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Arterial structure and function in vascular ageing: are you as old as your arteries? Dick H. J. Thijssen1,2 , Sophie E. Carter1 and Daniel J. Green1,3 1

Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, UK Radboud Institute for Health Sciences, Department of Physiology, Radboud University Medical Center, The Netherlands 3 School of Sports Science, Exercise and Health, The University of Western Australia, Western Australia

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Abstract Advancing age may be the most potent independent predictor of future cardiovascular events, a relationship that is not fully explained by time-related changes in traditional cardiovascular risk factors. Since some arteries exhibit differential susceptibility to atherosclerosis, generalisations regarding the impact of ageing in humans may be overly simplistic, whereas in vivo assessment of arterial function and health provide direct insight. Coronary and peripheral (conduit, resistance and skin) arteries demonstrate a gradual, age-related impairment in vascular function that is likely to be related to a reduction in endothelium-derived nitric oxide bioavailability and/or increased production of vasoconstrictors (e.g. endothelin-1). Increased exposure and impaired ability for defence mechanisms to resist oxidative stress and inflammation, but also cellular senescence processes, may contribute to age-related changes in vascular function and health. Arteries also undergo structural changes as they age. Gradual thickening of the arterial wall, changes in wall content (i.e. less elastin, advanced glycation end-products) and increase in conduit artery diameter are observed with older age and occur similarly in central and peripheral arteries. These changes in structure have important interactive effects on artery function, with increases in small and large arterial stiffness representing a characteristic change with older age. Importantly, direct measures of arterial function and structure predict future cardiovascular events, independent of age or other cardiovascular risk factors. Taken together, and given the differential susceptibility of arteries to atherosclerosis in humans, direct measurement of arterial function and health may help to distinguish between biological and chronological age-related change in arterial health in humans. (Received 1 May 2015; accepted after revision 29 June 2015; first published online 3 July 2015) Corresponding author D. Thijssen: Research Institute of Sport and Exercise Sciences, Liverpool John Moores University, Tom Reilly Building, L3 3AF, Liverpool, UK. Email: [email protected] Abbreviations AGE, advanced glycation end-products; CVD, cardiovascular disease; eNOS, endothelial nitric oxide synthase; ET-1, endothelin-1; FMD, flow mediated dilation; FRS, Framingham Risk Score; GTN, glyceryl trinitrate; IMT, intima-media thickness; L-NMMA, NG-monomethyl-L-arginine; NO, nitric oxide; sGC, soluble guanylyl cyclase; SNP, sodium nitroprusside.

The work by Professors Dick H. J. Thijssen and Daniel J. Green aims to understand the impact of exercise training and physical (in)activity in the context of (primary and secondary) prevention of cardiovascular disease. Specifically, we adopt novel imaging approaches to assess the effects of exercise training on micro and macrovessel function and structure, and explore factors that moderate these effects (e.g. older age). These benefits of exercise training on the arterial wall may contribute to the reduction in cardiovascular risk. Furthermore, our work has strongly focused on understanding (haemodynamic) stimuli, such as shear stress and arterial pressure, to mediate the effects of exercise training on the vasculature. In our work we encompasses the lifespan; from exercise training in the prevention of the development of atherosclerosis in obese children and adolescents, to research on the best combination of exercise and (non)pharmacological strategies in the management of patients with cardiovascular risk and/or disease.

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DOI: 10.1113/JP270597

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Introduction

Cardiovascular disease (CVD) remains the world’s leading cause of death. Predicting the occurrence of future CVD in apparently healthy asymptomatic subjects remains a major challenge in contemporary cardiovascular medicine. Age is a highly predictive risk factor for future CVD (Lakatta & Levy, 2003; Najjar et al. 2005). Several studies have reported that advancing age is associated with increased incidence of coronary heart disease, stroke and heart failure (Lakatta & Levy, 2003). Consequently, the use of chronological age is of great importance in the prediction of CVD (Kannel et al. 1961; Wilson et al. 1998; Mahmood et al. 2014). However, the mechanisms through which older age affects future development of CVD are not fully understood and the extent to which chronological age provides a surrogate for biological age of the vasculature remains obscure. In this review, we pose a number of questions to better understand the impact of chronological and biological age on the development of CVD in humans. Can we successfully predict cardiovascular events?

Several traditional and novel CV risk factors indepe ndently predict the future development of CVD (Kannel et al. 1961; Wilson et al. 1998; Wang et al. 2006; D’Agostino et al. 2008) and the combination of some of these risk factors predict future events. For example, the Framingham Risk Score (FRS) uses an algorithm, involving traditional CV risk factors like age, cholesterol, HDL, sex, smoking and systolic blood pressure to calculate the 10-year prediction of a CV event (Kannel et al. 1961; Wilson et al. 1998; Mahmood et al. 2014). A systematic review showed that the sensitivity and specificity of the FRS for future CV events, based on the area under the receiver operator curve, ranged between 0.60 and 0.86 (Siontis et al. 2012). This indicates that traditional CV risk factors fail to predict future CVD in a large proportion of cases (up to 50%; Naghavi et al. 2003). To support this view, traditional CV risk factors are poorly related to coronary atherosclerotic plaque burden (Johnson et al. 2009). Moreover, the observation of a receiver operator curve of only 0.53 in an older cohort of 85 years (de Ruijter et al. 2009) suggests that older age affects the ability of the FRS to predict future CVD using risk factors. The inability to accurately predict future events in individual patients is not specific for a single algorithm, since poor agreement is observed between multiple risk calculators when allocating subjects to risk categories (Allan et al. 2013). Taken together, the relative impact or importance of CV risk factors may differ between populations. Traditionally, the impact of age on CV risk has been ascribed to the time-dependent accumulation of harmful CV risk factors (Najjar et al. 2005). Nonetheless, older age predicts future CVD independently of other risk factors

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(Wilson et al. 1998; Mahmood et al. 2014), raising the question of direct impacts of ageing per se. Possibly, changes in the structure and function of the vasculature may directly explain age-related changes in CV risk. What is the impact of older age on vascular function?

Studies that examined the impact of age on vascular function have typically adopted a cross-sectional design, whereby healthy volunteers with a substantial age range were tested. Studies investigating coronary artery peak blood flow response to intracoronary infusion of acetylcholine, an endothelium-dependent dilator, demonstrate a progressive decline of the maximal response with age (Vita et al. 1990). Comparable observations can be made when examining coronary artery dilator responses to other vasoactive substances and/or manipulations (Zeiher et al. 1993). In peripheral vessels, measurement of conduit artery endothelial function using flow mediated dilatation (FMD), also demonstrates a progressive decline with age (Celermajer et al. 1994). The age-related impairment in conduit artery endothelial function has been reported in the brachial (Celermajer et al. 1994; Parker et al. 2006; Black et al. 2009), femoral (Thijssen et al. 2006) and popliteal (Parker et al. 2006) arteries. The onset of the age-related decline in conduit artery endothelial function is reported to occur at an earlier stage in males than females (Celermajer et al. 1994). However, females may exhibit accelerated functional descent after the onset of decline (Celermajer et al. 1994). Similar observations of an age-related, gradual decline in endothelial function can be observed when examining forearm resistance artery function by intra-arterial infusion of acetylcholine (Taddei et al. 1997, 2001; Higashi et al. 2006) and methacholine chloride (Gerhard et al. 1996). Finally, measures of endothelial function in the cutaneous microcirculation provide evidence that older age leads to an impaired microvascular dilatation response to local (Black et al. 2008; Tew et al. 2010) and systemic (Holowatz et al. 2003) heating, but also to local infusion of acetylcholine (Black et al. 2008; Tew et al. 2010). Taken together, these studies demonstrate a gradual, age-related decline in vascular function that occurs throughout the vascular tree, ranging from coronary arteries to peripheral conduit, resistance and microvessels in humans. These conclusions are supported by studies that adopt alternative measures of vascular function, such as measures of arterial compliance and stiffness (see later section; Tanaka et al. 2000; Moreau et al. 2003). Why is vascular function impaired with older age?

Given the potent anti-atherogenic properties of nitric oxide (NO), many studies have explored the role of  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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this vasodilator pathway in explaining age-related loss of vascular function. Some studies have indicated that older age is associated with diminished forearm vasoconstrictor response to infusion of NO-synthase inhibitor NG -monomethyl-L-arginine (L-NMMA), suggesting reduced NO bioavailability in resistance arteries (Taddei et al. 2000; Singh et al. 2002). Moreover, in older adults, supplementation of L-arginine, the pre-cursor for NO, improves skin blood flow responses to whole body heating (Holowatz et al. 2006) and coronary artery blood flow response to acetylcholine (Chauhan et al. 1996). NO mediates a significant component of the FMD response (Green et al. 2014); a measure that is typically reduced with older age. Age-related decreases in tetrahydrobiopterin (BH4) synthesis, an important co-factor in NO production, provides further support for the presence of impairment of the NO-pathway with older age (Pierce & Larocca, 2008). Indeed, BH4-supplementation in older humans enhanced brachial artery FMD (Eskurza et al. 2005) as well as the NO-dependent dilatation of forearm blood flow responses to acetylcholine (Higashi et al. 2006). Although previous reports have consistently reported the presence of lower NO bioavailability, somewhat conflicting results have been reported regarding endothelial nitric oxide synthase (eNOS) expression in vascular endothelial cells in older humans (Donato et al. 2009; Rippe et al. 2012). In addition to this focus on impaired vasodilator mechanisms, some studies have explored the impact of older age on vasoconstrictor pathways, in particular endothelin-1 (ET-1). For example, older men exhibit a greater lower limb vasodilatation response to ET-receptor blockade, indicative for a greater contribution of ET-1 to vascular tone with age (Thijssen et al. 2007). Comparable results were reported for the forearm resistance vascular bed, as older men demonstrate a bunted forearm blood flow response to exogenous ET-1 (Van Guilder et al. 2007). It was also demonstrated that an increased ET-1 expression in older men was inversely related to endothelial-dependent dilatation (Donato et al. 2009). Mechanistically, these changes in the contribution of ET-1 may relate to changes in sensitivity (and/or density) of ETA receptors (causing vasoconstriction) and/or ETB receptors (leading to vasodilatation). Previous work in animals demonstrated that the augmented vasoconstrictor response to ET-1 in older rats is mediated through an increased ETA pathway (Donato et al. 2005), whilst other work provided evidence for impairment in the ETB receptors (Asai et al. 2001). Impaired ETB receptors may also contribute to higher ET-1 plasma levels since these receptors contribute to ET-1 clearance. Future work is needed to confirm these hypotheses. In addition to an (age-related) altered endogenous bioavailability of vasoactive substances, impaired vascular  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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function with age may also relate to changes in smooth muscle function. Ultimately, (magnitude of) dilatation or constriction of vessels depends on the ability of the smooth muscle cells to alter tone. The capacity of conduit and resistance artery smooth muscle cells to dilate is typically assessed as the dilatory response following sublingual nitroglycerine (glyceryl trinitrate, GTN) or intra-arterial infusion of sodium nitroprusside (SNP), respectively. A recent meta-analysis demonstrated a small but significant age-related impairment in smooth muscle function in conduit and resistance arteries (Montero et al. 2015). These findings suggest that the age-related impairment in vascular function may, at least partly, be explained by attenuated smooth muscle cell function. A potential mechanistic explanation for this finding may relate to decreased expression of soluble guanylyl cyclase (sGC; the principal receptor of NO and mediating dilatation) on smooth muscle cells, such as observed in older animals (Chen et al. 2000; Kloss et al. 2000). The mechanisms underlying age-associated vascular dysfunction may relate to increased vascular oxidative stress and inflammation with older age, leading to pro-inflammatory phenotypical changes. In men, ageing is associated with increased endothelial cell oxidative stress and markers of inflammation, both of which are related to the age-related impairment in endothelial function (Donato et al. 2007; Rodriguez-Manas et al. 2009). Studies in older humans have found increased levels of inflammatory proteins, such as pro-inflammatory nuclear factor-κB and cytokines (e.g. IL-6, TNF-α, MCP-1; Donato et al. 2008; Rippe et al. 2012). A potential role for inflammation in age-related changes in vascular function is provided by the finding that inhibition of nuclear factor-κB improves brachial artery endothelial function in older individuals (Pierce et al. 2009; Seals et al. 2011). Support for a role of vascular oxidative stress is provided by studies reporting that older humans demonstrate increased markers of oxidative stress (e.g. nitrotyrosine; Gano et al. 2011), whilst this marker is inversely related with endothelial function (Donato et al. 2007). Furthermore, infusion of ascorbic acid (i.e. a reactive oxygen species scavenger) attenuated the impairment in brachial artery FMD (Eskurza et al. 2004) and increased acetylcholine-induced vasodilatation of the forearm vessels in older adults (Taddei et al. 2000). The mechanism of increased oxidative stress with older age may relate to elevated levels of reactive oxygen species combined with reduced (or absent compensatory improvements in) antioxidative capacity (Donato et al. 2015). These data suggest that older age is associated with increased oxidative stress and inflammation that, subsequently, may contribute to the impaired vascular function typically observed in older humans (Fig. 1).

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Why is vascular function impaired with older age: molecular pathways?

Pathways underlying the development of vascular dysfunction may relate to those involved in senescence (Kovacic et al. 2011), that is irreversible cell cycle arrest as a result of critically short telomeres or external stress (e.g. oxidative stress, DNA damage), possibly through the p53/p21 pathway (Erusalimsky, 2009; Donato et al. 2015). Senescent cells demonstrate an increased rate of apoptosis, impaired proliferation, eNOS inhibition and increases in mediators for inflammation (Rippe et al. 2012; Donato et al. 2015). Accumulation of senescent cells have been reported in atherosclerotic lesions (Minamino et al. 2002). Sirtuins, originally linked to metabolic health, may represent an important molecular regulator of vascular ageing (Cencioni et al. 2015; Paneni et al. 2015). For example, expression of SIRT1 (through p51 and p66Shc ) has been linked with protection against oxidative

What is the impact of older age on vascular structure: wall thickness?

When exploring the impact of older age on the structure of arteries, a common and frequently reported observation is the age-related increase in carotid artery intima-media thickness (IMT) (Homma et al. 2001; Tanaka et al. 2001; van den Munckhof et al. 2012; Engelen et al. 2013; Fig. 1). The increase in carotid IMT occurs linearly

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stress-induced cellular senescence/apoptosis, improved NO bioavailability and lower inflammation (Cencioni et al. 2015; Paneni et al. 2015). Nonetheless, the role of sirtuins in mediating age-related cardiovascular changes is under debate. Other molecular pathways, such as microRNAs and endothelial progenitor cells, may also contribute to the cellular changes with older age. Future studies should explore the potential role of (novel) molecular pathways related to (accelerated) cellular senescence.

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Figure 1. Proposed effects of older age on artery function and structure Schematic presentation of an endothelial cell and smooth muscle cell (top panel; including the most important pathways involved in theregulation of endothelial function) and structural aspects of an artery (lower panel; lumen size and wall thickness) in healthy young (left side) and older humans (right side). The difference in size of the various symbols between young and older humans relate to the impact of older age.

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(5 μm year–1 ) with older age, in both men and women (Homma et al. 2001; Engelen et al. 2013). Increased blood pressure, by virtue of exerting a greater distending force on the arterial walls, is suggested to importantly contribute to carotid artery thickening (Tanaka et al. 2001). To further support this link, 9-month anti-hypertensive treatment significantly decreased carotid IMT, with the magnitude of decrease in IMT being closely related to the decrease in carotid pulse pressure (Boutouyrie et al. 2000). Despite the strong scientific focus on the carotid artery, thickening of the arterial wall with age is also observed in upper and lower limb peripheral vessels (Dinenno et al. 2000; Green et al. 2010; van den Munckhof et al. 2012). Moreover, the magnitude of age-related thickening seems comparable between central and peripheral arteries (van den Munckhof et al. 2012). This is of particular importance, since the brachial artery is not considered atherosclerosis-prone (van den Munckhof et al. 2012), suggesting that thickening of the conduit arterial wall represents a systemic effect of ageing per se (van den Munckhof et al. 2012). To further support a common pathway for arterial thickening, systolic blood pressure (and therefore distending pressure) is positively correlated to brachial, superficial femoral and popliteal IMT (van den Munckhof et al. 2012), a finding similar to that observed for the carotid artery (Tanaka et al. 2001). What is the impact of older age on vascular structure: diameter?

Conduit artery diameter demonstrates a gradual increase with age in both central (Schmidt-Trucksass et al. 1999; van den Munckhof et al. 2012) and peripheral (Sandgren et al. 1998, 1999; van der Heijden-Spek et al. 2000; Green et al. 2010; van den Munckhof et al. 2012) vessels (Fig. 1). In fact, the relative increase in diameter with older age is comparable between central and (lower and upper limb) peripheral vessels (van den Munckhof et al. 2012). Cross-sectional analysis of the common carotid artery demonstrates a 0.017 mm year–1 increase in those without atherosclerosis, while a 0.03 mm year–1 increase is observed in those with pre-existing disease (Eigenbrodt et al. 2006). In lower limb vessels, popliteal and femoral diameter increases between 22 and 26% and between 12 and 21%, respectively, across a 40 year age span (25–67 years), equivalent to a 0.5% increase in diameter per year, despite maintenance of a constant body surface area (Sandgren et al. 1998, 1999). The mechanisms behind the age-related increase in diameter are unclear. It is suggested that lumen enlargement is a compensatory adaptation to plaque formation and/or increases in wall thickness, in order to maintain luminal area. Whilst evidence supports the presence of this adaptation in the carotid (Polak et al. 1996; Labropoulos et al. 1998) and peripheral (Labropoulos et al.  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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1998) vasculature, enlargement of lumen diameters can occur in the absence of plaque formation (Jensen-Urstad et al. 1999; Eigenbrodt et al. 2006), suggesting a compensatory enlargement cannot fully explain the increase in diameter with age. Alternatively, dilatation may occur due to age-related loss of elastic fibres (van der Heijden-Spek et al. 2000), fracturing of the elastic lamellae (O’Rourke & Hashimoto, 2007), and/or as an adaptation to stiffening of the vasculature (Schmidt-Trucksass et al. 1999). Especially the age-related loss of elastin may be of particular importance in the observed changes in diameter. With older age, elastin content decreases, elastin elongates and loses some of the elastic recoil properties (Fritze et al. 2012). Consequently, arteries rely more on the stiffer collagen in the arterial wall and become somewhat larger. What is the impact of older age on vascular structure: arterial stiffness?

Early studies demonstrated the impact of older age on pulse wave velocity (a measure reflecting stiffness of the arterial system; Avolio et al. 1985). Subsequent large-scale studies (e.g. Atherosclerosis Risk In Communities, Baltimore Longitudinal Study of Aging) reinforced the observation that older age is independently related to an increase in pulse wave velocity (AlGhatrif et al. 2013; Meyer et al. 2015). Interestingly, a steeper age-related increase in pulse wave velocity may be present in men compared to women (AlGhatrif et al. 2013) and/or in the presence of CV risk factors. The age-related increase in stiffness of (large and smaller-sized) vessels is likely to result from mechanical factors such as pressure, but also from pressure-independent factors (Blacher & Safar, 2005). Recent studies have suggested a potential role for age-related increases in the formation of advanced glycation end-products (AGEs) in the arterial wall that may contribute to vascular dysfunction (Zieman & Kass, 2004). AGEs represent the end-product of a non-enzymatic reaction with sugar derivates that lead to irreversible crosslinks with proteins (Brownlee, 1995). In the arterial wall, AGEs can bind to collagen, leading to changes in the mechanical properties of the vascular wall. However, the exact importance of these glycation end-products on arterial stiffness and function remains somewhat unclear (Oudegeest-Sander et al. 2013). Is there an interaction between vascular function and structure?

Vascular function and structure are often examined as independent characteristics. However, previous work has repeatedly demonstrated that conduit artery diameter is inversely correlated with vasodilator function (Celermajer et al. 1992; Silber et al. 2005; Thijssen et al. 2008). Whilst

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frequently reported for the brachial artery, the strong relation between diameter and dilator response is similarly present in different sized and located arteries and the relation remains present after correcting for the shear rate stimulus (Thijssen et al. 2008). The larger dilator response in smaller vessels may result from structural characteristics, since smaller vessels have more smooth muscle relative to elastic laminae, resulting in a larger wall-to-lumen ratio. Supporting this hypothesis, a positive relation was observed between wall-to-lumen ratio and endothelium-dependent and –independent vasodilator responses of upper and lower limb vessels (Thijssen et al. 2011). The presence of hyper-responsiveness in vessels with increased wall-to-lumen aligns with work of Folkow who, as early as in 1955, calculated that increased wall-to-lumen ratio would enhance vasodilator responsiveness (Folkow, 1978). Whilst the strong interaction between vascular function and structure is well-established, the impact of the ageing process on this relationship is an area for future investigation. Do measures of vascular function or structure predict future CV events?

Earlier sections have clearly demonstrated that older age, independent of other CV risk factors, can impact measures of vascular function and structure. Here, we summarise data to better understand whether changes in these vascular characteristics relate to changes in CV risk. Generally, studies have found evidence that measures of endothelial function are strongly related to the future development of CV events. For example, assessment of coronary artery function, either in response to intra-arterial infusion of drugs or in response to a sympathetic stimulus, is predictive for future CVD (Vita et al. 1990; Schachinger et al. 2000). Moreover, the predictive value of the coronary artery dilator response to intra-coronary infusion of acetylcholine is independent of traditional CV risk factors (Schachinger et al. 2000). Other measures of the peripheral vasculature, such as central or peripheral vascular stiffness, also demonstrate independent predictive capacity for future CV events (Vlachopoulos et al. 2010). For example, a 1 m s−1 increase in aortic pulse wave velocity was related to an age, sex and risk factor adjusted risk elevation of 14%, 15% and 15% for total CV events, CV mortality and all-cause mortality, respectively (Vlachopoulos et al. 2010). Even measurements of skin microvessel function correlate with FRS, with impaired microvascular function being related to increased CV risk (Vuilleumier et al. 2002; IJzerman et al. 2003). Given the relatively simple procedures, several studies have examined the predictive capacity of the brachial artery FMD and have shown, both in asymptomatic and symptomatic subjects, FMD can predict future CV events

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(Yeboah et al. 2009; Inaba et al. 2010; Green et al. 2011; Ras et al. 2013; Shechter et al. 2014). Importantly, these studies have typically found that the predictive capacity of the brachial artery FMD is independent of other CV risk factors. Combining brachial FMD with the FRS enhanced CV risk classification compared to the FRS in isolation (Yeboah et al. 2009). However, the predictive capacity of the FMD may be reduced in older populations (Yeboah et al. 2007). Age-related stiffening of the arteries and increases in diameter, and consequently attenuated dilator capacity, may contribute to these findings. In fact, the same authors found that brachial artery diameter (but not FMD) demonstrates independent predictive value for future CV events in older adults (Yeboah et al. 2007). Regarding other structural aspects of conduit arteries, studies have demonstrated that thickening of the arterial wall of carotid and peripheral vessels are both strong independent predictors of future CVD. A large number of studies have demonstrated that increased carotid (Lorenz et al. 2006, 2007, 2010; Polak et al. 2010) and peripheral artery (Weidinger et al. 2002) IMT is independently associated with future CV events. Specifically for carotid artery IMT, a meta-analysis showed that a 0.1 mm increase in IMT is associated with an increase in relative risk for myocardial infarction of 10–15% (Lorenz et al. 2007). Taken together, data provide strong evidence that functional and structural characteristics of peripheral and central arteries are strongly related to future CV events in humans. Vascular health to distinguish biological versus chronological age of arteries?

There is strong evidence that older age leads to a gradual change in vascular function and structure in several arterial beds, by various measures of function. This may explain why chronological age is strongly and independently related to CV risk. However, this does not necessarily inform us about the biological age of vessels. Repeated exposure to potentially harmful stimuli across the lifespan can ultimately lead to between-subject differences in vascular function, structure and health, with consequential dissociation between biological and chronological age. These differences may, in part, relate to inter-individual differences in susceptibility for vascular adaptations and/or atherosclerosis. An integrative measure of vascular structure and function, scaled for chronological age, could conceivably provide better prediction of the true biological age of human arteries. Conclusion

Advancing age has direct effects on the structure and function of the vasculature. More specifically, older age  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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results in a systemic, gradual loss of vascular endothelial function, possibly due to down-regulation of vasodilator pathways (e.g. NO) and/or up-regulation of vasoconstrictor pathways (e.g. ET-1). Increased oxidative stress and inflammation, possibly the development of cellular senescence and apoptosis, may contribute to age-related impairment in vascular function. Structurally, older arteries demonstrate thicker walls, which are stiffer and exhibit a larger diameter. Importantly, these functional and structural changes represent independent predictors of future CVD. These changes in the vasculature may provide a potential explanation for the age-related increase in CVD risk, especially since traditional risk factors cannot fully explain the impact of age on future CVD. Taken together, given the differential susceptibility of arteries to atherosclerosis and the predictive capacity of measures of vascular function and structure, direct assessment of vascular function and structure may help to distinguish between the biological and chronological age of arteries in humans in vivo. References AlGhatrif M, Strait JB, Morrell CH, Canepa M, Wright J, Elango P, Scuteri A, Najjar SS, Ferrucci L & Lakatta EG (2013). Longitudinal trajectories of arterial stiffness and the role of blood pressure: the Baltimore Longitudinal Study of Aging. Hypertension 62, 934–941. Allan GM, Nouri F, Korownyk C, Kolber MR, Vandermeer B & McCormack J (2013). Agreement among cardiovascular disease risk calculators. Circulation 127, 1948–1956. Asai K, Kudej RK, Takagi G, Kudej AB, Natividad F, Shen YT, Vatner DE & Vatner SF (2001). Paradoxically enhanced endothelin-B receptor-mediated vasoconstriction in conscious old monkeys. Circulation 103, 2382–2386. Avolio AP, Deng FQ, Li WQ, Luo YF, Huang ZD, Xing LF & O’Rourke MF (1985). Effects of aging on arterial distensibility in populations with high and low prevalence of hypertension: comparison between urban and rural communities in China. Circulation 71, 202–210. Blacher J & Safar ME (2005). Large-artery stiffness, hypertension and cardiovascular risk in older patients. Nat Clin Prac Cardiovasc Med 2, 450–455. Black MA, Cable NT, Thijssen DH & Green DJ (2009). Impact of age, sex, and exercise on brachial artery flow-mediated dilatation. Am J Physiol Heart Circ Physiol 297, H1109–H1116. Black MA, Green DJ & Cable NT (2008). Exercise prevents age-related decline in nitric-oxide-mediated vasodilator function in cutaneous microvessels. J Physiol 586, 3511–3524. Boutouyrie P, Bussy C, Hayoz D, Hengstler J, Dartois N, Laloux B, Brunner H & Laurent S (2000). Local pulse pressure and regression of arterial wall hypertrophy during long-term antihypertensive treatment. Circulation 101, 2601–2606. Brownlee M (1995). Advanced protein glycosylation in diabetes and aging. Annu Rev Med 46, 223–234.

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Additional information Competing interests The authors have no competing interests to disclose. Author contributions All authors have contributed to writing the manuscript. All authors have provided approval of the final version of the manuscript. Funding Professor Green is a National Health and Medical Research Council of Australia Principal Research Fellow and holds grants from that agency (APP1045204) and the National Heart Foundation of Australia (NHF G12P6417). Professor Thijssen is financially supported by the Netherlands Heart Foundation (E Dekker 2009T064).

 C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society