Targeting mitochondrial fitness as a strategy for healthy vascular aging

Cardiovascular diseases (CVD) are the leading cause of death worldwide and aging is the primary risk factor for CVD. The development of vascular dysfunction, including endothelial dysfunction and stiffening of the large elastic arteries (i.e., the aorta and carotid arteries), contribute importantly to the age-related increase in CVD risk. Vascular aging is driven in large part by oxidative stress, which reduces bioavailability of nitric oxide and promotes alterations in the extracellular matrix. A key upstream driver of vascular oxidative stress is age-associated mitochondrial dysfunction. This review will focus on vascular mitochondria, mitochondrial dysregulation and mitochondrial reactive oxygen species (ROS) production and discuss current evidence for prevention and treatment of vascular aging via lifestyle and pharmacological strategies that improve mitochondrial health. We will also identify promising areas and important considerations ('research gaps') for future investigation.
REVIEW ARTICLE| JUNE 25 2020 Targeting mitochondrial fitness as a strategy for healthy vascular aging Matthew J. Rossman ; Rachel A. Gioscia-Ryan ; Zachary S. Clayton ; Michael P. Murphy ; Douglas R. Seals Crossmark: Check for Updates Clin Sci (Lond) (2020) 134 (12): 1491–1519. https://doi.org/10.1042/CS20190559 Article history Split-Screen Views Icon Views PDF Link PDF Share Icon Share Cite Icon Cite Get Permissions Abstract Cardiovascular diseases (CVD) are the leading cause of death worldwide and aging is the primary risk factor for CVD. The development of vascular dysfunction, including endothelial dysfunction and stiffening of the large elastic arteries (i.e., the aorta and carotid arteries), contribute importantly to the age-related increase in CVD risk. Vascular aging is driven in large part by oxidative stress, which reduces bioavailability of nitric oxide and promotes alterations in the extracellular matrix. A key upstream driver of vascular oxidative stress is age-associated mitochondrial dysfunction. This review will focus on vascular mitochondria, mitochondrial dysregulation and mitochondrial reactive oxygen species (ROS) production and discuss current evidence for prevention and treatment of vascular aging via lifestyle and pharmacological strategies that improve mitochondrial health. We will also identify promising areas and important considerations (‘research gaps’) for future investigation. Keywords:arterial stiffness, endothelial function, mitophagy, oxidative stress, reactive oxygen species Subjects:Aging, Cardiovascular System & Vascular Biology, Translational Science Cardiovascular diseases (CVD) remain the largest contributor to morbidity and mortality in both developed and many developing nations [1,2]. Aging is by far the strongest risk factor for CVD, with >90% of all deaths occurring in adults 50 years of age and older [1,2]. Importantly, the changing demographics of aging characterized by a shift toward older populations [3] predicts a progressive, marked increase in prevalence of CVD in the absence of effective intervention [4]. A key mechanism by which aging increases CVD risk is the development of vascular dysfunction [5,6]. A number of adverse changes to the vasculature occur with aging, but two major clinically relevant expressions are endothelial dysfunction, as assessed by reduced arterial dilation in response to endothelium-derived nitric oxide (NO), and stiffening of the large elastic arteries (i.e., the aorta and carotid arteries) [5,6]. In combination, endothelial dysfunction and arterial stiffening contribute to a ‘vascular aging’ phenotype that drives much of the adverse effects of age on CVD. Vascular endothelial dysfunction The vascular endothelium is a single-cell layer lining the lumen of blood vessels. Endothelial cells play a critical role regulating vasomotor tone, metabolism, immune function, thrombosis and many other processes via synthesis and release of a variety of vasoactive molecules [7]. A major vasodilatory and largely vasoprotective molecule released by endothelial cells is NO, which is produced in response to mechanical (i.e., blood flow) and chemical (e.g., acetylcholine [ACh]) stimuli by the enzyme nitric oxide synthase (eNOS); eNOS catalyzes the generation of NO from L-arginine and oxygen, with NO subsequently diffusing to vascular smooth muscle cells where it induces vascular smooth muscle relaxation and vasodilation [7]. Endothelial dysfunction occurs with aging and is characterized by a decline in endothelium-dependent dilation (EDD), largely as a consequence of reductions in NO, although changes in concentrations of vasoactive factors such as prostaglandins, endothelin-1, norepinephrine and angiotensin II also contribute [7]. NO-mediated EDD can be determined in pre-clinical models by assessing changes in artery diameter in response to flow in vivo [8,9] or changes in diameter of isolated artery segments ex vivo in response to mechanical or pharmacological stimuli, such as ACh [10]. In humans, the gold-standard non-invasive assessment of NO-mediated EDD is brachial artery flow-mediated dilation (FMD), in which the change in brachial artery diameter in response to increases in blood flow is determined [10,11]. Brachial artery FMD primarily assesses macrovascular (conduit artery) function. Microvascular (resistance vessel) function can be determined by measuring changes in blood flow in response to intra-arterial infusions of ACh and is primarily assessed in the forearm [10,11]. These experimental approaches all demonstrate reduced endothelial function with aging in pre-clinical models and humans [12–17]. Endothelial dysfunction is the major antecedent of atherosclerosis [5,18] and both reduced brachial artery FMD and lower forearm blood flow responses to ACh are independent predictors of CV events and CVD in middle-aged and older adults free from clinical disease in large, community-based cohort studies [19–21]. Large elastic artery stiffening The aorta and carotid arteries expand and recoil as blood is ejected into the arterial system by the left ventricle during systole [22]. This action limits arterial pulsatile pressures by providing a dampening function and protects the downstream microvasculature from potentially damaging fluctuations in blood pressure and flow [23]. Moreover, the elastic recoil of the aorta aids in the propulsion of blood to the periphery and maintains perfusion of the heart during diastole [22]. With aging, aortic stiffening results in blood being ejected into a stiffer aorta, which augments central systolic blood pressure because the ejected pressure wave travels at a higher velocity in stiffer arteries and is reflected by points of impedance such that the returning pressure wave reaches the heart at mid-to-late systole [22,24]. In addition, the greater forward moving pressure wave amplitude (from systolic ejection, prior to the return of wave reflections) is a major contributor to the age-related increase in central systolic blood pressure after age 60, particularly in women, as a consequence of a plateau or decrease in reflected wave amplitude [25,26]. The augmented systolic blood pressure, in turn, contributes to isolated systolic hypertension and results in a loss of diastolic pressure augmentation, such that aortic pulse pressure is widened [22,24]. Aortic stiffening therefore increases left ventricular afterload during systole, promoting left ventricular hypertrophy and dysfunction, and compromises coronary perfusion during diastole because of the reduced augmentation of diastolic pressure [24,27]. The loss of pulsatility-dampening effects of the aorta and the carotid artery also allows for transmission of high pulsatile pressures to the delicate small vessels in the microcirculation, which is particularly harmful for high-flow, low-resistance organs such as the brain and kidney, and a potential causative factor in target organ damage [23]. Structural changes to arteries, functional influences (i.e., factors influencing vascular smooth muscle tone) and the stiffness of vascular smooth muscle cells contribute to large elastic artery stiffening with aging [28,29]. The primary structural changes mediating arterial stiffening occur in the extracellular matrix and include degradation/fragmentation of elastin (e.g., by matrix metalloproteinases), an increase in the deposition of collagen and formation of advanced glycation end products (AGEs), which cross-link collagen fibers, increasing their stiffness [5,30,31]. Increased vascular smooth muscle tone is a consequence of changes such as reductions in NO and increased sympathetic nervous system, endothelin-1 and renin–angiotensin aldosterone system activity [32–34]. These factors also influence the intrinsic stiffness of the vascular smooth muscle cells, which adds to the stiffness of the arterial wall [29]. The mechanical stiffness of the large elastic arteries can be determined ex vivo in pre-clinical models by directly measuring properties such as compliance by creating stress-strain curves [35,36]. In vivo, arterial stiffness can be assessed in pre-clinical settings and humans with pulse wave velocity (PWV), which is a measure of the (regional) speed of the pulse wave generated by the heart when blood is ejected into the arterial system [22]. Aortic PWV is the predominant measure in rodents and carotid-femoral PWV is the reference standard measure of aortic stiffness in humans [10,22]. Carotid-femoral PWV increases with aging and is a strong, independent predictor of CVD risk in older adults [37,38]. Moreover, consistent with aortic stiffness-associated end organ damage, growing evidence supports an association between elevated carotid-femoral PWV and other age-related clinical disorders such as cognitive decline, dementia, including Alzheimer’s disease, and decreases in renal function/chronic kidney disease [39–43]. The local distensibility of the carotid artery can also be determined in humans by measuring carotid artery compliance (the change in artery diameter for a given change in arterial pressure) and determining the carotid distensibility coefficient (i.e., changes in artery diameter normalized to diastolic lumen diameter) and/or carotid beta-stiffness index, which is largely independent of blood pressure [10,22]. Carotid artery compliance is associated with incident stroke, independent of aortic stiffness [44]. Mechanisms of vascular dysfunction with aging The primary molecular mechanisms of vascular aging are oxidative stress and chronic, low grade inflammation [45,46] (Figure 1). Excessive production of reactive oxygen species (ROS) in combination with unchanged or decreased abundance/activity of antioxidant enzymes (e.g., superoxide dismutase, SOD) results in the development of oxidative stress in arteries with aging [24,45]. Excess superoxide rapidly reacts with NO to form the secondary reactive species peroxynitrite (ONOO−), decreasing the bioavailability of NO [24,45], causing endothelial dysfunction. Peroxynitrite is also the primary molecule that reacts with and oxidizes tetrahydrobiopterin (BH4), an essential co-factor for NO production by eNOS [47]. Loss of BH4 leads to eNOS uncoupling, whereby eNOS produces more superoxide and less NO, exacerbating oxidative stress and decreasing bioavailable NO and endothelial cell function [47]. Excess ROS also can activate pro-inflammatory networks such as those regulated by the transcription factor nuclear factor kappa B (NF-kB), which up-regulates the production of pro-inflammatory cytokines that can impair vascular function and activate other ROS producing systems and enzymes, creating an adverse feed-forward (vicious) cycle of inflammation and oxidative stress [24,45]. Figure 1 Aging is associated with mitochondrial dysfunction-induced increases in reactive oxygen species (ROS) and oxidative stress and increases in pro-inflammatory cytokine signaling and chronic low-grade inflammation. Together, these processes induce vascular dysfunction, featuring: (lower left) large elastic artery stiffening mediated by degradation of elastin fibers (blue), increased deposition of collagen (brown), and greater cross-linking of structural proteins by advanced glycation end-products (dashed connecting lines); and (right) vascular endothelial dysfunction characterized by reduced nitric oxide (NO) bioavailability and endothelium-dependent dilation. These and other changes to arteries, in turn, increase the risk of developing cardiovascular diseases, chronic kidney disease, and Alzheimer’s disease and related dementias. VIEW LARGEDOWNLOAD SLIDE Mechanisms of age-associated vascular dysfunction and related clinical disorders Aging is associated with mitochondrial dysfunction-induced increases in reactive oxygen species (ROS) and oxidative stress and increases in pro-inflammatory cytokine signaling and chronic low-grade inflammation. Together, these processes induce vascular dysfunction, featuring: (lower left) large elastic artery stiffening mediated by degradation of elastin fibers (blue), increased deposition of collagen (brown), and greater cross-linking of structural proteins by advanced glycation end-products (dashed connecting lines); and (right) vascular endothelial dysfunction characterized by reduced nitric oxide (NO) bioavailability and endothelium-dependent dilation. These and other changes to arteries, in turn, increase the risk of developing cardiovascular diseases, chronic kidney disease, and Alzheimer’s disease and related dementias. This overall state of oxidative stress and inflammation also contributes to arterial stiffening with aging by altering the structural properties of the arterial wall. Production of collagen by fibroblasts is stimulated by superoxide-related oxidative stress [30,48,49]. Matrix metalloproteinases are up-regulated and elastin content is lower in aorta of SOD-deficient mice, consistent with the concept that elastin degradation is induced by oxidative stress [50]. Vascular oxidative stress also promotes transforming growth factor β signaling and this, in turn, stimulates inflammation, which further reinforces arterial stiffness via activation of the pro-oxidant enzyme, NADPH oxidase [48]. AGEs interact with the receptor for AGEs to activate NFkB-regulated pro-inflammatory pathways and oxidative stress, which ultimately perpetuates arterial stiffening and further increases production of AGEs [51]. Mitochondrial dysfunction is emerging as a key source of vascular oxidative stress and contributor to age-related vascular dysfunction. The remaining sections of this article will focus on mitochondrial dysfunction as a driver of vascular aging and review current evidence for prevention/treatment of age-associated vascular dysfunction via lifestyle and pharmacological strategies that improve mitochondrial health. We will also discuss current ‘research gaps’ and future directions for the field. Vascular mitochondria, mitochondrial dysregulation and ROS Mitochondria are cytoplasmic organelles that are present in the majority of cell types in the human body, including vascular endothelial and smooth muscle cells. Mitochondria are often referred to as the ‘powerhouse’ of the cell for their role in ATP production by oxidative phosphorylation, which occurs via a series of electron transfers through the respiratory chain in the mitochondrial inner membrane that is coupled to ATP synthesis by the FoF1-ATP synthase by the protonmotive force across the inner membrane. However, mitochondria are also vital for a number of additional cellular processes, including regulation of metabolism, calcium homeostasis, immune function, cell growth and stem cell function, and cell death pathways. Although mitochondrial density in vascular tissues is considerably lower than other tissues such as skeletal muscle, liver and heart [52,53], increasing evidence indicates that these organelles are critical for maintenance of cellular and tissue homeostasis in the vasculature. This topic has been reviewed in detail elsewhere [54–61], but below we briefly summarize some of the key roles of mitochondria in the vasculature. A first important distinction is to consider the vascular cell type in question, as the density and subcellular distribution of mitochondria vary between endothelial and vascular smooth muscle cells, and indeed even among the same cell types in different vascular beds [54,60]. In general, unlike in highly metabolically active tissues with greater ATP demand, the principal role of mitochondria in the vasculature appears to be cellular signaling rather than energy provision [54]. Cellular energy demand is quite low in endothelial cells, and ATP demand is met primarily via glycolysis. However, endothelial mitochondria are critical in the regulation of calcium homeostasis, apoptosis/necrosis, cellular response to stress, and immune and inflammatory pathways. An essential feature of these roles is the regulated production of signaling molecules including redox-active molecules (reactive oxygen, nitrogen and other species; mtROS), mitochondrial DNA, mitochondria-derived peptides and damage-associated molecular pattern molecules (DAMPs), which exert effects intra- and extra-cellularly [62]. Importantly, there is cross-talk between mitochondrial and nuclear signaling pathways, whereby mitochondria-derived signaling is both influenced by and can influence nuclear events including gene expression [63]. Similarly, in vascular smooth muscle cells, mitochondria have an important role in cellular signaling. Mitochondria are involved in signaling pathways for regulation of vascular smooth muscle cell growth and proliferation (e.g., TGF-β activity) [64], as well as maintenance of the dynamic balance among synthesis and breakdown of extracellular structural proteins, including collagen and elastin (e.g., matrix metalloproteinase enzyme activities) [65]. There is also emerging evidence demonstrating interplay between mtROS signaling and inflammatory pathways known to be important for regulating vascular smooth muscle cell function, including those involving NFkB and the NLRP3-inflammasome [66–69], further highlighting the crucial role of mtROS in vascular homeostasis. Mitochondrial ROS The signaling functions of vascular mitochondria are thought to be mediated in large part by the production of ROS at low, physiological levels. However, the dysregulation of this mtROS production also has the potential to lead to pathophysiological sequelae that disrupt other mitochondrial functions, cellular homeostasis, and ultimately vascular function. The production of ROS by mitochondria can occur at several sites (Figure 2), including but not limited to the electron transport proteins, and this topic has been reviewed in detail elsewhere [54,60,70]. The most important sites for ROS production within mitochondria appear to be complexes I and III. These ROS are thought to be critical transducers of signaling mediated by mitochondria, leading to post-transcriptional modification of proteins and interactions with immune and inflammatory cellular pathways, although the mechanistic details are still uncertain. In the vasculature, the proximal mtROS species is superoxide, which is generated primarily at the electron transport chain in the mitochondrial inner membrane via interaction between oxygen and unpaired electrons, influenced by the proton motive force and the redox state of the coenzyme Q pool and integrity of intrinsic electron transport chain proteins [54,58,60,70]. Superoxide is released into the matrix (complex I) or into both the matrix and intermembrane space (complex III); it can also undergo dismutation to hydrogen peroxide by the antioxidant enzyme manganese superoxide dismutase (MnSOD) [59,60,62,70]. Hydrogen peroxide is also generated de novo on the surface of the mitochondrial outer membrane or in the intermembrane space mitochondria by p66SHC, a growth factor adapter protein referred to as a sensor/marker and ‘master regulator’ of mitochondrial redox signaling whose activity is indicative of the rate of mtROS production [71]. In addition, NADPH oxidase 4 (NOX4) is viewed as a primarily mitochondrial isoform of the NOX monoamine oxidase family of enzymes that contributes to mitochondrial hydrogen peroxide generation [72], although more research is needed to confirm the mitochondrial specificity of NOX4. Figure 2 Aging is associated with dysregulated mitochondrial quality control featuring reduced mitochondrial biogenesis (upper left) and reduced mitophagy (upper right), increased mitochondrial fission (upper middle right), reduced mitochondrial fusion (lower middle right), reduced mitochondrial stress resistance (lower right), increased mitochondrial DNA damage (middle left of mitochondria image) and increased bioactivity of mitochondrial reactive oxygen species (e.g., superoxide and other reactive oxygen species [ROS], middle of mitochondria image) relative to antioxidant defenses (e.g., manganese superoxide dismutase [SOD], lower right of mitochondria). VIEW LARGEDOWNLOAD SLIDE Mechanisms of age-associated mitochondrial dysfunction Aging is associated with dysregulated mitochondrial quality control featuring reduced mitochondrial biogenesis (upper left) and reduced mitophagy (upper right), increased mitochondrial fission (upper middle right), reduced mitochondrial fusion (lower middle right), reduced mitochondrial stress resistance (lower right), increased mitochondrial DNA damage (middle left of mitochondria image) and increased bioactivity of mitochondrial reactive oxygen species (e.g., superoxide and other reactive oxygen species [ROS], middle of mitochondria image) relative to antioxidant defenses (e.g., manganese superoxide dismutase [SOD], lower right of mitochondria). Mitochondria as source and target of oxidative stress Mitochondria are not only a key source of cellular ROS production but also particularly vulnerable to potential damage caused by these molecules. The extensive lipid bilayer membranes, circular DNA lacking the protective histones of nuclear DNA, and numerous enzymes and proteins that characterize mitochondria all represent potential targets for ROS-induced damage, which, in turn, has adverse effects on mitochondrial function [73]. Although mitochondria have endogenous antioxidant defense mechanisms, including MnSOD, catalase, the glutathione/glutathione peroxidase systems and the thioredoxin/peroxiredoxin pathway [74], excessive levels of mtROS can overwhelm these defense systems, resulting in mitochondrial oxidative stress. As such, oxidative damage to mitochondria results in an abundance of less healthy mitochondria. Importantly, mitochondrial quality control mechanisms exist to degrade dysfunctional mitochondria/mitochondrial components by mitophagy (organelle-specific form of autophagy) and generate new mitochondria by mitochondrial biogenesis (e.g., by PGC-1α-regulated processes) [75]. In addition, a balance in the mitochondrial dynamics processes of fission and fusion is critical for maintaining mitochondrial health/function and regulating mtROS production, at least in part by effects on bioenergetic function and mitochondrial membrane potential (e.g., reducing hyperpolarization) [76]. Indeed, dysregulation of mitochondrial dynamics processes—characterized by excess fission relative to fusion—promotes endothelial inflammation in an NFkB-dependent manner [77] and is necessary for the cellular senescence-associated inflammatory phenotype induced by angiotensin II [78]. All of these mitochondrial stress response/defense and quality control pathways become impaired in settings of excess mtROS such as aging, ultimately resulting in a pool of less healthy mitochondria, which may perpetuate mitochondrial dysfunction in part by further increasing mtROS [79]. In summary, due to the fundamental role of certain key mitochondrial processes as drivers of excess mtROS, they may be considered hallmarks of impaired mitochondrial health. Notable examples of this include: increased production of superoxide and other mtROS; mitochondrial DNA damage; decreased endogenous antioxidant defenses (e.g., MnSOD content/activity); dysregulated mitochondrial quality control (e.g., impaired mitophagy, decreased mitochondrial biogenesis and related PGC-1α signaling); impaired bioenergetic function with uncoupling of electron transport from ATP production; altered balance of mitochondrial dynamics resulting in excessive fission/insufficient fusion; and an overall reduction in the ability of mitochondria to adequately respond to stress (Figure 2). Therefore, interventions that target these processes and attenuate excessive mtROS have the potential to improve overall mitochondrial quality or ‘fitness’, with associated wide-ranging salutary effects on overall cellular function. Stress response as an indicator of mitochondrial fitness Loss of the ability to respond to stress is a common feature of the aging process [80] and many disease states. Mitochondria are vital for the ability of cells to maintain or restore homeostasis following exposure to stress—termed resistance and resilience, respectively [81]. Robust cellular stress resistance mediated by mitochondria is well established in cardiac tissues in the setting of cardioprotection against ischemia/reperfusion injury, for example [82]. Mitochondria are also vital for cellular adaptation following exposure to mild stressors through a process termed ‘mitohormesis’, as in the case of exercise training [83]. In contrast, dysregulation of mitochondrial health can lead to inadequate stress response, resulting in cellular damage or death. Indeed, impairment of mitochondrial stress resistance may be an integrative hallmark of mitochondrial dysregulation (Figure 2). Known vascular stressors, including hypoxia, inflammation, hyperglycemia, hyperlipidemia, oxidized low-density lipoprotein, and cigarette smoke, stimulate ROS production in mitochondria, activating signaling pathways that allow mitochondria-mediated adaptation to stress or initiation of cell death events [59,61]. Importantly, robust mitochondrial stress response appears to be a feature of healthy vascular function, whereas vascular disease is characterized by impaired mitochondrial stress response [60,84,85]. Functional implications of vascular mitochondrial dysfunction and excessive mtROS Although vascular mitochondrial production of ROS at physiological levels is critical for maintenance of cellular homeostasis, excessive levels of mtROS have detrimental effects on key aspects of vascular physiology. Endothelial function Excessive production of mitochondria-derived superoxide may contribute to vascular oxidative stress and reduce the bioavailability of NO, either directly via formation of peroxynitrite or indirectly by uncoupling of eNOS. These events are further propagated by peroxynitrite-mediated inhibition of an appropriate up-regulation of the mitochondrial antioxidant enzyme MnSOD [86]. Decreased NO bioavailability leads to impairments in endothelial function (as described above) but may also contribute to further mitochondrial dysregulation. NO has a key regulatory role in PGC-1α signaling and mitochondrial biogenesis [87]. Moreover, NO acts as a tonic inhibitor of complex IV of the mitochondrial respiratory chain; as such, decreases in NO bioavailability may also augment mitochondrial superoxide production by the electron transport chain as this tonic inhibition is removed [60]. Mitochondria-derived hydrogen peroxide is a key signaling molecule in the vasculature, as a compensatory vasodilatory mechanism for reduced NO bioavailability in the microvasculature and coronary arterioles in atherosclerotic heart disease [60]. However, as with superoxide, excessive levels of hydrogen peroxide production, either de novo or as a result of superoxide dismutation, can disrupt vascular homeostasis, including activation of NFkB with resultant prothrombotic and proinflammatory effects [60,88]. Arterial stiffening The majority of evidence suggests that oxidative stress is a critical upstream mechanism driving arterial stiffening with aging, although there is some indication for potential sex differences in the role of oxidative stress in this process [89,90]. Regardless, there is growing evidence that mtROS are a key source of this oxidative stress [72,91,92]. Excessive mtROS in vascular smooth muscle cells may induce aberrant signaling in growth factor (e.g., transforming growth factor β1) and proteolytic enzyme (e.g., matrix metalloproteinase) pathways that leads to overproduction of collagen and accelerated elastin degradation [50,65,93]. Further, mtROS are now recognized as important activators of pro-inflammatory signaling [67,68] in vascular smooth muscle cells that is also implicated in mediating structural changes in arteries [88,91,94]. Finally, excessive levels of mtROS may also contribute to oxidative stress-driven formation of AGEs and subsequent cross-linking of collagen in the arterial wall [95]. Vascular disease is characterized by excessive mtROS production and altered mitochondrial health Given the current understanding of mitochondrial biology and function specifically in vascular cells, the concept that excess mtROS and associated alterations in mitochondrial health may play an important causative role in vascular dysfunction and disease is compelling. In the following section, we outline current experimental evidence supporting a link between mitochondrial dysregulation and vascular dysfunction across a range of experimental settings and disease states, with a focus on vascular aging. The first line of evidence of an association between mitochondria and vascular dysfunction comes from cross-sectional studies in which chronic disease states characterized by vascular dysfunction are accompanied by elevated mtROS and/or markers of altered vascular mitochondrial health. Mitochondrial DNA damage is elevated in arteries from apolipoprotein E-null (to promote atherosclerosis) mice [96] as well as in plaques from human patients with atherosclerosis [97] and in circulating cells from patients with diabetes mellitus and atherosclerotic CVD [98]. Consistent with these observations, mitochondrial bioenergetics and mitophagy are impaired in naturally aged and aged atherosclerosis-susceptible mice [99], and excessive mtROS levels and disruption of mitochondrial dynamics are evident in endothelial cells from mice [100] and patients with diabetic vascular disease [101]. Moreover, rodent models of diabetic vascular disease demonstrate an impaired mitochondrial stress response to exercise [84,85]. These associations between mitochondrial oxidative stress and vascular dysfunction are corroborated by experimental approaches involving pharmacological manipulation and genetic knockout approaches to alter mtROS ex vivo and in vivo. For example, chronic low doses of angiotensin-II in mice elevate mtROS and induce endothelial dysfunction, at least in part by hyperacetylation-mediated impairment of MnSOD secondary to inactivation of the mitochondrial NAD+-dependent deacetylase sirtuin 3 [102,103]. Heterozygous knockout of the key mitochondrial antioxidant MnSOD to experimentally increase mtROS results in endothelial dysfunction [104] and acceleration of arterial stiffening with age [50]. Knockouts of p66SHC [105] and NOX4 [72], which recapitulate settings of decreased mtROS, exhibit preserved vascular function. Mice expressing a defective mitochondrial DNA polymerase with resulting excessive mitochondrial DNA damage exhibit accelerated aging, including development of vascular dysfunction [106]. In contrast, treating arteries ex vivo or supplementing rodents in vivo with mitochondria-targeted antioxidants to decrease mitochondrial oxidative damage ameliorates vascular endothelial dysfunction in spontaneously hypertensive rats and rats with angiotensin II-induced hypertension [107,108]. Similarly, mitochondria-targeted antioxidant administration improves cutaneous microvascular function in patients with chronic kidney disease [109] and EDD of arterioles isolated from adipose tissue biopsies in patients with Type 2 diabetes [110]. Taken together, the evidence from multiple experimental approaches, including genetically manipulated rodents, disease models and clinical populations indicates that mitochondrial oxidative stress may be a key upstream mechanism underlying vascular dysfunction. Primary vascular aging is accompanied by elevated mtROS and reduced mitochondrial fitness Accumulating evidence also indicates that mitochondrial oxidative stress and associated impairments in mitochondrial fitness underlie the vascular dysfunction accompanying primary aging in the absence of clinical disease. Arterial mtDNA quantity decreases with aging in mice and is associated with reduced mitochondrial respiration and arterial stiffening [106]. Excessive mtROS and activation of p66SHC in the face of reduced or unchanged abundance of MnSOD have been observed in vascular tissues from aged rodents (Figure 3) with corresponding impairments in vascular function [111,112], decreased mitophagy, evidence of reduced mitochondrial quality control [112,113], and greater susceptibility to acute mitochondrial stress [111,113,114]. In humans, expression of MnSOD is lower in vascular endothelial cells obtained by endovascular biopsies from older adults with impaired FMD compared with a young adult reference group [115], and impaired mitochondrial bioenergetics, elevated mtROS and impairments in EDD have been observed in biopsied arterial segments from older versus young adults [116,117]. More direct evidence for excessive mtROS-mediated suppression of vascular function with primary aging comes from studies in which acute scavenging of mtROS ex vivo in arteries from old mice and older adults or in vivo (humans) with the mitochondria-targeted antioxidant MitoQ reverses age-related endothelial dysfunction [111,117,118]. Figure 3 Vascular (A) mitochondrial superoxide production and (B) p66SHC are higher in old (Old Control) relative to young (Young Control) mice. The mitochondrial isoform of superoxide dismutase (SOD), manganese SOD (MnSOD), is lower in (C) aorta from old control compared with young control mice and (D) arterial endothelial cells from healthy older adults relative to young adult controls. Data are mean ± SEM. *P2 standard deviations from the group mean of young subjects in the Framingham Heart Study [148]), but had no effect in subjects with normal (low) levels of aortic stiffness [118] (Figure 7). The aortic de-stiffening effects of MitoQ were associated with an attenuation of declines in aortic elastin content and function in old mice. It is unlikely that structural changes in the arterial wall would occur over 6 weeks in humans, suggesting mechanisms other than changes in elastin content contributed to the decrease in aortic stiffness in humans. Rather, reduced vascular smooth muscle tone and/or stiffness secondary to enhanced NO signaling, i.e. effects on ´functional’ determinants of arterial stiffening, presumably mediated these beneficial effects in older humans. As mitochondrial ROS appear to stimulate sympathetic vasoconstrictor nerve activity [149], it is possible that MitoQ may also have decreased aortic stiffness through reductions in α-adrenergic mediated vascular smooth muscle tone. Collectively, these observations suggest that MitoQ and potentially other strategies directly targeting mitochondrial oxidative stress may be viable options for treating vascular dysfunction with aging; however, the pilot study findings must first be confirmed in a larger and longer duration clinical trial. Figure 7 Data from our laboratory demonstrating that oral MitoQ supplementation reduces arterial stiffness in (A) old mice and (B) older healthy humans with normal age-related increases in aortic stiffness (PWV > 7.6 m/s; right-hand panels [148], red shading) but not in young mice or older adults without normal age-related aortic stiffening at baseline (green shading) (left-hand panels). Data are mean ± SEM. *P 7.6 m/s; right-hand panels [148], red shading) but not in young mice or older adults without normal age-related aortic stiffening at baseline (green shading) (left-hand panels). Data are mean ± SEM. *P