Thomas J. LaRocca

Short-term calorie restriction reverses vascular endothelial dysfunction in old mice by increasing nitric oxide and reducing oxidative stress.

To determine if short‐term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n = 30) and young (Y, n = 10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, approximately 30%) for 8 weeks. Ex vivo carotid artery endothelium‐dependent dilation (EDD) was impaired in old ad libitum (OAL) vs. young ad libitum (YAL) (74 ± 5 vs. 95 ± 2% of maximum dilation, P < 0.05), whereas old calorie‐restricted (OCR) and YCR did not differ (96 ± 1 vs. 94 ± 3%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P < 0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P < 0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P < 0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (SOD) mimetic that restored EDD in OAL (to 97 ± 2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese SOD activity and sirtuin1 expression were reduced in OAL (P < 0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P < 0.05), but unaffected by CR. Carotid artery endothelium‐independent dilation did not differ among groups. Short‐term CR initiated in old age reverses age‐associated vascular endothelial dysfunction by restoring NO bioavailability, reducing oxidative stress (via reduced NADPH oxidase–mediated superoxide production and stimulation of anti‐oxidant enzyme activity), and upregulation of sirtuin‐1.

Translational evidence that impaired autophagy contributes to arterial ageing

•  Advancing age is the major risk factor for the development of cardiovascular diseases. •  Arterial endothelial dysfunction, characterized by impaired endothelium‐dependent dilatation (EDD), is a key antecedent to age‐associated clinical cardiovascular disease. •  We tested the hypothesis that changes in autophagy, the process by which cells recycle damaged biomolecules, may be an underlying cause of the age‐related reduction in EDD. •  We show that autophagy is impaired in arteries of older humans and mice with reduced EDD, and that enhancing autophagy restores EDD by reducing superoxide‐dependent oxidative stress and inflammation, and increasing nitric oxide bioavailability. •  Our results identify impaired autophagy as a potential cause of age‐related arterial dysfunction and suggest that boosting autophagy may be a novel strategy for the treatment of arterial endothelial dysfunction and prevention of cardiovascular diseases with ageing.

MicroRNA changes in human arterial endothelial cells with senescence: relation to apoptosis, eNOS and inflammation.

A senescent phenotype in endothelial cells is associated with increased apoptosis, reduced endothelial nitric oxide synthase (eNOS) and inflammation, which are implicated in arterial dysfunction and disease in humans. We tested the hypothesis that changes in microRNAs are associated with a senescent phenotype in human aortic endothelial cells (HAEC). Compared with early-passage HAEC, late-passage HAEC had a reduced proliferation rate and increased staining for senescence-associated beta-galactosidase and the tumor suppressor p16(INK4a). Late-passage senescent HAEC had reduced expression of proliferation-stimulating/apoptosis-suppressing miR-21, miR-214 and miR-92 and increased expression of tumor suppressors and apoptotic markers. eNOS-suppressing miR-221 and miR-222 were increased and eNOS protein and eNOS activation (phosphorylation at serine1177) were lower in senescent HAEC. Caveolin-1 inhibiting miR-133a was reduced and caveolin-1, a negative regulator of eNOS activity, was elevated in senescent HAEC. Inflammation-repressing miR-126 was reduced and inflammation-stimulating miR-125b was increased, whereas inflammatory proteins were greater in senescent HAEC. Development of a senescent arterial endothelial cell phenotype featuring reduced cell proliferation, enhanced apoptosis and inflammation and reduced eNOS is associated with changes in miRNAs linked to the regulation of these processes. Our results support the hypothesis that miRNAs could play a critical role in arterial endothelial cell senescence.

Habitually exercising older men do not demonstrate age-associated vascular endothelial oxidative stress

We tested the hypothesis that older men who perform habitual aerobic exercise do not demonstrate age‐associated vascular endothelial oxidative stress compared with their sedentary peers. Older exercising men (n = 13, 62 ± 2 years) had higher (P < 0.05) physical activity (79 ± 7 vs. 30 ± 6 MET hours per week) and maximal exercise oxygen consumption (42 ± 1 vs. 29 ± 1 mL kg−1 per minute) vs. sedentary men (n = 28, 63 ± 1 years). Brachial artery flow‐mediated dilation (FMD), a measure of vascular endothelial function, was greater (P < 0.05) in the exercising vs. sedentary older men (6.3 ± 0.5 vs. 4.9 ± 0.4%Δ) and not different than young controls (n = 20, 25 ± 1 years, 7.1 ± 0.5%Δ). In vascular endothelial cells sampled from the brachial artery, nitrotyrosine, a marker of oxidative stress, was 51% lower in the exercising vs. sedentary older men (0.38 ± 0.06 vs. 0.77 ± 0.10 AU). This was associated with lower endothelial expression of the oxidant enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p47phox subunit, 0.33 ± 0.05 vs. 0.61 ± 0.09 AU) and the redox‐sensitive transcription factor nuclear factor kappa B (NFκB) (p65 subunit, 0.36 ± 0.05 vs. 0.72 ± 0.09 AU). Expression of the antioxidant enzyme manganese superoxide dismutase (SOD) (0.57 ± 0.13 vs. 0.30 ± 0.04 AU) and activity of endothelium‐bound extracellular SOD were greater (6.4 ± 0.5 vs. 5.0 ± 0.6 U mL−1 per minute) in the exercising men (both P < 0.05), but differences no longer were significant after correcting for adiposity and circulating metabolic factors. Overall, values for the young controls differed with those for the sedentary, but not the exercising older men. Older men who exercise regularly do not demonstrate vascular endothelial oxidative stress, and this may be a key molecular mechanism underlying their reduced risk of cardiovascular diseases.

Mitochondria-targeted antioxidant (MitoQ) ameliorates age-related arterial endothelial dysfunction in mice

The development of age‐related arterial endothelial dysfunction, a key antecedent of increased cardiovascular disease (CVD) risk, is mediated largely by reduced nitric oxide bioavailability as a consequence of oxidative stress. Mitochondria are critical signalling organelles in the vasculature, which, when dysregulated, become a source of excessive reactive oxygen species; the role of mitochondria‐derived oxidative stress in age‐related vascular dysfunction is unknown. We show that a mitochondria‐targeted antioxidant, MitoQ, ameliorates vascular endothelial dysfunction in old mice and that these improvements are associated with the normalization of mitochondria‐derived oxidative stress and markers of arterial mitochondrial health. These results indicate that mitochondria‐derived oxidative stress is an important mechanism underlying the development of age‐related vascular endothelial dysfunction and therefore may be a promising therapeutic target. Mitochondria‐targeted antioxidants represent a novel strategy for preserving healthy vascular endothelial function in primary ageing and preventing age‐related CVD in humans.

The autophagy enhancer spermidine reverses arterial aging.

Arterial aging, characterized by stiffening of large elastic arteries and the development of arterial endothelial dysfunction, increases cardiovascular disease (CVD) risk. We tested the hypothesis that spermidine, a nutrient associated with the anti-aging process autophagy, would improve arterial aging. Aortic pulse wave velocity (aPWV), a measure of arterial stiffness, was ~20% greater in old (O, 28 months) compared with young C57BL6 mice (Y, 4 months, P<0.05). Arterial endothelium-dependent dilation (EDD), a measure of endothelial function, was ~25% lower in O (P<0.05 vs. Y) due to reduced nitric oxide (NO) bioavailability. These impairments were associated with greater arterial oxidative stress (nitrotyrosine), superoxide production, and protein cross-linking (advanced glycation end-products, AGEs) in O (all P<0.05). Spermidine supplementation normalized aPWV, restored NO-mediated EDD and reduced nitrotyrosine, superoxide, AGEs and collagen in O. These effects of spermidine were associated with enhanced arterial expression of autophagy markers, and in vitro experiments demonstrated that vascular protection by spermidine was autophagy-dependent. Our results indicate that spermidine exerts a potent anti-aging influence on arteries by increasing NO bioavailability, reducing oxidative stress, modifying structural factors and enhancing autophagy. Spermidine may be a promising nutraceutical treatment for arterial aging and prevention of age-associated CVD.

Physiological geroscience: targeting function to increase healthspan and achieve optimal longevity

Most nations of the world are undergoing rapid and dramatic population ageing, which presents great socio‐economic challenges, as well as opportunities, for individuals, families, governments and societies. The prevailing biomedical strategy for reducing the healthcare impact of population ageing has been ‘compression of morbidity’ and, more recently, to increase healthspan, both of which seek to extend the healthy period of life and delay the development of chronic diseases and disability until a brief period at the end of life. Indeed, a recently established field within biological ageing research, ‘geroscience’, is focused on healthspan extension. Superimposed on this background are new attitudes and demand for ‘optimal longevity’ – living long, but with good health and quality of life. A key obstacle to achieving optimal longevity is the progressive decline in physiological function that occurs with ageing, which causes functional limitations (e.g. reduced mobility) and increases the risk of chronic diseases, disability and mortality. Current efforts to increase healthspan centre on slowing the fundamental biological processes of ageing such as inflammation/oxidative stress, increased senescence, mitochondrial dysfunction, impaired proteostasis and reduced stress resistance. We propose that optimization of physiological function throughout the lifespan should be a major emphasis of any contemporary biomedical policy addressing global ageing. Effective strategies should delay, reduce in magnitude or abolish reductions in function with ageing (primary prevention) and/or improve function or slow further declines in older adults with already impaired function (secondary prevention). Healthy lifestyle practices featuring regular physical activity and ideal energy intake/diet composition represent first‐line function‐preserving strategies, with pharmacological agents, including existing and new pharmaceuticals and novel ‘nutraceutical’ compounds, serving as potential complementary approaches. Future research efforts should focus on defining the temporal patterns of functional declines with ageing, identifying the underlying mechanisms and modulatory factors involved, and establishing the most effective lifestyle practices and pharmacological options for maintaining function. Continuing development of effective behavioural approaches for enhancing adherence to healthy ageing practices in diverse populations, and ongoing analysis of the socio‐economic costs and benefits of healthspan extension will be important supporting goals. To meet the demands created by rapid population ageing, a new emphasis in physiological geroscience is needed, which will require the collaborative, interdisciplinary efforts of investigators working throughout the translational research continuum from basic science to public health.

You're Only as Old as Your Arteries: Translational Strategies for Preserving Vascular Endothelial Function with Aging

Endothelial dysfunction develops with age and increases the risk of age-associated vascular disorders. Nitric oxide insufficiency, oxidative stress, and chronic low-grade inflammation, induced by upregulation of adverse cellular signaling processes and imbalances in stress resistance pathways, mediate endothelial dysfunction with aging. Healthy lifestyle behaviors preserve endothelial function with aging by inhibiting these mechanisms, and novel nutraceutical compounds that favorably modulate these pathways hold promise as a complementary approach for preserving endothelial health.

Reduced vascular tetrahydrobiopterin (BH4) and endothelial function with ageing: is it time for a chronic BH4 supplementation trial in middle‐aged and older adults?

Ageing is associated with a reduction in endothelium-dependent dilatation (EDD) of both resistance and conduit arteries in humans and rodents (Eskurza et al. 2005; Delp et al. 2008). This is partly the result of a reduction in endothelial-derived nitric oxide (NO) bioavailability. The decrease in NO bioavailability probably is mediated by several possible mechanisms including a decrease in synthesis of NO by the enzyme endothelial NO synthase (NOS), increased degradation of NO by reactive oxygen species such as superoxide anion (O2·−), or a combination of both. A decrease in synthesis of NO is not likely to be mediated by a decrease in NOS protein expression or activity as there is no consistent evidence for such changes with ageing, and indeed, the opposite has been reported. Rather it is more likely that impaired NO synthesis is explained by enhanced oxidation or decreased synthesis of endothelial tetrahydrobiopterin (BH4), a key cofactor for the NOS enzymes that is obligatory for NO synthesis. BH4 is a naturally occurring enzyme cofactor which is synthesized de novo from guanosine 5′-triphosphate (GTP) and is controlled by the rate limiting enzyme GTP cyclohydrolase I (GTPCH I). Conversely, BH4 can also be generated via a ‘salvage pathway’ by the enzymes sepiapterin reductase and dihydrofolate reductase; however, the salvage pathway cannot compensate for a reduction in GTPCH I-mediated de novo synthesis of BH4. Interestingly, BH4 is a powerful reducing agent and is highly susceptible to oxidation by reactive oxygen species such as peroxynitrite. O2·− produced from NADPH oxidase and/or other enzymatic sources (xanthine oxidase, mitochondria, uncoupled NOS) reacts with NO and forms peroxynitrite before being inactivated by the enzymatic antioxidant superoxide dismutase. Because vascular oxidative stress develops with ageing, it is plausible that BH4 becomes oxidized leading to uncoupling of endothelial NOS with subsequent increased production of O2·−. However, direct experimental evidence to support this hypothesis is still lacking. A reduction in l-arginine as a result of increased degradation by the enzyme arginase I has been advanced as an alternative mechanism of endothelial NOS uncoupling and reduced NO-mediated EDD with ageing (Santhanam et al. 2007). Because the Km value of arginase I is significantly higher than that of NOS enzymes, under normal physiological conditions arginase I and NOS should not be in competition for l-arginine. However, Berkowitz and colleagues recently extended previous findings of the role of increased arginase I activity in the age-related impairment of EDD by demonstrating that arginase I is post-translationally modified in aged vessels by S-nitrosylation of two cysteine residues (Santhanam et al. 2007). They reported that NO produced from inducible NOS nitrosylates arginase I decreasing its Km value ∼6-fold, therefore increasing its affinity for l-arginine and supporting the idea that a reduction in l-arginine secondary to an increase in arginase I activity contributes to impaired NO bioavailability and EDD of vessels of aged rodents. In a recent study in The Journal of Physiology, Delp et al. (2008) addressed several important questions related to the contribution of l-arginine and BH4 bioavailability to impaired flow-induced EDD of the skeletal muscle microcirculation with vascular ageing. They first asked if an increase in arginase I activity in resistance arteries (soleus 1A arterioles) of aged rats leads to a decrease in EDD as reported in large conduit (aorta) arteries (Santhanam et al. 2007). Delp and colleagues demonstrated that ex vivo administration of the arginase inhibitor Nω-hydroxy-nor-l-arginine or administration of exogenous l-arginine to the bath did not improve EDD of arterioles in the older rodents. Consistent with these observations, they found no difference in l-arginine content in the arterioles between old and young rats. These findings indicate that a reduction in l-arginine as a result of either increased arginase I activity or some other mechanism does not contribute to impaired EDD of 1A arterioles in older rats. The reason for the divergent results between these studies is unclear, but it is possible that they reflect differences in regulation of vascular l-arginine between conduit and resistance arteries with ageing in rodents. Delp and colleagues next asked if the decrease in EDD is a result of a reduction in vascular BH4 content. Indeed, Delp et al. reported that total BH4 content of soleus 1A arterioles was reduced ∼60% in the older compared with the young rats. This is in contrast to findings reporting that BH4 content was not reduced in aortas of older compared with young mice (Blackwell et al. 2004). The explanation for these conflicting results is not clear, but differences in the arterial segments investigated (aorta versus arterioles) and/or species differences (rats versus mice) may be involved. Delp and colleagues also demonstrated that ex vivo administration of sepiapterin, a precursor for BH4, in isolated soleus 1A arterioles resulted in a 52% improvement in flow-induced EDD in old compared with young rats, although the age-related reduction in EDD was not restored to levels of the young animals. Moreover, improvement in EDD was abolished by administration of the NOS inhibitor Nω-monomethyl-l-arginine so that EDD was not different in old compared with young rats in the absence of NO production. These observations suggest that the ∼50% improvement in EDD from sepiapterin administration (i.e. BH4) in the older rats was NO mediated, but that additional mechanisms may be involved. In humans, Eskurza et al. (2005) demonstrated that a single high dose of oral BH4 restored EDD of the brachial conduit artery in sedentary older adults to that of young adults. That BH4 administration fully restored EDD in older humans, but not in the study by Delp and colleagues obviously could have been explained by inherent differences between humans and rodents. However, because intracellular levels of BH4 were not measured in the arterioles after administration of sepiapterin in the study by Delp et al. it is unknown if sepiapterin restored the vascular BH4 levels in the older rodents. Use of sepiapterin (as opposed to BH4) to increase intracellular BH4 levels depends on the activity of the salvage pathway enzymes sepiapterin reductase and dihydrofolate reductase. It is possible that the intracellular BH4 synthesis was limited in the older animals by reduced content and/or activities of these enzymes. Nonetheless, Delp and colleagues provide the first evidence that BH4 content (and not l-arginine) is reduced in vascular tissue of skeletal muscle arterioles with ageing in rodents and that exogenous administration of a BH4 precursor partially restores the age-related impairment of EDD. Several important questions remain to be answered concerning the role of BH4 and endothelial dysfunction with ageing. Is the decrease in BH4 with ageing mediated by increased oxidation of BH4 to BH2 and other biopterins, or is BH4 synthesis decreased (e.g. via a reduction in GTPCH I activity). Delp and colleagues measured only total BH4 and not the ratio of oxidized to reduced biopterins, and did not measure GTPCH I content or activity. Therefore, the mechanism for the age-related reduction in vascular BH4 remains to be determined. Next, is the decrease in vascular BH4 with ageing associated with reductions in BH4 content of endothelial cells, smooth muscle cells, or both? Delp and colleagues studied whole arterioles and therefore it is unclear which vascular cell type is responsible for the observed reduction in BH4. Lastly, is BH4 (or sepiapterin) administration acting as an antioxidant or restoring intracellular BH4 levels and ‘recoupling’ endothelial NOS? Some insight into this question was provided by Heitzer et al. (2000), who administered tetrahydroneopterin (NH4), a compound with similar antioxidant properties as BH4 but without the influence of endothelial NOS coupling, into the brachial arteries of chronic smokers. They demonstrated that in contrast to augmentation of impaired forearm EDD by brachial artery infusion of BH4, NH4 had no effect on EDD. Furthermore, results from studies using electron paramagnetic resonance spin suggest that O2·− scavenging by BH4 is not a major reaction in vivo (Vasquez-Vivar et al. 2003). Thus, these findings support the idea that administration of BH4 improves EDD by recoupling endothelial NOS and not by antioxidant actions. Perhaps most importantly, a final question to be answered is whether BH4 content and GTPCH I content/activity are reduced in conduit and/or resistance arteries with ageing in humans. However, although endothelial cell sampling techniques in humans are available, this will still be technically challenging because primary culturing of endothelial cells from humans is difficult and basal BH4 and GTPCH I protein expression are barely detectable in cultured endothelial cells. In conclusion, the study by Delp et al. (2008) provides further support for the potentially important role of reduced BH4 in mediating the age-associated impairments in EDD. However, regardless of whether endothelial BH4 content is reduced with ageing or the mechanism by which acute exogenous BH4 administration improves EDD, it may be time to determine the efficacy of chronic BH4 supplementation for restoring vascular endothelial dysfunction in middle-aged and older adults.

Dietary rapamycin supplementation reverses age‐related vascular dysfunction and oxidative stress, while modulating nutrient‐sensing, cell cycle, and senescence pathways

Inhibition of mammalian target of rapamycin, mTOR, extends lifespan and reduces age‐related disease. It is not known what role mTOR plays in the arterial aging phenotype or if mTOR inhibition by dietary rapamycin ameliorates age‐related arterial dysfunction. To explore this, young (3.8 ± 0.6 months) and old (30.3 ± 0.2 months) male B6D2F1 mice were fed a rapamycin supplemented or control diet for 6–8 weeks. Although there were few other notable changes in animal characteristics after rapamycin treatment, we found that glucose tolerance improved in old mice, but was impaired in young mice, after rapamycin supplementation (both P < 0.05). Aging increased mTOR activation in arteries evidenced by elevated S6K phosphorylation (P < 0.01), and this was reversed after rapamycin treatment in old mice (P < 0.05). Aging was also associated with impaired endothelium‐dependent dilation (EDD) in the carotid artery (P < 0.05). Rapamycin improved EDD in old mice (P < 0.05). Superoxide production and NADPH oxidase expression were higher in arteries from old compared to young mice (P < 0.05), and rapamycin normalized these (P < 0.05) to levels not different from young mice. Scavenging superoxide improved carotid artery EDD in untreated (P < 0.05), but not rapamycin‐treated, old mice. While aging increased large artery stiffness evidenced by increased aortic pulse‐wave velocity (PWV) (P < 0.01), rapamycin treatment reduced aortic PWV (P < 0.05) and collagen content (P < 0.05) in old mice. Aortic adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation and expression of the cell cycle‐related proteins PTEN and p27kip were increased with rapamycin treatment in old mice (all P < 0.05). Lastly, aging resulted in augmentation of the arterial senescence marker, p19 (P < 0.05), and this was ameliorated by rapamycin treatment (P < 0.05). These results demonstrate beneficial effects of rapamycin treatment on arterial function in old mice and suggest these improvements are associated with reduced oxidative stress, AMPK activation and increased expression of proteins involved in the control of the cell cycle.