losartan (100 uM) and BQ 123. In conclusion, ET-1 induces ROS production primarily through an ETA-ERKl/2 NOX2 pathway, additionally, Ang II-induced ROS production also involves an ETa pathway.

The roles of ERK1/2 and PI3K in abnormal vascular functions in angiotensin II-infused hypertensive rats

Background: Ang II plays a major role in cardiovascular regulation. Recently, it has become apparent that vascular superoxide anion may play an important role in hypertension development. Treatment with antisense NAD(P)H oxidase or SOD decreased BP in Ang II-infused rats. Wang et al recently reported mice which lack one of the subunits of NAD(P)H oxidase developed hypertension at a much lower extent when compared to the wild type animals infused with Ang II, indicating that superoxide anion contributes to elevation in BP in the Ang II-infused hypertensive model. In the Ang II-infused hypertensive model, altered reactivity of blood vessels is often associated with the elevation of systolic blood pressure. We have observed abnormal tension development and impaired endothelium-dependent relaxation in the isolated aorta of Ang II-infused and DOCA-salt hypertensive rats. Recently, several other cellular signal molecules, including ERK1I2 and PI3K, have been determined to play important roles in the regulation of smooth muscle contraction and relaxation. ERKl/2 and PI3K pathways are also reported to contribute to Ang II induced cell growth, hypertrophy, remodeling and contraction. Moreover, these signaling pathways have shown ROS-sensitive properties. Therefore, the aim of the present study is to investigate the roles of ERKl12 and PI3K in vascular oxidative stress, spontaneous tone and impaired endothelium relaxation in Ang II-infused hypertensive model. Hypothesis: We hypothesize that the activation of ERKl12 and PI3K are elevated in response to an Ang II infusion for 6 days. The elevated activation of phospho-ERKl/2 and PI3K mediated the increased level of vascular superoxide anion, the abnormal vascular contraction and impaired endothelium-dependent vascular relaxation in Ang II-infused hypertensive rats. Methods: Vascular superoxide anion level is measured by lucigenin chemiluminescence. Spontaneous tone and ACh-induced endothelium-dependent relaxation was measured by isometric tension recording in organ chamber. The activity of ERK pathway will be measured by its Western blot of phosphorylation of ERK. PI3K activity was evaluated indirectly by Western blot of the phosphorylation of PDKl, a downstream protein of PI3K signaling pathway. The role of each pathway was also addressed via comparing the responses to the specific inhibitors. Results: Superoxide anion was markedly increased in the isolated thoracic aorta from Ang II-infused rats. There was spontaneous tone developed in rings from Ang II-induced hypertensive but not sham-operated normotensive rats. ACh-induced endothelium-dependent relaxation function is impaired in Ang II-infused hypertensive rats. Superoxide dismutase and NAD(P)H oxidase inhibitor, apocynin, inhibited the abnormal spontaneous tone and ameliorated impaired endothelium-dependent relaxation. The expression of phopho-ERKII2 was enhanced in Ang II-infused rats, indicating the activity of ERK1I2 could be increased. MEK1I2 inhibitors, PD98059 and U126, but not their inactive analogues, SB203580 and U124, significantly reduced the vascular superoxide anion in aortas from Ang II-infused rats. The MEK1I2 inhibitors reduced the spontaneous tone and improved the impaired endothelium-dependent relaxation in aorta of hypertension. These findings supported the role of ERKII2 signaling pathway in vascular oxidative stress, spontaneous tone and impaired endothelium-dependent relaxation in Ang II-infused hypertensive rats. The amount of phospho-PDK, a downstream protein of PI3K was increased in Ang II rats indicating the activity of PI3K activity was elevated. Strikingly, PI3K significantly inhibited the increase of superoxide anion level, abnormal spontaneous tone and restored endothelium-dependent relaxation in Ang II-infused hypertensive rats. These findings indicated the important role of PI3K in Ang II-infused hypertensive rats. Conclusion: ERKII2 and PI3K signaling pathways are sustained activated in Ang II-infused hypertensive rats. The activated ERKII2 and PI3K mediate the increase of vascular superoxide anion level, vascular abnormal spontaneous tone and impaired endothelium-dependent relaxation.

Aging policy: a case study of co-ordination in Canadian governments

ABSTRACT Canada is an aging society. The number of people aged sixty-five and over is rising, while the number of people under twenty is declining. These two concurrent changes in the age structure have produced a sh~ft in the demographic composition of Canada which is commonly referred to as the aging phenomenon. Regardless of whether or not the number of people under twenty continues to decline, the number of elderly in Canada will almost double over the next twenty years. This rapidly growing elderly clientele will doubtless have an impact on Canadian governments. Federal, provincial and municipal governments are presently providing a variety of programs that have a special bearing on the aged and most senior citizens are beneficiaries of one or more of these programs. The ramifications of a rapidly growing elderly clientele are obvious. In order to cope with the impact of a significant increase in the number of elderly persons, the development and implementation of aging policy must be co-ordinated at each level of government and between and among levels of government. If aging policy is not co-ordinated, the results are likely to be: inappropriate policy decisions; duplication and overlap; and, ineffective and irresponsive services. No one benefits from these results. The need for co-ordination is apparent. The purpose of this thesis is to examine existing governmental efforts to co-ordinate policy in the field of aging. These efforts are examined by focusing on interactions directed at co-ordination between and among major actors in aging policy. A framework is used to structure the description and analysis of these interactions. The variables of formalisation and intensity and the concept of power are instrumental in analysing interactions for co-ordination. The underlying intent of this thesis is to discover some of the main gaps in existing governmental efforts to co~ordinate aging policy. Gaps are, in fact, discovered. Several explanations for the existence of gaps in interactions for co-ordination are discussed. A major hypothesis involving a relationship between a bureaucratic form of organisation and interactions for coordination is suggested. Finally, three recommendations for improving co-ordination in aging policy are offered.

The association between blood pressure and vascular characteristics in children

Hypertension is thought to exist in up to five percent of children. A select number of studies have investigated the role elevated blood pressure plays in pediatric atherosclerotic progression. However these studies contain significant methodological flaws and fail to recognize important confounding factors. Therefore, the influence of elevated blood pressure on arterial health in children remains to be clearly understood. The purpose of this study was to investigate the association between blood pressure (BP) and arterial thickness and stiffuess in children. Common carotid artery (CCA) intima-media thickness (IMT) and distensibility (Dist), as well as systemic pulse wave velocity (PWV) were measured in 21 elevated blood pressure (EBP; BP ~ 95th percentile) and 83 normal blood pressure (NBP; BP < 90th percentile) children 11-14 years of age. Both EBP and NBP groups demonstrated BP within the normal clinical range, but EBP showed significantly elevated BP as compared to the NBP group. Independent t-tests failed to show significant differences between the EBP and NBP groups for CCA IMT (0.43 ± 0.05 mm and 0.42 ± 0.06 mm, respectively) and Dist (0.0058 ± 0.0024 mmHg-1 and 0.0064 ± 0.0019 mmHil respectively). In contrast, a significantly elevated PWV (p

Electrocortical indices of cognitive control in working memory : exploring the effects of proactive interference, cognitive load, and aging

Cognitive control involves the ability to flexibly adjust cognitive processing in order to resist interference and promote goal-directed behaviour. Although frontal cortex is considered to be broadly involved in cognitive control, the mechanisms by which frontal brain areas implement control functions are unclear. Furthermore, aging is associated with reductions in the ability to implement control functions and questions remain as to whether unique cortical responses serve a compensatory role in maintaining maximal performance in later years. Described here are three studies in which electrophysiological data were recorded while participants performed modified versions of the standard Sternberg task. The goal was to determine how top-down control is implemented in younger adults and altered in aging. In study I, the effects of frequent stimulus repetition on the interference-related N450 were investigated in a Sternberg task with a small stimulus set (requiring extensive stimulus resampling) and a task with a large stimulus set (requiring no stimulus resampling).The data indicated that constant stimulus res amp ling required by employing small stimulus sets can undercut the effect of proactive interference on the N450. In study 2, younger and older adults were tested in a standard version of the Sternberg task to determine whether the unique frontal positivity, previously shown to predict memory impairment in older adults during a proactive interference task, would be associated with the improved performance when memory recognition could be aided by unambiguous stimulus familiarity. Here, results indicated that the frontal positivity was associated with poorer memory performance, replicating the effect observed in a more cognitively demanding task, and showing that stimulus familiarity does not mediate compensatory cortical activations in older adults. Although the frontal positivity could be interpreted to reflect maladaptive cortical activation, it may also reflect attempts at compensation that fail to fully ameliorate agerelated decline. Furthermore, the frontal positivity may be the result of older adults' reliance on late occurring, controlled processing in contrast to younger adults' ability to identify stimuli at very early stages of processing. In the final study, working memory load was manipulated in the proactive interference Sternberg task in order to investigate whether the N450 reflects simple interference detection, with little need for cognitive resources, or an active conflict resolution mechanism that requires executive resources to implement. Independent component analysis was used to isolate the effect of interference revealing that the canonical N450 was based on two dissociable cognitive control mechanisms: a left frontal negativity that reflects active interference resolution, , but requires executive resources to implement, and a right frontal negativity that reflects global response inhibition that can be relied on when executive resources are minimal but at the cost of a slowed response. Collectively, these studies advance understanding of the factors that influence younger and older adults' ability to satisfy goal-directed behavioural requirements in the face of interference and the effects of age-related cognitive decline.

Autonomic and Electrophysiological Correlates of Cognitive Control in Aging

This thesis tested a model of neurovisceral integration (Thayer & Lane, 2001) wherein parasympathetic autonomic regulation is considered to play a central role in cognitive control. We asked whether respiratory sinus arrhythmia (RSA), a parasympathetic index, and cardiac workload (rate pressure product, RPP) would influence cognition and whether this would change with age. Cognitive control was measured behaviourally and electrophysiologically through the error-related negativity (ERN) and error positivity (Pe). The ERN and Pe are thought to be generated by the anterior cingulate cortex (ACC), a region involved in regulating cognitive and autonomic control and susceptible to age-related change. In Study 1, older and younger adults completed a working memory Go/NoGo task. Although RSA did not relate to performance, higher pre-task RPP was associated with poorer NoGo performance among older adults. Relations between ERN/Pe and accuracy were indirect and more evident in younger adults. Thus, Study 1 supported the link between cognition and autonomic activity, specifically, cardiac workload in older adults. In Study 2, we included younger adults and manipulated a Stroop task to clarify conditions under which associations between RSA and performance will likely emerge. We varied task parameters to allow for proactive versus reactive strategies, and motivation was increased via financial incentive. Pre-task RSA predicted accuracy when response contingencies required maintenance of a specific item in memory. Thus, RSA was most relevant when performance required proactive control, a metabolically costly strategy that would presumably be more reliant on autonomic flexibility. In Study 3, we included older adults and examined RSA and proactive control in an additive factors framework. We maintained the incentive and measured fitness. Higher pre-task RSA among older adults was associated with greater accuracy when proactive control was needed most. Conversely, performance of young women was consistently associated with fitness. Relations between ERN/Pe and accuracy were modest; however, isolating ACC activity via independent component analysis allowed for more associations with accuracy to emerge in younger adults. Thus, performance in both groups appeared to be differentially dependent on RSA and ACC activation. Altogether, these data are consistent with a neurovisceral integration model in the context of cognitive control.