Mechanisms of Dysfunction in the Aging Vasculature and Role in Age-Related Disease

Abstract

Advancing age promotes cardiovascular disease (CVD), the leading cause of death in the United States and many developed nations. Two major age-related arterial phenotypes, large elastic artery stiffening and endothelial dysfunction, are independent predictors of future CVD diagnosis and likely are responsible for the development of CVD in older adults. Not limited to traditional CVD, these age-related changes in the vasculature also contribute to other age-related diseases that influence mammalian healthspan and potential lifespan. This review explores mechanisms that influence age-related large elastic artery stiffening and endothelial dysfunction at the tissue level via inflammation and oxidative stress, and at the cellular level via Klotho and energy sensing pathways (AMPK, Sirtuins, mTOR). We also discuss how long-term calorie restriction, a healthspan and lifespan extending intervention, can prevent many of these age-related vascular phenotypes through the prevention of deleterious alterations in these mechanisms. Lastly, we discuss emerging novel mechanisms of vascular aging, including senescence and genomic instability within cells of the vasculature. As the population of older adults steadily expands, elucidating the cellular and molecular mechanisms of vascular dysfunction with age is critical to better direct appropriate and measured strategies that utilize pharmacological and lifestyle interventions to prevent against CVD risk within this population.

Cardiovascular Disease, Arterial Stiffness, Endothelial Function and Aging.

Cardiovascular diseases (CVDs), broadly defined as stroke, coronary artery disease (CAD), heart failure, and cardiac arrest, are the predominant killers of Americans, accounting for ~774,000 deaths per year according to current statistics from the Centers for Disease Control¹. CVDs cause ~32% of all deaths for Americans 65 years of age and older, making them the leading causes of death in this age group¹. Furthermore, with advancing age, the prevalence of CVDs among Americans increases progressively, from ~5.5% in 25–44 year-olds to ~41% in people 65 years of age and older². Thus, CVDs can be considered true diseases of aging.

Arterial dysfunction plays an important causal role in the vast majority of CVDs^{3, 4}. Age-related alterations in arteries ranging in size from the large elastic arteries to the conduit and small resistance arteries, and down to the microcirculation are thought to lead to a dysfunctional vascular phenotype that precedes CVDs^{3, 5}. Interestingly, age-related arterial dysfunction is present in the absence of clinical CVD and conventional CVD risk factors⁶, supporting the concept that age-related arterial dysfunction is a primary effect of advancing age that may be a precursor to the development of clinical CVD. Although many functional and morphological characteristics of arteries are altered by advancing age (e.g. increase in the size of large and feed arteries, blunted angiogenesis, loss of vascular density⁷ and a diminished endothelial glycocalyx⁸), large longitudinal studies have demonstrated that two specific age-associated arterial phenotypes are potent risk factors for future CVD diagnosis and CVD-related morbidity and mortality: increased stiffness of the large elastic arteries and endothelial dysfunction^{5, 9}. Figure 1 is the accumulated and normalized vascular function data from large cross-sectional studies of healthy (no frank CVD) humans. These data show that by the 5^th decade of life, small artery endothelial dilation and pulse wave velocity (PWV; an indicator of arterial stiffness) are already altered, but that conduit artery endothelium dependent dilation (EDD) and systolic blood pressure (SBP) are maintained close to youthful levels, but decline substantially thereafter.

Figure 1. Age-Associated Cardiovascular Changes.

Predicted percent-change from youth in markers of vascular function: brachial systolic blood pressure (SBP)¹³; carotid-to-femoral pulse wave velocity (cfPWV)³⁰⁵; maximal forearm blood flow to acetylcholine (FBF)³⁰⁶; brachial flow-mediated dilation (FMD)^307–309. All lines are the percent of the predicted fold-change from reference values at 20 yr. Reference values: SBP 110 mmHg; cfPWV 5.9 m/sec; FBF 897 Δ%; FMD 9%.

Large artery stiffening.

An important, early change seen with advancing age is stiffening of the large elastic arteries. For the purpose of this review, large artery stiffness is defined as increased conducted wave velocity and/or changes in the passive mechanical properties of the artery that reduce compliance. Longitudinal studies have shown a progressive increase in large elastic artery stiffness with aging (Figure 1) that contributes to CVD risk and is associated with pathophysiological conditions such as hypertension, stroke, left ventricular hypertrophy, sub-endocardial ischemia and cardiac fibrosis¹⁰. Large artery stiffening is postulated to impact CVDs in several ways. First, an important function of large elastic arteries (such as the aorta) is to act as a capacitance vessel to dampen the increase in pressure caused by the ejection of blood from the left ventricle during systole. The elasticity of the aorta allows it to distend as blood is pumped into it during systole, then to recoil, facilitating continuous blood flow and maintaining pressure during diastole. In this way, the elasticity of the aorta helps it to act as a secondary pump of blood during elastic recoil and to prevent large pulse pressures being conducted to the smaller blood vessels, the so called “Windkessel Effect”. As we age, the aorta loses elasticity through multiple mechanisms, becoming stiffer, and the capacitance function becomes impaired, increasing the systolic blood pressure per unit blood ejected from the left ventricle. Second, increased large artery stiffness leads to a greater velocity of the incident, systolic pressure waveform down the arterial tree, resulting in a quicker return of its reflected waveform¹¹. In stiff arteries, the reflected waveform superimposes on the incident waveform, thereby augmenting systolic pressure and aortic impedance¹², which increase the work imposed on the left ventricle during systole. Last, large artery stiffening leads to higher systolic pressure, a reduction in diastolic pressure and a concomitant widening of pulse pressure¹³. The widening of pulse pressure has also been hypothesized to augment transmission of pulsatility along the arterial tree. This has been measured in mid-sized and smaller conduit arteries, and pulsatility and higher pressure have been measured directly from the capillaries in the skin of older adults¹⁴. Increased pulsatility in muscular and resistance arteries with advancing age will deleteriously affect their ability to distribute blood flow and appropriately alter vascular resistance, a function necessary to reduce pressure in the microcirculation. These alterations could contribute to peripheral organ damage and dysfunction with aging¹⁵, particularly in vulnerable regions like the cerebral^16–20 and renal circulations¹⁰.

The most commonly used method to measure large elastic artery stiffness in long term epidemiological studies is carotid-to-femoral PWV (cfPWV), due to its ease of use and high reproducibility²¹. Other techniques, like augmentation index (AI) and direct measurements of arterial distensibility, have also been utilized, depending on the questions of interest²². Higher cfPWV is indicative of greater large elastic artery stiffness and is a strong predictor of several age-related CVD outcomes^{23, 24} and cognitive impairment²⁵. Many of the factors that alter aortic stiffness with age are plastic, such as modulators of arterial tone (i.e., vasodilators vs. vasoconstrictors), and can rapidly induce changes in arterial stiffness in different physiological states, such as in response to acute stress²⁶. Other factors that affect aortic stiffness are less plastic and require more time (months to years). These include accumulation of advanced glycation end products (AGEs), alterations to collagen and elastin content and/or arrangement, remodeling and structural changes in the extracellular matrix. Importantly, changes in large artery stiffness with age not only affect end organ systems, but also modulate the cells within the vasculature, such as the endothelium, which in turn can further exacerbate large artery stiffness and subsequent global vascular dysfunction in older adults.

Endothelial dysfunction.

The arterial endothelium is extremely dynamic, performing many vital functions that vary from one segment of the arterial tree to another, as well as from one organ system to another^{27, 28}. The vascular endothelium releases molecules that act in an autocrine and paracrine manner to regulate the function and health of the vascular network. These functions include the maintenance of blood in a fluid state, exchange of fluid and molecules between the blood and surrounding tissues, creation of new vascular networks, participation and facilitation of the immune response, and the control of vascular resistance in response to changes in blood flow through the regulation of arterial tone in resistance vessels^{27, 28}. A healthy vascular endothelium must constantly maintain a balance between oxidants and antioxidants, vasodilators and vasoconstrictors, pro- and anti-inflammatory molecules, and pro- and anti-thrombotic signals. A dysfunctional endothelium has lost this tightly regulated balance, and displays oxidant, vasoconstrictor, pro-inflammatory and pro-thrombotic properties. Age-related dysfunction of the vascular endothelium is defined in this review as an age-associated impairment in flow- or agonist-induced vasodilation and/or a reduction in the critical endothelium-derived vasodilator, nitric oxide (NO). One hallmark of vascular endothelial dysfunction is impaired EDD, which is predictive of future CVD events^{3, 4}. Increased age is the strongest independent correlate of EDD²⁹, and the age-related decline in endothelial function in both the microcirculation and larger arteries (Figure 1) contributes to a host of hemodynamic changes. These include augmented large and resistance arterial tone, induction of greater oscillatory shear stress, and further elevations in large artery stiffness^{3, 30}. By altering hemodynamics, EDD contributes to increases in the prevalence of hypertension¹³ and atherosclerosis⁴ with advancing age.

Interaction of arterial stiffness and endothelial dysfunction.

Although endothelial dysfunction and large artery stiffening are independent predictors of CVD, each of these can exacerbate the other, producing a cycle of damage and dysfunction in advanced age. For example, in vitro evidence suggests that increased matrix stiffness may impair flow-induced dilation by reducing the ability of the endothelium to effect a response to laminar shear stress³¹. Impaired EDD can also contribute to arterial stiffening by increasing the active tone in arteries³² and they may share common underlying mechanisms like oxidative stress and inflammation. As such, the mechanisms that underlie age-related arterial dysfunction provide an opportunity to develop and test interventions that may restore arterial function, prevent CVD and improve healthspan in middle-aged and older adults.

Goals of the Review.

Having considered the importance of age-related arterial stiffening and endothelial dysfunction on CVD the goals of this review are: first, we review two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction in healthy older adults and animal models. Next, we discuss some of the cellular and molecular events that induce and contribute to the sustained cycle of inflammation and oxidative stress in the aged vasculature. We also review mechanistic insight gained from studies of calorie restriction (CR) and their relevance and how traditional aging and longevity pathways also appear to modulate arterial function with advancing age. Furthermore, we highlight important signaling and genomic changes with aging that impact cellular and tissue processes, ultimately leading to the functional vascular aging phenotype (Figure 2). Lastly we describe the role of the vasculature in the aging process (lifespan, healthspan), together with examples of non-canonical age-associated CVDs, specifically metabolic syndrome and Alzheimer’s disease (AD)/dementia.

Figure 2. Hallmarks of Vascular Aging.

In addition to increases in genomic instability that are associated with increases in DNA damage, an inadequate DNA damage response (DDR) and telomere dysfunction, dysregulation of signaling pathways, including the growth factor signaling pathway, klotho, and the nutrient sensing pathways, mammalian Target of Rapamycin (mTOR), adenosine monophosphate protein kinase (AMPK), and sirtuin-1 (SIRT-1) will induce increases in cellular senescence and the dysregulation of autophagy that contribute to increased tissue oxidative stress and inflammation that ultimately result in the age-related arterial phenotype characterized by endothelial dysfunction and arterial stiffening. (Illustration Credit: Ben Smith).

Mechanisms of Vascular Dysfunction with Aging and Their Prevention by Calorie Restriction.

Reductions in EDD and increases in arterial stiffness can be reversed by lifestyle interventions such as CR, habitual exercise^{33, 34}, sodium restriction^{35, 36}, and weight loss³⁷. Among these interventions, CR is the one most consistently associated with increases in lifespan. As such, insight from studies aimed at elucidating the molecular and cellular mediators of CRs beneficial effects on lifespan may provide novel targets for pharmacological intervention in the setting of CVD. In animal studies, CR is typically a lifelong intervention, initiated after sexual maturity, in which caloric intake is restricted by 40% of ad libitum (AL) intake without inducing malnutrition. CR improves maximal and/or median lifespan, as well as physiological function in rodents and non-human primates³⁸. It has been associated with a dramatic reduction in CVD in non-human primates³⁹ and protection against cardiac ischemia⁴⁰. In addition, there is recent evidence for a vasoprotective effect of CR such that EDD and NO bioavailability are preserved^{41, 42} and increases in large artery stiffness are prevented⁴³ in aged rodents. Although the translatability of CR to humans has often come in to question, the results of the CALERIE trial, in which non-obese young and middle-aged (21–51 yrs) adults were randomized to 25% CR for two years, indicated that CR was able to slow biological aging⁴⁴ and reduce CVD risk factors⁴⁵. Although the mechanisms underlying age-related arterial dysfunction and their prevention by CR are still being elucidated, the most widely explored mechanisms are oxidative stress and inflammation with modulation of autophagy emerging as a potential candidate mechanism.

Oxidative Stress.

Elevations in plasma markers of oxidative stress in older adults⁴⁶ suggest that aging is a state of chronic, systemic oxidative stress⁴⁷, a condition that ensues when the bioavailability of reactive oxygen species (ROS) is increased relative to antioxidant defenses^{48, 49}. Oxidative stress in the vasculature is a primary mechanism underlying age-associated reductions in EDD and NO bioavailability^{46, 50, 51} as well as increases in large artery stiffness^{46, 50}, and is characterized by increases in arterial ROS, including superoxide anion (O₂^-)⁵² and hydrogen peroxide (H₂O₂)⁵³.

O₂^- reduces NO bioavailability^{49, 54} by interacting with NO to produce peroxynitrite (ONOO^-). ONOO^- is itself a ROS that nitrates cellular proteins to form nitrotyrosine (NT), a robust but reversible, marker of cellular oxidative stress and ONOO^- production^55–57. Increased abundance of arterial NT has been demonstrated in both animal models^{34, 58, 59} and ECs of humans⁶⁰. In addition, markers of lipid peroxidation such as 4-hydroxynonenal (4-HNE) or malondialdehyde (MDA) and other oxidative stress markers such as glutathionylation at cysteine residues are also observed in aged arteries^{61, 62}. Functionally, greater superoxide-mediated suppression of NO bioavailability leads to reductions in EDD^{34, 46, 50, 59} and contributes to arterial stiffening in rodent models^{63, 64} and older women⁶⁵ (Figure 3). CR attenuates age-related elevations in arterial O₂^- production^{41–43, 52}, which contributes to reduced vascular oxidative stress, as evidenced by lower arterial NT abundance after CR compared to age-matched AL controls^{42, 43}.

Figure 3. Impact of Aging on Endothelial Cells.

In younger endothelial cells (Upper Panel), endothelial nitric oxide synthase (eNOS) has adequate cofactor availability, e.g., tetrahydrobiopterin (BH₄), and produces nitric oxide (NO) through the conversion of L-arginine to L-citrulline. Reactive oxygen species (ROS), e.g., superoxide (O₂⁻) and hydrogen peroxide (H₂O₂), produced by the mitochondrial electron transport chain (ETC) or cytosolic oxidant enzymes, such as NADPH oxidase (NOX), are quenched by endogenous antioxidant enzymes (superoxide dismutase [SOD] and catalase). In older endothelial cells (Lower Panel), ROS produced in the mitochondria increase NOX mediated O₂⁻, this quenches NO bioavailability, through its conversion to peroxynitrite (ONOO⁻), as well as uncouple eNOS by reducing BH₄ availability. In the face of unchanged antioxidant defenses, these effects lead to a reduction in NO bioavailability and a pro-oxidant phenotype in the aged endothelium. (Illustration Credit: Ben Smith).

O₂^- is produced by oxidant enzyme systems such as NADPH oxidase-p67 phox, xanthine oxidase, cytochrome P450 2C9, and uncoupled eNOS^{48, 49, 66}. This O₂^- acts to reduce NO bioavailability and impair EDD. With aging, there is an increased production of O₂^- that is associated with both elevated expression and activity of NADPH oxidase³⁴ as well as with increased eNOS uncoupling⁶⁷. NADPH oxidase derived O₂^- directly contributes to age-associated endothelial dysfunction, as evidenced by the restoration of EDD and NO after in vitro inhibition of this enzyme by apocynin in arteries from aged mice³⁴. Likewise, eNOS uncoupling appears to play a role in age-related arterial dysfunction, as tetrahydrobiopterin (BH₄) treatment, which reduces eNOS uncoupling, leads to improvements in EDD in older adults that are associated with a reduction in the oxidative stress-mediated suppression of EDD⁶⁸ (Figure 3). Further supporting a role for eNOS uncoupling in age-related elevations in arterial O₂^-, it has been demonstrated that ex vivo administration of BH₄ reduces O₂^- in arterioles from old rats⁶⁹. In contrast to NADPH oxidase, other oxidant enzyme systems may be less critical to age-related arterial oxidative stress. Indeed, a preponderance of the data examining vascular function in NIA supported models of aged mice, rats (Fisher 344) and humans have found no age-related increase in expression and/or activity of xanthine oxidase or cytochrome P450^{34, 60, 70, 71}, nor has pharmacological inhibition of xanthine oxidase or cytochrome P450 2C9 been found to be effective in improving EDD in older adults^{70, 72}. Still, there is a single study that has suggested that xanthine oxidase is a source of augmented O₂^- in aged Sprague Dawley rats⁷³ and there is also evidence for a critical role of xanthine oxidase in pathological settings such as heart failure^{74, 75}. In summary, it is likely that NADPH oxidase and uncoupled eNOS are the major enzymatic sources of O₂^- in aged arteries and endothelial cells (ECs).

Reductions in O₂^- appear to be an important mechanism underlying the functional benefits of CR, as treatment of arteries with the superoxide scavenger TEMPOL improves EDD and NO bioavailability in old AL fed mice³⁴, but not in old mice after CR^{43, 52}, indicating that CR attenuates the superoxide-mediated suppression of EDD and NO^{42, 43, 52}. This CR-mediated reduction in O₂^- may result from reduced production, as CR is also associated with reductions in NADPH oxidase expression and activity^{43, 52, 76}. Although there is no direct evidence for a role of reduced eNOS uncoupling in the vascular benefits afforded by CR, association has been made between eNOS uncoupling and reductions in sirtuin (SIRT)-1, a deacetylase closely associated with CR-mediated lifespan extension⁷⁷. Nevertheless, it remains to be determined if reductions in eNOS uncoupling or other oxidant enzymes play a role in the beneficial effects of CR.

In addition to the enzymatic production of O₂^-, mitochondrial O₂^- is another important source of arterial ROS in aging. Although O₂^- can be produced in abundance in the mitochondria at complex 1 and 3⁷⁸, it may be confined to the mitochondria. Mitochondrial O₂^- produced at complex 1 and 3 are released into the mitochondrial matrix or intermembrane space, respectively, and due to its highly reactive nature, O₂^- is unlikely to migrate to the cytosol or extracellular space without being transformed to a less reactive form of ROS, such as H₂O₂. Nevertheless, mitochondrial ROS, in the form of H₂O₂⁷⁹, contributes to the ROS production/spillover in arteries from old rodents and likely contributes to age-associated endothelial dysfunction. Indeed, genetic or pharmacologic targeting of mitochondrial O₂^- reduces arterial O₂^- and leads to improvements in NO and EDD^{80, 81}. Furthermore, reducing mitochondrial ROS may also underlie the vasoprotective effects of CR, as elevations in mitochondrial O₂^- production are not present in aged primary cerebral microvascular ECs from aged mice after lifelong CR⁷⁶.

To maintain an appropriate redox balance, elevations in superoxide production with aging should signal an increase in antioxidant defenses. However, total antioxidant capacity is reduced⁸² and the expression and/or activity of critical antioxidant enzymes do not increase in arteries of old rodents³⁴. Thus, combined with the enhanced oxidant production, inadequate antioxidant defenses also contribute to age-related arterial oxidative stress and reductions in critical antioxidant enzymes, superoxide dismutases (SODs), catalase and glutathione (GSH), have been implicated in age-associated oxidative stress.

SODs are critical anti-oxidant enzymes that convert superoxide to H₂O₂, and thus play a primary role in ROS removal⁸³. In the face of elevated superoxide production with aging, neither copper zinc SOD (CuZnSOD), the intracellular isoform, nor manganese SOD (MnSOD), the mitochondrial isoform^{34, 43, 52}, increase in arteries of old mice^{34, 43}. Although the expression of extracellular SOD (ecSOD) has been shown to be either unchanged^{43, 84} or increased³⁴, there is no increase in activity of any of these SOD isoforms in arteries with advancing age^{34, 43, 52}. Interestingly, there is experimental evidence that a lack of appropriate SOD response to O₂^- overproduction may have clinical significance, as these antioxidants play a critical role in the setting of CVD. For example, MnSOD may play a cardioprotective role. Evidence for this derives from genetic models in which deletion of MnSOD results in cardiomyopathy and heart failure⁸⁵; whereas overexpression of ecSOD improves endothelial function in the setting of hypertension⁸⁶. Likewise, old MnSOD haploinsufficient mice have impaired EDD compared with old wildtype mice⁸⁷, further supporting the importance of this antioxidant and mitochondrial O₂^- in the function of aged arteries.

Interestingly, the inadequate response in SOD expression and activity with aging may contribute not only to the excess O₂^- that quenches NO bioavailability, but may also contribute to impaired vasodilation via reduced H₂O₂ production⁸⁸. While at high concentrations H₂O₂ is itself a ROS that can impair vascular function⁸⁹. H₂O₂ produced through the actions of SOD also promotes vasodilation via direct actions as an endothelium derived hyperpolarizing factor⁸⁸, as well as by indirectly inducing eNOS expression, the enzyme responsible for the production of NO⁹⁰. Interestingly, the importance of H₂O₂ as a vasodilator differs across the lifespan and with the emergence of CAD, such that the primary mediator of flow–induced dilation in coronary arteries shifts from prostacyclin in youth to NO in healthy adulthood and is again altered in the presence of CAD to favor H₂O₂⁹¹.

Like SODs, aortic expression of catalase, the enzyme responsible for the reduction of H₂O₂ to oxygen and water, also fails to increase with advancing age in mice⁴³. In advanced age, the lack of an increase in catalase combined with increased superoxide production may lead to excess accumulation of H₂O₂ and contribute to the impairments in vasodilation⁸⁹. Still another antioxidant implicated in age-associated oxidative stress is GSH, an intracellular thiol that reduces disulfide bonds and acts as an electron donor, the expression of which is reduced in aortic lysates of old rats⁴¹. Although exogenous treatment with a GSH-depleting drug was without effect on EDD in aged rats⁹², evidence for deleterious effects of inadequate GSH expression can be found in disease states. Indeed, impaired GSH-related antioxidant effects are associated with CVD, such that atherosclerotic plaques demonstrate depressed antioxidant GSH effects⁹³ and low serum GSH is associated with family history of CVD⁹⁴. Furthermore, there is evidence that GSH supplementation can improve EDD in patients with atherosclerosis⁹⁵.

In addition to lowering ROS production, reductions in oxidative stress after CR are also associated with improvements in antioxidant defenses. In contrast to AL aging, total antioxidant capacity in the serum of old CR rats does not differ from that of young rats⁸², and arterial expression of the antioxidants, MnSOD, CuZnSOD and ecSOD^{43, 52}, as well as the activity of total SOD⁵² and catalase⁴³ are all increased in aged mice after CR. Likewise, CR has also been demonstrated to increase GSH activity in human subjects⁹⁶. Although it is not known if CR alters the reliance on H₂O₂ as a vasodilator, it does appear that a reduction in oxidative stress, which is mediated by both decreased oxidant production and improved antioxidant defenses, is a primary mechanism underlying the beneficial effects of CR on the vasculature.

Inflammation.

Chronic, low grade inflammation has been implicated in age-associated impairments in endothelial function and increases in large artery stiffness. Age-associated sterile inflammation is characterized by increases in circulating C-reactive protein (CRP), as well as pro-inflammatory cytokines and adhesion molecules including tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and vascular cell adhesion molecule (VCAM)-1^{60, 71}. These circulating markers of inflammation, in particular CRP and IL-6, have been positively related to aortic stiffness⁹⁷ and inversely related to EDD in older adults⁹⁸. While some inflammatory mediators are derived from vascular cells per se, including endothelial-derived TNF-α⁹⁹, immune cells infiltrating the arterial adventitia may also be an important source of both inflammatory cytokines and ROS production¹⁰⁰. This is supported by data demonstrating increased accumulation of both macrophages and lymphocytes in the adventitia of aged arteries¹⁰¹ and a role for adventitial-derived ROS in arterial inflammation in the setting of CVDs¹⁰².

Activation of the ROS-sensitive, pro-inflammatory transcription factor, Nuclear factor kappa B (NFκB), is believed to play a critical role in age-related vascular inflammation, with increased expression of NFκB observed in ECs from older human subjects^{60, 71}. Through its interaction with the inhibitory protein, IκB-α, NFκB resides in the cytoplasm¹⁰³. In response to ROS¹⁰⁴ or other inflammatory stimuli¹⁰³, IκB-α is phosphorylated by the inhibitor of NFκB kinase subunit beta (IKKβ) that will then release its inhibition on NFκB, leading to the nuclear translocation of NFκB and the activation of gene transcription of pro-inflammatory target genes such as TNF-α and IL-6¹⁰³. Age-associated increases in NFκB activity have been directly implicated in arterial dysfunction in older rodents and humans, as NFκB inhibition by salicylate (salsalate in humans) improves EDD in aged mice¹⁰⁵ and in overweight/obese middle-aged and older adults¹⁰⁶. Furthermore, both salsalate treatment¹⁰⁷ and inhibition of the NFκB-target TNF-α¹⁰⁸ improve aortic stiffness in older adults.

Both vascular inflammation, via the cytokines IL-1β and TNF-α, and oxidative stress influence the primary mechanisms of extracellular matrix changes in the vessel wall with aging (Figure 4). Implicated in age-related arterial remodeling are an imbalance between matrix metalloproteinases (MMP) and their inhibitors, MMP-mediated arterial remodeling, elastin breakdown and fragmentation, differential changes in isoform expression and glycation of collagens, transforming growth factor-beta (TGF-β)-associated collagen accumulation¹⁰⁹, and VSMC migration, proliferation and senescence (a more complete discussion of the specific role of the VSMC in arterial aging can be found in a recent review¹¹⁰). While the VSMCs of large arteries also directly impact arterial structure and stiffness by synthesizing and secreting ECM proteins, the interaction of the VSMCs and ECM components play a critical role in determining arterial stiffness. In addition to the VSMC per se, plasma MMP-9 is also associated with increased large elastic artery stiffness in both healthy individuals and individuals with isolated systolic hypertension¹¹¹.

Figure 4. Mechanisms of Age-Associated Arterial Dysfunction.

The vascular aging phenotype is characterized by increased large artery stiffness and pulse pressure and reduced endothelium dependent dilation (EDD) and nitric oxide (NO) bioavailability. Oxidative stress, caused by increased production of reactive oxygen species (ROS) in the absence of an adequate antioxidant defense, and inflammation are two interconnected mechanisms that underlie arterial dysfunction in advanced age. Increases in oxidant production with aging are associated with increased NADPH oxidase (NOX)- and mitochondrial-produced ROS, such as superoxide (O₂-) and hydrogen peroxide (H₂O₂). These ROS act to (1) quench bioavailable NO, thus limiting EDD, (2) induce inflammatory signaling through nuclear factor kappa B (NFκB) activation and (3) induce matrix metalloproteinase (MMP)-9 and transforming growth factor beta (TGF-β) that contribute to alterations in the extracellular matrix (ECM) including increases in collagen content and decreases or fragmentation of elastin that contribute to increases in arterial stiffening. In a vicious cycle, increased NFκB activation also exacerbates both oxidative stress and inflammation by transcribing oxidant enzymes such as NOX, pro-inflammatory cytokines and adhesion molecules, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and monocyte chemoattractant protein (MCP)-1, and this contributes to downstream increases in MMP-9 and TGF-β expression. Increased adhesion molecule expression contributes to the infiltration of immune cells such as T cells and macrophages (Mφ) to the perivascular tissues and these immune cells further exacerbate inflammation and oxidative stress through the production of cytokines, such as interferon(INF)-γ and TNF-α, as well as O₂-. (Illustration Credit: Ben Smith).

MMP-9 can be directly transcribed via NFκB¹¹² or indirectly via ROS-mediated activation of NFκB¹¹³. Interestingly, our group has demonstrated a substantial increases in MMP-9 in mouse aortic tissue with aging and life-long CR normalizes MMP-9 to near young values⁴³. Experimentally, the simple viral overexpression of TGF-β is sufficient to induce fibrosis¹¹⁴. With aging, arterial expression of TGF-β is elevated in aortas of mice and this is associated with greater collagen I and III content in the adventitia and reduced medial elastin content in the aorta, as well as increased arterial superoxide¹¹⁵. In addition to modulation by oxidative stress and inflammation, both MMPs and TGF-β also appear to be influenced by NO bioavailability. For example, inhibition of NO via in vivo treatment with the NOS inhibitor, L-NAME, increased both markers of inflammation in the heart and coronary arteries of rats¹¹⁶. Likewise, it was demonstrated that shear stress-induced NO down-regulates MMP-2 expression in cultured ECs and further that treatment of ECs with an NO donor under static culture conditions will mimic this shear stress-induced reduction in MMP-2¹¹⁷. In addition, inducible NOS (iNOS)-associated NO inhibits MMP-9, as activity of this MMP is induced after iNOS inhibition and inhibited by SMC-produced NO¹¹⁸. Conversely, NO derived from macrophages can suppress tissue inhibitor of matrix metalloproteinases (TIMP)-1, leading to increased MMP-9 and VSMC migration when assessed in an in vitro wound healing assay¹¹⁹, supporting a role of immune cell derived ROS in vascular remodeling. Thus, increases in oxidative stress and inflammation and decreases in NO bioavailability with aging may act in concert to alter both TGF-β and MMPs, important determinants of the structural components of the artery, thereby influencing arterial stiffness. Thus, all of the three major layers of the artery; the intima, media and adventitia, contribute to oxidative stress and inflammation that impair vascular function (in-depth reviews of each of these vascular components are available^{110, 120, 121}).

Interestingly, oxidative stress and inflammation act in a vicious cycle to negatively impact arterial function with aging. While ROS will act on NFκB to stimulate pro-inflammatory signaling, pro-inflammatory signaling itself can stimulate the local production of O₂^- by inducing transcription of redox-sensitive genes, such as those encoding subunits of NADPH oxidase¹²², and through the local recruitment of immune cells, which exacerbate the presence of inflammatory cytokines and also produce ROS to further promote a pro-oxidant environment. Indeed, advancing age is associated with increases in T and B cells as well as macrophages in the perivascular adipose tissue of both the aorta and mesenteric vascular arcade. Furthermore, aged splenic immune cells from these same mice demonstrate increased pro-inflammatory cytokine expression compared to cells isolated from young mice. Importantly, both the elevations in immune cell infiltration and the pro-inflammatory immune cell phenotype are prevented by CR¹²³. Interestingly, both gene expression and activity of NFκB are also reduced in nuclear extracts of arteries and ECs from CR compared to AL fed old mice^{41, 76}. Likewise, while the activity of the NFκB inhibitor IκB-α is decreased with aging^{105, 124}, this disinhibition of NFκB is attenuated after CR¹²⁴. Thus, a reduction in NFκB-associated inflammatory signaling may also contribute to improved vascular dysfunction in aged mice after CR. Taken together, it appears that inflammatory and oxidative pathways interact with advancing age to induce and perpetuate arterial dysfunction in a vicious cycle that can be broken by CR.

Autophagy.

Autophagy is a cellular housekeeping mechanism that helps to maintain homeostasis. Basal levels of autophagy are necessary to protect tissues from oxidative stress and inflammation by promoting the destruction of damaged proteins and organelles¹²⁵. However, inadequate autophagy will lead to the accumulation of damaged proteins and promote further oxidative stress and inflammation. Flux through autophagic processes is stimulated by intra- and extracellular cues and is aimed at promoting survival and minimizing destruction of required proteins and organelles. Intriguingly, autophagy is reduced in animal models of aging¹²⁶ and has recently been implicated as a modulator of longevity, with overexpression of an autophagy-related gene 5 (Atg5) leading to the induction of autophagy and an increase lifespan in mice¹²⁷. Furthermore, nutrient deprivation is a well-established stressor that stimulates autophagy¹²⁸, leading to the speculation that the effects of CR may be mediated by an induction of autophagy. This idea is supported by the finding that there is a lack of lifespan extension in response to dietary restriction in autophagy deficient, Beclin-1 knockdown nematodes¹²⁹. Thus, there appears to be a critical role for autophagy in CR-mediated lifespan extension, although the role of autophagy in age-related vascular dysfunction or in the vasoprotection afforded by CR has only recently begun to be explored.

Within the vasculature, autophagy has been implicated in diverse physiological and patho-physiological processes including angiogenesis, paracrine responses in the endothelium, vascular wall calcification, and atherosclerosis (reviewed in Nussenzeig et al.¹³⁰). In autophagy deficient, Atg3 knockdown ECs, there is a blunting of shear-induced eNOS activation and NO production that is associated with increases in both oxidative stress and inflammation¹³¹, providing evidence for a direct link between autophagy and endothelial function. Pharmacological evidence also supports this link, as inhibition of autophagy reduces, and activation enhances, the expression of eNOS in ECs¹³². Autophagy has also been associated with the modulation of eNOS uncoupling¹³³ and NO appears to regulate EC mitophagy (mitochondrial specific autophagy)¹²⁸.

Markers of autophagic flux are lower in both ECs of older adults and arteries of aged mice¹³⁴. Induction of autophagy after treatment with trehalose restores autophagic flux, and is associated with improvements in EDD and NO bioavailability as well as reductions in markers of oxidative stress and inflammation in the arteries of aged mice¹³⁴. Likewise, induction of autophagy with spermidine reduces arterial stiffness and improves EDD and NO bioavailability in aged mice. These vascular benefits are associated with a reduction in arterial O₂^-135. Although far from definitive, indirect evidence of a role for increased autophagy after CR comes from a study in cultured ECs. It was found that ECs treated with the CR mimetic, resveratrol, were protected against TNF-α induced inflammation through the induction of autophagy¹³⁶. Although it remains unknown if the vasoprotection afforded by CR is dependent on increases in autophagy, there is growing evidence that impaired autophagic flux plays a critical role in age-related vascular dysfunction, oxidative stress and inflammation.

Countermeasures to Vascular Aging

mTOR. Rapamycin originally was discovered in a soil sample from Easter Island and used as an antifungal metabolite¹³⁷. Through its inhibition of the mammalian target of rapamycin (mTOR), rapamycin also has anti-proliferative and immunosuppressant properties¹³⁸. mTOR is a highly conserved serine/threonine kinase that responds to changes in energy balance and regulates many cellular functions, including translation, transcription, protein turnover, metabolism, and stress responses¹³⁹. There are currently two known mTOR signaling complexes: the rapamycin “sensitive” mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2); the latter is less sensitive to rapamycin and less studied within the vasculature. As such, in this review we refer to either mTORC1 signaling or general mTOR activity¹⁴⁰.

mTOR inhibition reduces mRNA translation and protein synthesis by activating ribosomal S6 protein kinase (S6K1) and inhibiting eukaryotic translation factor binding protein (4E-BP1)^{137, 141}. In early studies, yeast, flies, and nematodes demonstrated lifespan extension through genetic manipulation or rapamycin treatment that inhibited TOR signaling¹⁴². Harrison et al. reported similar findings in mammals, with rapamycin-induced mTOR inhibition leading to lifespan extension and a delay in age-associated phenotypes¹⁴³. These findings were further supported by a genetic model in which S6K1 knockout led to increased maximal lifespan and resistance to age-associated pathologies in mice¹⁴⁴. Taken together, these studies implicate mTOR signaling in aging and its associated diseases.

While traditionally used as an immunosuppressant in organ transplant patients, rapamycin was recently found to act as a tumor suppressant in certain cancers¹⁴⁵ and appears to have cardioprotective benefits^146–149. To the best of our knowledge, the first evidence of rapamycin’s cardioprotective benefits in humans were found in kidney transplant patients, in which a lower incidence of hypertension has been reported in patients treated with rapamycin for immunosuppression^{146, 147}. In a subsequent study, endothelial function, assessed by radial artery flow-mediated dilation, was also found to be greater in transplant patients treated with rapamycin¹⁴⁸. In the setting of advancing age, our group has found increased mTOR activation in arteries of old mice that is accompanied by impaired EDD and reduced NO bioavailability^{43, 84}, and that this can be reversed by 6 weeks of dietary rapamycin treatment⁸⁴. We also observed reductions in superoxide production and NADPH oxidase expression after rapamycin treatment in arteries of old mice⁸⁴. Thus, mTOR inhibition may exert some endothelial-protective effects by altering redox balance and/or inflammation in advanced age. Indeed, rapamycin treatment has also been shown to reduce liver mitochondrial ROS production in middle-aged mice¹⁵⁰, as well as increased oxidative stress response gene expression of the antioxidants CuZnSOD, SOD, and GSH reductase¹⁵¹. Moreover, in a rat model of Parkinson’s disease, rapamycin treatment was shown to be associated with lower oxidative stress and increased glutathione peroxidase activity¹⁵². Lastly, rapamycin reduced the ratio of reduced/oxidized glutathione (GSH/GSSG) in type 2 diabetic rat hearts, indicative of an improvement in oxidative stress after mTOR inhibition¹⁵³. In addition to its favorable effects on redox balance, rapamycin treatment also results in downregulation of the inflammatory cytokine, TNF-α¹⁵¹. These findings were further supported by pathway analysis that revealed an upregulation of free radical scavenging genes and a downregulation of NFκB signaling genes after rapamycin treatment¹⁵¹. Thus, it appears that inhibition of mTOR by rapamycin can impact both oxidative stress and inflammation, which may be one mechanism underlying greater NO bioavailability and EDD in rapamycin treated old mice (Figure 5).

Figure 5. Summary of the Interdependence of Klotho and Nutrient Sensing Pathways in the Face of Advancing Age and Nutrient Excess, and Strategies to Reverse Age-Related Arterial Phenotypes.

↓ represents a reduction, ↑ represents an increase, ↔ represents weak or conflicting evidence and? Represents a lack of available data for the indicated outcome. Black arrows indicate pathway and drug interactions, with arrow points indicating induction and ovals indicating inhibition. The yellow box indicates that these pathways respond in a similar direction to the interacting pathway indicated by the black lines. AICAR, aminoimidazole carboxamide ribonucleotide; AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; NMN, nicotamide mononucleotide; SIRT, sirtuin; STACs, sirtuin activating compounds.

mTOR inhibition via rapamycin treatment also exerts a beneficial effect on central arterial stiffness. Rapamycin treatment in kidney transplant patients results in lower central arterial stiffness, assessed by PWV and carotid AI, compared with patients that receive cyclosporine treatment for immunosuppression¹⁴⁹. Lower central arterial stiffness in these patients was also accompanied by a reduction in blood pressure in the peripheral (brachial) and central (carotid) arteries¹⁴⁹. In old mice, we have shown that age-related aortic stiffening is attenuated following 6 weeks of dietary rapamycin treatment⁸⁴. In contrast to human data, rapamycin-mediated reductions in aortic stiffness in old mice were independent of blood pressure changes⁸⁴. The reductions in aortic stiffness after mTOR inhibition in mice are likely related to structural changes in the aorta that are independent of blood pressure, as reductions in aortic stiffness in old mice following 6-weeks of rapamycin treatment were accompanied by lower aortic collagen and AGEs content, although no difference in aortic elastin content was present between groups⁸⁴. Additionally, there may be other cardiac benefits associated with mTOR inhibition that may influence arterial stiffness, as rapamycin is a potential therapy for cardiac hypertrophy¹⁵⁴ and vascular restenosis¹⁵⁵. Although the results of studies utilizing mTOR inhibition to improve vascular aging phenotypes are promising, studies that directly target mTOR in the vasculature are scarce. Further study is warranted to determine if chronic inhibition of the mTOR pathway via rapamycin or other rapamycin analog treatments as well as whether more direct targeting of mTOR signaling in vascular cell types are efficacious to improve cardiovascular health in older adults.

AMPK.

AMPK is a highly conserved heterotrimeric serine-threonine kinase that is an important energy sensing signaling protein that integrates energy balance, metabolism, and stress resistance¹⁵⁶. AMPK is made up of a catalytic α subunit, a structural β subunit and the AMPK binding site containing γ subunit¹⁵⁷. AMPK activation requires phosphorylation of its α subunit, and occurs in response to increased AMP:ATP ratio, as well as physiological stimuli such as shear stress, heat shock, exercise, and hypoxia¹⁵⁷. Interestingly, blunting age-related physiological dysfunction after mTOR inactivation also results in increased AMPK activity¹⁴⁴; whereas AMPK activation results in direct mTOR inhibition via phosphorylation of Raptor, a critical mTOR adaptor protein^{158, 159}. Additionally, AMPK signaling has been shown to be upregulated in S6K1 knockout mice that have reduced mTOR signaling¹⁴⁴. Pharmacological AMPK activation can be achieved by aminoimidazole carboxamide ribonucleotide (AICAR) or metformin¹⁵⁷. AICAR is a direct activator of AMPK via phosphorylation at the α2 subunit¹⁵⁷, whereas metformin inhibits Complex I of the respiratory chain¹⁶⁰, indirectly elevating the AMP:ATP ratio¹⁶¹. Importantly, indirect activation of AMPK by metformin also stimulates glycolysis, which may result in numerous AMPK-independent effects that impact the transcription factors and kinases involved in other cell cycle and metabolic pathways (p53, p38, MAPK, PKC and Akt)^162–164. Therefore, metformin-mediated effects cannot solely be attributed to AMPK. Importantly, increased AMPK and decreased mTOR signaling are implicated in the longevity and physiological benefits associated with long term CR in mammals^{143, 144} and thus may be efficacious therapeutic targets to improve arterial function in advanced age.

AMPK activity has been shown to be reduced in the aorta¹⁶⁵ and cerebral arteries¹⁶⁶ of old rodents (Figure 5). In cultured ECs, AMPK is an activator of eNOS^{167, 168} that contributes to NO production via direct phosphorylation of eNOS^{169, 170}, as well as through Rac1 and Akt signaling ^{168, 171}. Using metformin treatment, AMPK activation has been shown to augment endothelial function in type 1 and 2 diabetic rodents^{172, 173}. Similarly, AICAR treatment in old mice increases EDD, although this improvement is not mediated by increased NO bioavailability¹⁶⁵. An NO independent effect of AICAR was also found after acute in vitro administration to isolated aortic rings¹⁷⁴. AMPK activators may impact endothelial function through transcriptional regulation of proteins involved in inflammation, mitochondrial biogenesis, fatty acid and cholesterol synthesis, glucose metabolism, cell growth and oxidative stress signaling¹⁵⁷. Indeed, after AMPK activation there is a reduction in inflammatory cytokines that is associated with blunted NFκB signaling in ECs^{175, 176}. Thus, AMPK activators appear to improve endothelial function, but likely do so in an NO independent mechanism. There are few studies that have investigated the beneficial effects of AMPK activation on central arterial stiffness. In response to enhanced AMPK activity after metformin treatment, reductions in aortic PWV were observed in premenopausal women with polycystic ovary syndrome¹⁷⁷. Although the effects of directly augmenting AMPK activity in old mice are unknown, accelerated high fat diet (HFD)-induced arterial stiffening and elevated collagen I expression are attenuated by AICAR in a mouse model deficient in the putative anti-aging gene, Klotho¹⁷⁸. Taken together, these findings suggest that augmenting AMPK signaling increases bioavailability of NO and vasodilatory responses^{168, 171, 179}, and although there is less direct evidence to suggest that increased AMPK activity lowers central arterial stiffness in advanced age, further study is warranted (Figure 5).

SIRTs.

SIRTs are a family of transcriptional regulators that play a major role in longevity¹²⁹ and inflammation¹⁸⁰. SIRTs were originally discovered in a screen for gene silencing factors in yeast and, therefore, given the name Sir2 (silent information regulator 2). Little research was conducted on the Sir2 family until these proteins were identified as critical regulators of longevity¹⁸¹. Thereafter, the mammalian Sir2 homologues, SIRTs 1–7, were quickly identified as nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases¹⁸² and ADP-ribosyltransferases. SIRTs 1–4 have been implicated in the control of cellular metabolism with SIRT-2, 3 and 4 expressed in the mitochondria and SIRT-1 expressed predominately in the nucleus with some cytoplasmic expression¹⁸³. The majority of the physiological benefits of CR on longevity can be attributed to the SIRT family and, specifically, SIRT-1^{183, 184}. SIRT-1 acts by deacetylating histone and non-histone proteins¹⁸⁵ to modify nuclear transcription factors, co-regulators and proteins to adapt gene expression in response to the cellular energy state and provide “stress resistance” by modulation of pro-inflammatory and oxidative stress pathways via deacetylating histone and non-histone proteins^{180, 185}. Evidence for a critical role of SIRT-1 on longevity comes from studies in genetic models in which SIRT-1 deletion abolishes the lifespan extension afforded by CR¹⁸⁶, while SIRT-1 overexpression recapitulates the CR phenotype¹⁸⁷. These longevity-associated properties of SIRTs can also be ascribed to SIRT-6, which is also a NAD-dependent nuclear deacetylase that regulates lifespan¹⁸⁸ and has been shown to be elevated in rats after CR¹⁸⁹. Although emerging evidence suggests SIRT-6 appears to play a role in endothelial function¹⁹⁰, the majority of vascular aging studies have focused on SIRT-1, which will be discussed further.

SIRT-1 activity has been associated with reductions in oxidative stress, inflammation and pro-apoptotic signaling, as well as improvements in insulin sensitivity, DNA damage repair, and telomere stability¹⁹¹. Age-related decreases in SIRT-1 expression are present in a multitude of tissues¹⁹², and our group has demonstrated age-related reductions in SIRT-1 expression and activity in the vasculature of humans and mice^{43, 52, 192, 193}, which are associated with endothelial dysfunction in old mice^{43, 192}. However, reduced SIRT-1 expression and endothelial dysfunction can be prevented by short and long-term calorie restriction^{41, 43, 52}. Additionally, lifelong transgenic whole-body overexpression of SIRT-1 prevents the age-related decline in endothelium-dependent vasodilation¹⁹⁴. SIRT-1 has been reported to modulate NO and endothelial function in small and large arteries directly via deacetylation and subsequent activation of eNOS^{192, 195}. To investigate mechanisms for which activation of SIRT-1 prevents/ameliorates age-related vascular dysfunction, several SIRT-1 activators have been investigated, with the majority of studies utilizing the naturally occurring polyphenol, resveratrol¹⁹⁶. Extremely high doses of resveratrol (2400 mg/kg of food) have been reported to improve EDD in isolated arteries from middle-aged animals after 1 year of treatment¹⁹⁷. Although there appears to be an improvement in the vascular aging phenotype after resveratrol treatment¹⁹⁸, there was no effect on lifespan^{197, 199, 200}. Additionally, resveratrol activates up to 15 other unique pathways, and is a potent antioxidant and phytoestrogen^{196, 201, 202}. Therefore, as with metformin, the pleiotropic resveratrol and other non-specific agents are not optimal to investigate mechanisms underlying the beneficial effects of SIRT-1, but these agents do afford insights as potential therapeutic agents.

Supplementation with nicotinamide mononucleotide (NMN), a key NAD⁺ intermediate, has also been shown to increase SIRT-1 activation²⁰³, resulting in reversal of mitochondrial dysfunction in advanced age²⁰⁴. In old mice, chronic NMN supplementation increases SIRT-1 activity and reverses age-related endothelial dysfunction and oxidative stress²⁰⁵, and has also recently been shown to increase skeletal muscle angiogenesis and blood flow leading to augmented endurance exercise capacity²⁰⁶. However, in middle-aged and older adult humans, augmented NAD⁺ activity via 6 weeks of supplementation with the NAD⁺ precursor, nicotinamide riboside, was not effective in improving EDD, as determined by brachial artery flow-mediated dilation²⁰⁷. In addition, direct activation of SIRT-1 can be achieved by treatment with the small molecule, SRT-1720, which has been shown to increase lifespan and preserve glucose tolerance in aged rodents²⁰⁸. In addition, our group has demonstrated that four weeks of SRT-1720 treatment restores SIRT-1 activity and reduces age-related NFκB acetylation, arterial inflammation and oxidative stress in old mice¹⁹³. Yet surprisingly, SRT-1720 improvements in EDD are mediated not by increases in NO, but by increased reliance on COX-2 vasodilators¹⁹³. Therefore, despite age-related reductions in vascular SIRT-1 that accompany reduced NO bioavailability and endothelial dysfunction, increased activation of SIRT-1 with SRT-1720 improves endothelial function and reduces inflammation and oxidative stress, but these effects are not associated with a restoration of NO.

SIRT-1 also plays a role in central arterial stiffness, as age-related reductions in aortic SIRT-1 expression are accompanied by elevated aortic stiffness in old mice (Figure 5)^{43, 192}. Moreover, transgenic whole-body overexpression of SIRT-1 prevents aortic stiffening in old mice¹⁹⁴, and, recently, our laboratory has observed lower aortic stiffness in all age groups (i.e., ages 3–24 mo), as well as a slower rate of aortic stiffening with advancing age in transgenic SIRT-1 overexpressing mice²⁰⁹. We also observed that a youthful aortic elastin:collagen ratio is maintained in 24 mo old transgenic SIRT-1 overexpressing mice, indicating that SIRT-1 overexpression prevents adverse structural changes in arteries, possibly through its negative regulation of MMP-9 and/or a positive regulation of TIMP-1^{210, 211}. Age-related reductions in aortic SIRT-1 expression and the associated aortic stiffness^{43, 192} can be prevented by CR^{41, 43, 52}. Pharmacologic SIRT-1 activation has also been shown to be beneficial for aortic stiffening. In non-human primates, high-fat and high-sucrose diet-induced increases in aortic stiffness can be prevented by resveratrol²¹². NMN supplementation-mediated increases in arterial SIRT-1 activity can reverse age-related aortic stiffening and normalize aortic collagen and elastin content in old mice²⁰⁵. Importantly, reductions in aortic stiffness, as well as blood pressure, have been reported in middle-age and older adults after 6 weeks of nicotinamide riboside supplementation²⁰⁷. Taken together these data suggest that SIRT-1 activation may hold significant promise for ameliorating endothelial dysfunction and aortic stiffening with aging or CVD. Future studies utilizing small molecule activators of SIRTs are needed to determine if they can ameliorate age-related CVD pathologies.

Klotho.

The Klotho gene was discovered in 1997 and named after Clotho, one of the three Fate sisters that was responsible for spinning the thread of human life in ancient Greek mythology. In the initial study, Kuro-o et al demonstrated that inactivation of Klotho resulted in a prematurely aged phenotype in mice characterized by infertility, arteriosclerosis, skin atrophy, osteoporosis, emphysema, as well as a short lifespan, with homozygous Klotho knockout (Klotho^−/−) mice dying at 8–9 weeks of age²¹³. Importantly, Klotho^−/− mice were indistinguishable from wild-type mice until 3–4 weeks old, indicating that Klotho deletion-related effects were not due to incomplete development²¹³. In follow up studies, transgenic overexpression of Klotho was shown to extend lifespan in mice by 20–30%²¹⁴, promote cardioprotection²¹⁵, and reduce oxidative stress²¹⁶. The Klotho protein appears to exist in two forms, as a transmembrane protein that is bound to fibroblast growth factor receptor and as a circulating protein following cleavage from its transmembrane form²¹⁷. As a circulating protein, Klotho appears to exert anti-aging effects by suppressing growth factor signaling and oxidative stress^{214, 216}. In general, Klotho declines with advancing age²¹⁸, resulting in a two-fold reduction in circulating Klotho concentrations from age 40 to 70 years in humans²¹⁹. Additionally, genetic variants of Klotho can affect its secretion, as heterozygous expression of the KL-VS Klotho variant results in greater Klotho secretion²²⁰ and is associated with longevity and cardiovascular health^{221, 222}. However, for unknown reasons, homozygous KL-VS Klotho variant results in lower Klotho secretion²²³, and is inversely associated with longevity and risk of CVD^{221, 222, 224}. Still other Klotho genetic variants are associated with a greater risk of CVD²²⁵. Although Klotho variants are common and appear to have some relevance in aging and CVD by altering Klotho secretion, further discussion on the intricacies of functional Klotho variants is beyond the scope of this review.

Whether Klotho is expressed within the arterial vasculature remains a highly controversial topic²²⁶, but there are reports that Klotho is expressed in both human and mouse arteries^227–229. Despite the controversy regarding arterial Klotho expression, whole-body Klotho haplodeficiency (Klotho^+/−), which results in lower circulating Klotho protein²³⁰, reduces aortic eNOS expression ²³⁰ and impairs aortic and resistance arterial endothelial function in mice²³¹ (Figure 5). Accordingly, Klotho deficiency-associated endothelial dysfunction can be reversed by parabiosis between wild-type and Klotho^+/− mice²³¹. Additionally, in Otsuka Long-Evans Tokshuma fatty (OLETF) rats, a model of type-2 diabetes and obesity, adenovirus-mediated Klotho gene delivery reverses aortic endothelium-mediated vasorelaxation and augments NO production²³². In addition to its endothelial protective role, Klotho appears to also exert hypotensive effects, as Klotho gene delivery in OLETF rats led to a lower systolic blood pressure and prevented medial hypertrophy in the aorta²³². Although this blood pressure lowering effect was not consistent between rodent models, as no reduction in blood pressure was observed after Klotho gene delivery in spontaneously hypertensive rats²³³, the underlying physiological mechanisms for elevated blood pressure in the spontaneously hypertensive rat may limit the generalizability of findings from that model. Nevertheless, there is an apparent inverse relationship between Klotho and aortic stiffness, as serum Klotho concentrations are ~45% lower in patients with elevated arterial stiffness and hypertension²³⁰. In mice, Klotho^+/− results in greater aortic stiffness and blood pressure that are accompanied by a reduced elastin: collagen ratio, as well as elevated MMP-2 and MMP-9 expression^{230, 234}. Despite equivocal findings on the ability of Klotho gene delivery to lower blood pressure, augmenting Klotho does appear to increase antioxidant defenses in rats and mice, and this is a likely mechanism underlying the improvements in endothelial function²³³.

In previous reviews we have commented on the interdependence of mTOR, AMPK, and SIRT-1 as they relate to the aging vasculature²³⁵, illustrating that decreased AMPK activity reduces SIRT-1 responses to low energy states²³⁶, whereas SIRT-1 directly interacts with the mTORC1 complex and SIRT-1 inhibition via nicotinamide increased mTOR activation²³⁷. We now expand on those interactions, update with recent findings, and also include a discussion of Klotho, as it appears to have some interaction with and/or governance over these pathways (Figure 5). Interestingly, Klotho^+/− mice have reduced aortic SIRT-1 and AMPK expression²³⁰. Consequently, HFD-induced arterial stiffening and elevated collagen I expression are attenuated by AICAR in Klotho^+/− mice¹⁷⁸, while Klotho deficiency-induced aortic stiffness and hypertension are ameliorated after SRT-1720 treatment in Klotho^+/− mice²³⁰. Moreover, vascular calcification, associated with increased mTOR activation, results in lower Klotho expression that can be reversed by rapamycin treatment²³⁷. Alternatively, rapamycin treatment is ineffective at reducing vascular calcification when Klotho is deleted in Klotho^−/− mice²²⁹. Collectively, these studies suggest that Klotho deficiency downregulates AMPK and SIRT-1 expression, which may contribute to age-related arterial stiffening²³⁰. Further, they suggest that while Klotho is required for some of the vascular benefits afforded by mTOR inhibition, mTOR activity also impacts Klotho expression.

Cellular Senescence in Vascular Aging.

Cellular senescence is defined as irreversible cell cycle arrest, and occurs in response to critically short telomeres, excessive mitogenic signals²³⁸, increases in extracellular or intracellular stressors like oxidative stress²³⁹, chromatin disruptions²⁴⁰, and DNA damage ²⁴¹. First described by Leonard Hayflick as an in vitro serial passage phenomenon in human fibroblasts, it is hypothesized that senescence develops in order to provide a critical tumor suppressive mechanism that prevents passing damaged DNA to daughter cells^{242, 243}. Senescence is induced by the two major tumor suppressor pathways, known as the tumor suppressor protein, p53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) and cyclin-dependent kinase inhibitor 2A p16/retinoblastoma-like protein 1 (pRB) pathways²⁴⁴. The preference toward one pathway versus another appears to be cell type-specific^{244, 245} with variation across species²⁴⁶. Currently, a preponderance of data suggests that senescence via the p53/p21 pathway is activated primarily by DNA damage and telomere dysfunction, while the p16/pRB pathway is linked primarily to mitogenic stress, chromatin disruptions, or general cellular stress^{244, 245, 247}, although exceptions can be found. Senescent cells are characterized by a pro-inflammatory, oxidant senescence-associated secretory phenotype (SASP)²⁴² that occurs within a few days of senescence induction and appears to be irreversible due to stable chromatin modifications around clusters of SASP genes^{248, 249}. The release of classical SASP inflammatory mediators such as, IL-6, IL-1, IL-8, TNF-α, and monocyte chemoattractant protein-1 (MCP-1), as well as ROS production occurs when vascular cells are passaged to senescence^{250, 251} and can be found in ECs sampled from older adults⁴³. The SASP likely reinforces cell cycle arrest in an autocrine fashion and activates immune cell surveillance of senescent cells^{242, 252}. Furthermore, several studies support a major role for elevated NFκB activation in the induction and maintenance of the SASP in human and rodent cells, which, as previously mentioned, is a major inflammatory hub in age-related inflammation^{253, 254}. Within the vascular compartment, senescent cells have been associated with a host of CVD states, such as atherosclerotic plaque²⁵⁵, abdominal aortic aneurysm²⁵⁶ and hypertension²⁵⁷. Furthermore, we and others have recently shown that senescent cells accumulate in normal arterial tissue over the lifespan of humans^{258, 259}. We demonstrated that markers of senescence are increased in ECs taken from arteries and veins of older healthy adults. Importantly, there was a strong negative correlation between a marker of endothelial function (brachial artery flow-mediated dilation) and cyclin dependent kinase inhibitor, p16, such that higher expression of endothelial senescence markers was negatively associated with endothelial function. This demonstrated, for the first time, that in the absence of atherosclerosis, humans accumulate senescent ECs in vivo, which are in turn linked with attenuated endothelial function. This relation has been scientifically demonstrated in aged mice, where the clearance of senescent cells by genetic manipulation and removal of p16 positive cells or senolytic treatment to clear senescent cells leads to improved vascular endothelial function²⁶⁰.

Taken together, it is clear that vascular cell senescence is sufficient to blunt vascular function with advancing age. Conceptually, vascular cell senescence likely contributes to the spread of vascular inflammation and oxidative stress from senescent cells to healthy non-senescent neighboring cells via the SASP (Figure 6). Thus, promoting chronic low-grade inflammation and subsequent oxidative stress in non-senescent vascular cells. In summation, these alterations blunt NO and endothelial function and could lead to elevations in large artery stiffness through chronic matrix remodeling. Lastly, this chronic inflammation and oxidative stress could persist and accumulate indefinitely in the vasculature until removal of the senescent cells. Therefore, until clear mechanisms of age-related vascular senescence are elucidated, therapeutic options targeting senescence mediated dysfunction will be limited to senolytics or to the chronic treatment of the associated inflammation or oxidative stress pathways. While the precise mechanisms of cellular senescence induction are still unclear, several proof of concept hypotheses have been tested in relation to genomic instability which will be discussed further in the next section.

Figure 6. DNA Damage with Aging.

(A) With advancing age, DNA damage resulting from reactive oxygen species (ROS) or mechanical stress accumulates leading to mutations or chromosomal instability that ultimately contributes to cellular senescence or altered gene expression that drives the age-related pro-inflammatory and pro-oxidant vascular phenotype. This cellular dysfunction acts in an autocrine and paracrine manner affecting the local milieu and exacerbating endothelial dysfunction. (B) Indeed, DNA damage induced by ionizing radiation leads to cellular senescence and vascular dysfunction. Likewise, experimental manipulation genes involved in telomere capping, such as telomeric repeat binding factor (TRF)-2, and those involved in both nuclear DNA repair, including excision repair cross-complementation group (ERCC)-1/2 and ataxia-telangiectasia mutated (ATM) kinase, as well as in mitochondrial (Mt) DNA repair, such as polymerase gamma (POLG), will lead to increases in cellular senescence, vascular dysfunction and atherogenesis. (Illustration Credit: Ben Smith).

Age related Genomic Instability: Inducer of vascular senescence and dysfunction

One emerging molecular mechanism that explains the occurrence of senescent vascular smooth muscle cells and ECs in older adults is genomic instability. Genomic instability, or DNA damage acquired over time, is an important mechanism that may underlie the age-related accumulation of senescent cells reported in arterial tissues^{258, 259}. Age-related genomic instability in vascular cells can occur from a variety of genotoxic insults, including oxidative stress^{261, 262} and mechanical stress²⁶³, resulting in potentially harmful mutations or chromosomal instability. It has been well described that augmented DNA damage via ionizing radiation leads to a succession of events including vascular apoptosis, cellular senescence, vascular dysfunction and subsequent end organ damage (reviewed in detail by Wang et al.²⁶⁴). In recent years, numerous studies have highlighted the role of dysregulated DNA damage repair pathways in the development of vascular cell senescence, dysfunction and/or pathology. Specifically, reduced expression of a major double stranded DNA (dsDNA) break repair protein, ataxia-telangiectasia mutated (ATM) protein kinase, via haploinsufficiency, accelerates the progression of atherosclerosis in an ApoE model²⁶⁵. This atheroprone phenotype is associated with elevated mitochondrial ROS production, highlighting the interplay between genomic damage and mitochondrial function²⁶⁵. Furthermore, mutations in two other DNA damage repair factors result in more subtle phenotypes typical of those seen in aged mice. Deficiency of the DNA excision repair proteins 1 and 2 (ERCC 1 and 2) and advanced age in mice both result in profound increases in senescent cells within vasculature, increases in large artery stiffening, systolic blood pressure and endothelial dysfunction²⁶⁶. Interestingly, the emergence of these phenotypes only occurs with chronological aging of the mice, evidence that it likely takes time for the DNA mutations and damage to accumulate, and it is through their accumulation that senescence and subsequent vascular dysfunction is induced. Similarly, mutations in mitochondrial DNA can influence vascular function. Accumulation of mitochondrial DNA damage in mice that lack a critical mitochondrial polymerase gamma (POLG), a catalytic unit of mitochondrial DNA polymerase, have accelerated atherogenesis in comparison to wild type POLG/APOE deficient mice²⁶⁷. This finding has been supported by human studies in which atheromas are more severe in arteries that display greater amounts of mitochondrial DNA damage²⁶⁷. Taken together, these data suggest that DNA damage is associated with vascular aging and associated CVDs, and that inducing DNA damage in rodent models is sufficient to produce an arterial aging phenotype.

Lastly, telomere dysfunction is another form of genomic instability that may lead to cellular senescence with advancing age. Telomere dysfunction, defined here as dsDNA damage response occurring at the telomere, can occur in several ways and will end in repair (if possible) or cell cycle arrest. First, telomere dysfunction can occur in response to critically short telomeres that are generated by numerous divisions, extensive genotoxic damage to the telomere, or by the cell being formed with very short telomeres. Some investigators estimate the critically short telomere length to be around 300–400 bp^{268, 269}. A critically short telomere will elicit a dsDNA damage signal due the substantially shortened telomere being unable to form the T-loop structure at the end of the chromosome. Breakdown of the T-loop structure gives the appearance of a dsDNA break, which in turn elicits a response from the DNA damage machinery²⁷⁰. This is the mechanism hypothesized to induce senescence in primary cells that are serially passaged²⁷¹. A second mechanism of telomere dysfunction is the occurrence of true dsDNA breaks at the telomeric DNA. Telomeric DNA is particularly guanine rich, making it particularly susceptible to oxidation²⁷². Therefore, it is feasible that this may directly promote dsDNA breaks and the subsequent response. Last, alterations in the shelterin proteins that form and maintain the T-loop may be damaged, dysfunctional, or present in insufficient amounts. This would also result in uncapping of the T-loop and a dsDNA response at the telomeric DNA. Importantly, we do not define shortening of telomeres per se as dysfunctional. Telomere length may provide important information about the proliferation status of immune cells with aging or possibly vascular cells in atherogenesis, but it provides little information on the status of the canonical signaling pathway necessary to induce cellular senescence. Furthermore, data suggest that mean telomere length in the vasculature is not associated with classic cellular senescence signaling via p53 and p21²⁵⁹. Therefore, when examining mechanistic determinants of cellular senescence via telomere dysfunction, telomere length alone is not sufficient, and a secondary measure of DNA damage at the telomere (IF-FISH, ChIP assay etc.) is likely necessary to capture the complete picture of telomere dysfunction within a cell or tissue.

Currently, while several studies have demonstrated telomere attrition in the vasculature with aging and age-related diseases, only one has shown that healthy aging results in age-related telomere dysfunction with dsDNA breaks localized to telomeric DNA²⁵⁹. Furthermore, in arteries from hypertensive patients, telomere length was similar to normotensive patients, but the arteries of hypertensive patients demonstrated greater telomeric dsDNA damage. Recent proof of concept studies suggest that telomere dysfunction is sufficient to induce arterial dysfunction, as critically short telomeres may induce endothelial dysfunction²⁷³. However, these effects may be confounded by the established beneficial effects of telomerase on vascular function that is mediated through reductions in mitochondrial ROS, rather than through direct effects on telomere length²⁷⁴. Furthermore, a recent study that utilized inducible deletion of the shelterin protein, telomeric repeat binding factor 2 (TRF2), suggests that telomere dysfunction and subsequent vascular senescence is sufficient to induce large and small artery endothelial dysfunction, loss of NO, and increases in systolic blood pressure²⁷⁵. Similarly, an atherogenic mouse model of TRF2 loss of function in vascular smooth muscle cells demonstrated increased atherosclerotic plaque formation and markers of senescence²⁷⁶. Therefore, induction of telomere dysfunction is sufficient to promote atherogenesis as well as age-related arterial phenotypes.

Taken together, mouse models of augmented DNA damage, blunted DNA damage repair (genomic or mitochondrial) and dysfunctional telomeres demonstrate age like vascular phenotypes which include senescence, inflammation, oxidative stress, endothelial dysfunction, large artery stiffness and elevated blood pressure. While this is promising, these animal models still do not provide mechanistic proof that these genomic changes are responsible for arterial changes seen in older animals or humans. Future studies need to provide data to demonstrate that manipulation of these pathways can attenuate or reverse age-related changes.

Role of the Vasculature in the Rate of Biological Aging.

While age-related arterial dysfunction is heavily tied to CVD, emerging research is also interested in elucidating the role of ageing in different vascular cells types (e.g., ECs and vascular smooth muscle cells) in diseases beyond traditional CVDs. The potential for vascular cells to influence age-related non-CVDs and lifespan can be appreciated by realizing that the human vasculature is expansive, residing in nearly every tissue of the body. It is also estimated that ECs represent 2.1% of the total cell number (6×10¹¹ total number of ECs) in the human body, making it one of the highest of the non-circulating cell types. For comparison, adipocytes are estimated at 0.2%, hepatocytes at 0.8% and red blood cells at ~84% of the total cells in the body²⁷⁷. Therefore, it is reasonable that the phenotype of ECs may independently influence the surrounding tissue, via alterations in blood flow, nutrient delivery, immune cell infiltration, permeability etc. and through their function as a secretory tissue, releasing bioactive molecules that could then alter the cellular homeostasis of the surrounding tissues. The latter is an interesting concept that we will explore in the next paragraphs.

Supporting a vascular-centric theory of biological aging, Hasegawa et al.²⁷⁸ reported reduced markers of cellular senescence (i.e. β galalactosidase staining) after reducing the pro-inflammatory NFκB pathway in ECs using the endothelial-specific (Tie2 Cre) dominant negative I kappa B-alpha (IkBα) in normal chow fed old mice. The mice also demonstrated blunted adhesion molecule expression and greater systemic NO bioavailability, and prevention of age-related increases in blood pressure. Interestingly, lifespan also increased, and while the group size was small (N=21–25) for a lifespan cohort study, these are promising data that should stimulate future studies to better understand the role of endothelial inflammation, senescence and NO in lifespan. An increased lifespan in this animal model is particularly compelling as the cause of death in aged rodents is not typically cardiovascular in nature, therefore implying that this manipulation may alter cancer and kidney diseases which are the major causes of death in laboratory rodents²⁷⁹. Hasegawa et al. also examined the role of attenuated endothelial NFκB signaling in high fat diet-induced insulin resistance, an expression of metabolic dysfunction that is similar to that which occurs as a consequence of aging in mouse models²⁷⁸.

Role of Vascular Aging in Metabolic Dysfunction.

Genetic blockade of pro-inflammatory signaling in the endothelium prevented obesity- and age-associated systemic insulin resistance, adipose inflammation, and was associated with increases in skeletal muscle blood flow²⁷⁸. These studies provide direct evidence of a role of endothelial function in the control of metabolic homeostasis and add to the evidence that dysfunction of the arteries contributes to age-associated metabolic syndrome/insulin resistance. The prevalence of metabolic syndrome, increases with advancing age, is associated with peripheral insulin resistance²⁸⁰ and increases patient risk for diabetes and CVDs, as well as CVD and all-cause mortality²⁸¹.

In the setting of metabolic syndrome²⁸² and aging²⁸³, the adipose is a metabolically important endocrine organ, contributing significantly to systemic inflammation. With aging and obesity, immune cells infiltrate adipose tissue and become important sources of circulating cytokines and adipokines that alter systemic insulin sensitivity. Because the endothelium is the site of immune cell adhesion, and is itself a source of inflammatory cytokine and adhesion molecules, the vascular endothelium plays a critical role in the maintenance of metabolic homeostasis via modulation of tissue inflammation²⁸⁴. In the setting of aging, reduced EDD and impaired angiogenic sprouting in adipose tissue have been associated with metabolic dysfunction and adipose tissue inflammation and oxidative stress²⁸⁵, suggesting that endothelial function in the adipose tissue may be an important determinant of metabolic homeostasis with advancing age.

Adipose tissue blood flow also influences metabolic and endocrine dysfunction in insulin resistant states. Both fasted and postprandial blood flow to adipose tissue is impaired in obesity, and may contribute to altered lipid metabolism and increased CVD risk²⁸⁶. There is also tight coupling between the vascular and metabolic functions in the adipose tissue, with adipose tissue blood flow and lipolysis increasing in response to β-adrenergic stimulation, and vasoconstriction and suppression of lipolysis in response to α-adrenergic stimulation²⁸⁷. Thus, adrenergic modulation of vascular tone and lipolysis has an important role in the normal control of metabolism. Taken together, these studies provide evidence that systemic metabolic homeostasis is importantly modulated by vascular control of tissue blood flow and by inflammation.

Influence of Vascular Aging on Alzheimer’s Disease and Cognitive Function.

AD pathology is another condition in which the role of vascular dysfunction is becoming increasingly appreciated. The pathology of AD has been well characterized with the accumulation of both amyloid beta (Aβ) plaques, neurofibrillary tangles (aggregates of Tau) and inflammation in neurons and in the neurovascular unit²⁸⁸. Although the vascular contribution to non-familial AD progression was overlooked for A long time, it is now clear that the vasculature and CVD risk factors influence the pathological progression of AD²⁸⁹. A recent meta-analysis demonstrated that AD risk is increased in patients who have had a prior stroke²⁹⁰, but stroke alone may not completely explain vascular links to AD. For example, autopsies performed on deceased patients in the Baltimore Longitudinal Study of Aging revealed that intracranial atherosclerosis increased dementia risk that was independent of the presence of AD-related pathologies or cerebral infarcts²⁹¹. Furthermore, age-related elevations in large elastic artery stiffness are associated with greater cerebral artery pulse pressure, cognitive impairments, and AD diagnosis^292–294. Taken together, these studies suggest that two of the major age-related arterial changes, large artery stiffness and cerebrovascular endothelial dysfunction, may play a critical role in the development of cognitive impairment and AD, both through increased stroke risk as well as through other, as of yet unknown, factors.

Emerging data that support the role of age-related large elastic artery stiffness and enhanced pulsatility in the development of cerebrovascular dysfunction and subsequent cognitive impairment and AD pathology has recently been reviewed in detail²⁹⁵. Briefly, increased large artery stiffness may propagate pulsatility to the cerebral circulation, disturbing auto-regulation via impaired myogenic tone²⁹⁶, with subsequent alterations in regional cerebral perfusion and impaired neurovascular coupling²⁹⁷. Other consequences include endothelial dysfunction, impairments in blood brain barrier function, altered angiogenesis, and perfusion-related outcomes^{298, 299}. Age-related changes in moment-to-moment cerebral blood flow regulation have been associated with altered neuronal activity²⁹⁷, and abnormal neurovascular coupling is associated with cognitive decline with aging³⁰⁰. Furthermore, a direct link between impaired neurovascular coupling, functional hyperemia and measures of spatial and recognition memory has been shown in a mouse model³⁰¹.

While all of these changes may underlie increasing neurovascular inflammation and neuronal damage, small artery cerebral endothelial dysfunction in particular may be involved in many etiologies involved in cognitive decline and AD. Indeed, endothelial dysfunction in the small cerebral arteries likely influences inflammatory/oxidative stress in the neurovascular unit, blood flow regulation via altered resistance artery tone, blood brain barrier function, and adhesion and/or infiltration of immune cells. Additionally, cerebral artery endothelial function may directly influence the β-amyloid precursor protein processing and the activity of critical Aβ production enzymes via endothelial NO production (for review, see³⁰²). Thus, age-related impairments in cerebral vascular function, due to enhanced large artery stiffness and endothelial dysfunction, are a likely initiation site for increased Tau and Aβ accumulation, neuroinflammation, and neurodegeneration, thereby contributing to future progression of cognitive impairment and AD.

Taking the evidence from metabolic syndrome and AD together, vascular dysfunction appears to be an important component of some chronic, age-related disease states in addition to those more traditionally thought of as CVDs. Gaining a better appreciation of the underlying mechanisms and regional differences in age-related vascular dysfunction may lead to improvements in treatments for these disorders with concomitant improvements in healthspan.

Conclusions

In summary, we have described the role of arterial aging in the progression of clinical CVD, which has a major societal impact via morbidity and mortality in the US, as well as the vast majority of developed nations. Also presented were several poignant examples of how the vasculature impacts non-CVD states, as well as evidence that the vasculature not only plays a role in mammalian healthspan but in lifespan as well. However, the role of the vasculature in non-CVD age-related diseases and lifespan is insufficiently understood, reinforcing the importance of research on the biological aging processes in this dynamic tissue. Age-related oxidative stress and inflammation in arteries interact to suppress vascular function, but this state can be prevented by lifestyle intervention to ultimately extend healthspan and lifespan. Emerging evidence suggests several promising pathways such as Klotho or energy-sensing pathways, e.g., SIRTs, mTOR and AMPK, that are attractive pharmacological targets for interventions, the manipulation of which have been demonstrated, in some cases, to restore normal youthful pathway activity in older mammals (Figure 2,5). While anti-aging therapies per se were not a sole focus of the present review, the reader is directed to a recent review expanding on this area of research³⁰³. Unfortunately, we as a research community still do not understand the critical initiating event(s) that alter the cellular machinery to induce the well described “vicious cycle” of oxidative stress and inflammation. While some focus on the effects of acute alterations in circulating lipids, glucose, or circulating factors associated with sedentary aging, others seek to define the role of molecular events as potential key initiators, as we described in detail with genomic instability and cellular senescence. Importantly, it is likely that these areas are related, suggesting the necessity for strong cross-field collaborations to best answer this critical question. Finally, treating older adults with prescription drugs to ameliorate age-related vascular dysfunction to prevent future CVD is not currently accepted in the absence of clinical disease. While clinical guidelines continue to be altered to allow earlier treatment (most recently, the hypertensive cutoff for systolic blood pressure being reduced from 140 to 130 mmHg³⁰⁴), the ethical and moral obligations to society to treat arterial dysfunction are complicated, as a single drug treatment or lifestyle intervention may not always be effective in reversing this state. Overall, despite impressive advancements in understanding the biology of the aging vasculature, as aging researchers, we must strive to better elucidate pathways to identify and develop appropriate countermeasures against age-related arterial dysfunction. Ultimately, to quote the phrase popularized by Sir William Osler in the late 19^th century, “you are only as old as your arteries”.

Abbreviations

4E-BP1

Eukaryotic translation factor binding protein

4-HNE

4-hydroxynonenal

AD

Alzheimer’s disease

AGE

Advanced glycation end product

AI

Augmentation index

AICAR

Aminoimidazole carboxamide ribonucleotide

AL

Ad libitum

Atg5

Autophagy-related gene 5

ATM

Ataxia-telangiectasia mutated

Aβ

Amyloid β

BH₄

Tetrahydrobiopterin

CAD

Coronary artery disease

cfPWV

Carotid-to-femoral pulse wave velocity

CR

Caloric restriction

CRP

C-reactive protein

CuZnSOD

Copper zinc superoxide dismutase

CVD

Cardiovascular disease

dsDNA

Double stranded DNA

EC

Endothelial cell

ecSOD

Extracellular superoxide dismutase

EDD

Endothelium dependent dilation

eNOS

Endothelial nitric oxide synthase

ERCC

DNA excision repair protein

GSH

Glutathione

GSH/GSSG

Reduced/oxidized glutathione

H₂O₂

Hydrogen peroxide

HFD

High fat diet

IKKβ

Inhibitor of nuclear factor kappa B kinase subunit beta

IkBα

I kappa B-alpha

IL

Interleukin

iNOS

Inducible nitric oxide synthase

MCP1

Monocyte chemoattractant protein-1

MDA

Malondialdehyde

miR

microRNA

MMP

Matrix metalloproteinases

MnSOD

Manganese superoxide dismutase

mTOR

Mammalian target of rapamycin

mTORC1

Mammalian target of rapamycin complex 1

mTORC2

Mammalian target of rapamycin complex 2

NAD+

Nicotinamide adenine dinucleotide

NFkB

Nuclear factor kappa B

NMN

Nicotinamide mononucleotide

NO

Nitric oxide

NT

Nitrotyrosine

O2-

Superoxide anion

OLETF

Otsuka Long-Evans Tokshuma Fatty

ONOO^-

Peroxynitrite

p21

Cyclin-dependent kinase inhibitor 1A

p53

Tumor suppressor protein p53

POLG

Mitochondria polymerase gamma

pRB

Cyclin-dependent kinase inhibitor 2A p16/retinoblastoma-like protein 1

PWV

Pulse wave velocity

RAAS

Renin-angiotensin-aldosterone system

ROS

Reactive oxygen species

S6K1

S6 protein kinase

SASP

Senescence associated secretory phenotype

SBP

Systolic blood pressure

SIRT

Sirtuin

SOD

Superoxide dismutase

TGF-β

Transforming growth factor-beta

TIMP

Tissue inhibitor of matrix metalloproteinases

TNF-α

Tumor necrosis factor alpha

TRF2

Telomere repeat binding factor 2

VCAM

Vascular cell adhesion molecule