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.

Life-long caloric restriction reduces oxidative stress and preserves nitric oxide bioavailability and function in arteries of old mice.

Aging impairs arterial function through oxidative stress and diminished nitric oxide (NO) bioavailability. Life-long caloric restriction (CR) reduces oxidative stress, but its impact on arterial aging is incompletely understood. We tested the hypothesis that life-long CR attenuates key features of arterial aging. Blood pressure, pulse wave velocity (PWV, arterial stiffness), carotid artery wall thickness and endothelium-dependent dilation (EDD; endothelial function) were assessed in young (Y: 5-7 month), old ad libitum (Old AL: 30-31 month) and life-long 40% CR old (30-31 month) B6D2F1 mice. Blood pressure was elevated with aging (P < 0.05) and was blunted by CR (P < 0.05 vs. Old AL). PWV was 27% greater in old vs. young AL-fed mice (P < 0.05), and CR prevented this increase (P < 0.05 vs. Old AL). Carotid wall thickness was greater with age (P < 0.05), and CR reduced this by 30%. CR effects were associated with amelioration of age-related changes in aortic collagen and elastin. Nitrotyrosine, a marker of cellular oxidative stress, and superoxide production were greater in old AL vs. young (P < 0.05) and CR attenuated these increase. Carotid artery EDD was impaired with age (P < 0.05); CR prevented this by enhancing NO and reducing superoxide-dependent suppression of EDD (Both P < 0.05 vs. Old AL). This was associated with a blunted age-related increase in NADPH oxidase activity and p67 expression, with increases in superoxide dismutase (SOD), total SOD, and catalase activities (All P < 0.05 Old CR vs. Old AL). Lastly, CR normalized age-related changes in the critical nutrient-sensing pathways SIRT-1 and mTOR (P < 0.05 vs. Old AL). Our findings demonstrate that CR is an effective strategy for attenuation of arterial aging.

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.

SIRT-1 and vascular endothelial dysfunction with ageing in mice and humans.

We tested the hypothesis that reductions in the cellular deacetylase, sirtuin-1 (SIRT-1), contribute to vascular endothelial dysfunction with ageing via modulation of endothelial nitric oxide synthase (eNOS) acetylation/activation-associated nitric oxide (NO) production. In older (30 months, n = 14) vs. young (5-7 months, n = 16) B6D2F1 mice, aortic protein expression of SIRT-1 and eNOS phosphorylated at serine 1177 were lower (both P < 0.05), and acetylated eNOS was 6-fold higher (P < 0.05), whereas total eNOS did not differ (P = 0.65). Acetylcholine (ACh)-induced peak endothelium-dependent dilatation (EDD) was lower in isolated femoral arteries with ageing (P < 0.001). Incubation with sirtinol, a SIRT-1 inhibitor, reduced EDD in both young and older mice, abolishing age-related differences, whereas co-administration with l-NAME, an eNOS inhibitor, further reduced EDD similarly in both groups. Endothelium-independent dilatation to sodium nitroprusside (EID), was not altered by age or sirtinol treatment. In older (64 ± 1 years, n = 22) vs. young (25 ± 1 years, n = 16) healthy humans, ACh-induced forearm EDD was impaired (P = 0.01) and SIRT-1 protein expression was 37% lower in endothelial cells obtained from the brachial artery (P < 0.05), whereas EID did not differ. In the overall group, EDD was positively related to endothelial cell SIRT-1 protein expression (r = 0.44, P < 0.01). Reductions in SIRT-1 may play an important role in vascular endothelial dysfunction with ageing. SIRT-1 may be a key therapeutic target to treat arterial ageing.

Voluntary wheel running restores endothelial function in conduit arteries of old mice: direct evidence for reduced oxidative stress, increased superoxide dismutase activity and down-regulation of NADPH oxidase.

Habitual aerobic exercise is associated with enhanced endothelium-dependent dilatation (EDD) in older humans, possibly by increasing nitric oxide bioavailability and reducing oxidative stress. However, the mechanisms involved are incompletely understood. EDD was measured in young (6-8 months) and old (29-32 months) cage control and voluntary wheel running (VR) B6D2F1 mice. Age-related reductions in maximal carotid artery EDD to acetylcholine (74 vs. 96%, P < 0.01) and the nitric oxide (NO) component of EDD (maximum dilatation with ACh and l-NAME minus that with ACh alone was -28% vs. -55%, P < 0.01) were restored in old VR (EDD: 96%, NO: -46%). Nitrotyrosine, a marker of oxidative stress, was increased in aorta with age, but was markedly lower in old VR (P < 0.05). Aortic superoxide dismutase (SOD) activity was greater (P < 0.01), whereas NADPH oxidase protein expression (P < 0.01) and activity (P = 0.05) were lower in old VR vs. old cage control. Increasing SOD (with 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl) and inhibition of NADPH oxidase (with apocynin) improved EDD and its NO component in old cage control, but not old VR mice. VR increased endothelial NO synthase (eNOS) protein expression (P < 0.05) and activation (Ser1177 phosphorylation) (P < 0.05) in old mice. VR did not affect EDD in young mice. Our results show that voluntary aerobic exercise restores the age-associated loss of EDD by suppression of oxidative stress via stimulation of SOD antioxidant activity and inhibition of NADPH oxidase superoxide production. Increased eNOS protein and activation also may contribute to exercise-mediated preservation of NO bioavailability and EDD with ageing.

Nuclear factor-{kappa}B activation contributes to vascular endothelial dysfunction via oxidative stress in overweight/obese middle-aged and older humans.

BACKGROUND: We tested the hypothesis that nuclear factor-kappaB (NF-kappaB) activity contributes to vascular endothelial dysfunction with aging and obesity in humans. METHODS AND RESULTS: We conducted a randomized, double-blind, placebo-controlled crossover study in 14 nondiabetic overweight or obese (body mass index > or =25 kg/m(2)) middle-aged and older (age 52 to 68 years) adults. Salsalate (nonacetylated salicylate, 4500 mg/d), a compound that inhibits NF-kappaB activity, or placebo was administered for 4-day periods. Plasma salicylate concentrations reached the midtherapeutic range (21.8+/-1.1 mg/100 mL, P< or =0.0001 versus placebo) by day 4 of salsalate treatment. Salsalate increased expression of the inhibitor of NF-kappaB and reduced total and nuclear expression of NF-kappaB in endothelial cells obtained from the subjects (all P<0.05). Salsalate increased brachial artery flow-mediated dilation by 74% (from 4.0+/-0.4% to 6.6+/-0.5%, P<0.001) but did not affect endothelium-independent dilation (P=0.83). The change in brachial artery flow-mediated dilation with salsalate was inversely related to baseline flow-mediated dilation (r=-0.77, P<0.01). Infusion of vitamin C increased brachial artery flow-mediated dilation during placebo (P<0.001) but not after salsalate (P=0.23). Salsalate reduced nitrotyrosine (P=0.06) and expression of NADPH oxidase p47(phox) (P<0.05) in endothelial cells obtained from the subjects but did not influence circulating or endothelial cell inflammatory proteins. CONCLUSIONS: Our findings provide the first direct evidence that NF-kappaB, in part via stimulation of oxidative stress, plays an important role in mediating vascular endothelial dysfunction in overweight and obese middle-aged and older humans.

Weight loss alone improves conduit and resistance artery endothelial function in young and older overweight/obese adults.

Obesity is associated with vascular endothelial dysfunction, as indicated by impaired endothelium-dependent dilation. Presently there is no direct evidence that energy intake-restricted weight loss alone improves conduit or resistance artery endothelium-dependent dilation, the mechanisms involved, or whether improvements differ with patient age. A total of 40 overweight or obese (body mass index: >or=25<40 kg/m(2)) nondiabetic men and women aged 21 to 69 years completed 12 weeks of reduced energy intake (n=26; 15 male) or attention control (n=14; 9 male) and 4 weeks of weight maintenance (randomized trial). Energy intake restriction reduced estimated total energy intake (33%), body weight (10.5%), total and abdominal body fat, plasma leptin, oxidized low-density lipoprotein, and improved several metabolic risk factors. Brachial artery flow-mediated dilation was increased by 30% (6.0+/-0.7% versus 7.9+/-0.7%Delta; P=0.01; n=17). Peak forearm blood flow during intrabrachial artery infusion of acetylcholine was increased by 26% (16.8+/-1.4 versus 21.1+/-1.9 mL/100 mL per minute; P<0.05; n=15); this was inversely related to the reduction in the abdominal visceral:subcutaneous fat ratio (r=-0.46; P<0.05) and was abolished by inhibition of NO synthesis with N(G)-monomethyl-L-arginine. Improvements in endothelium-dependent dilation were not related to age: mean increases in subjects >50 years of age were similar to or greater than those <50 years of age. Energy intake-restricted weight loss alone is an effective intervention for improving peripheral conduit and resistance artery endothelial function in young and older overweight/obese adults. The improvements in resistance artery function are mediated by an increase in NO bioavailability and are related to reductions in abdominal visceral fat.

Gender and bilateral differences in single-leg countermovement jump performance with comparison to a double-leg jump.

Expectations may be for both legs to function identically during single- and double-leg vertical jumps. However, several reasons might prevent this from occurring. The goals of this investigation were twofold: assess the presence of side-to-side jump height differences during single-leg jumps in a homogenous group of healthy subjects and determine if those with a jump height asymmetry possessed consistent biomechanical differences during single-and double-leg jumps. Thirteen men and 12 women with competitive volleyball experience volunteered for the study. Significance was assessed at p < 0.05. The men jumped significantly higher than the women in all conditions and possessed differences in several anthropometric, kinematic, and kinetic parameters. Based on a three-jump average, all subjects had one leg that they could jump higher with (the dominant leg, DL). The men generated significantly greater maximum ground reaction forces and ankle joint powers on their DL whereas the women had no differences during the single-leg jumps. The only side-to-side differences that existed during the double-leg jumps were in the average ground reaction forces during propulsion. These findings suggest that equality of single-leg jump performance is the exception rather than the norm, with identification of consistent biomechanical attributes difficult within a group.

Lower-extremity bilateral differences during step-close and no-step countermovement jumps with concern for gender.

The goals of this investigation were to characterize gender differences in step-close (SC) and no-step (NS) countermovement jumps, examine biomechanical differences of the lead leg (LL) and trail leg (TL) during the SC jump, contrast the LL and TL of the SC to those of the NS jump, and determine whether bilateral asymmetries of the SC jump transfer to NS jump performance. The SC jump differs from the NS jump by a lead-in step that is continuous with the ensuing countermovement. Recreationally competitive volleyball players (12 men and 12 women) volunteered for the study. Three maximal-effort attempts in each condition were analyzed. Ground reaction forces were measured with force platforms and lower-extremity kinematics with optical capture. Ground reaction force as well as anatomical flexion and extension plane joint angle, moment, and power maximum (or minimum) and average values during the propulsion phase were analyzed with significance assessed at p < 0.05. Differences existed between the men and women in anthropometrics and jump height, as well as in many of the joint angles and body weight-normalized kinetic parameters, suggesting that women would benefit from increased strength and power at the ankle, knee, and hip. Differences also existed in many of the parameters between the LL and TL of the SC jump. Subjects jumped higher in the SC condition with greater demands placed on the LL, with the TL often acting similarly to its behavior in the NS condition. A few asymmetries of the LL and TL in the SC jump at the ankle and knee were also present in the NS jump. Strength and conditioning programs should include activities, such as plyometric jumps, that incorporate a step-close technique to optimize the development of this jump style. To minimize the development of functional asymmetries, the LL should be alternated by sets or repetitions.

Bilateral asymmetries in max effort single-leg vertical jumps.

While asymmetries in the lower extremity during jumping may have implications during rehabilitation, it is not clear if healthy subjects should be expected to jump equivalently on each leg. Therefore, the goal of this study was to determine if asymmetries exist in maximal effort single-leg vertical jumps. After obtaining university-approved informed consent, 13 men and 12 women with competitive volleyball playing experience and no injuries of the lower-extremity that would predispose them to asymmetries participated. After thorough warm-up, five maximal effort vertical jumps with countermovement were performed on each leg (random order) with ground reaction forces and lower extremity kinematics recorded. The best three jumps from each leg were analyzed, assigning the leg with the highest jump height average as the dominant side. Asymmetry was assessed by determining statistical significance in the dominant versus non-dominant sides (p < 0.05). A significant interaction existed between side and gender for thigh length and peak vertical ground reaction force. Women had a significantly shorter thigh and men a greater peak vertical ground reaction force on their dominant side. All other parameters were assessed as whole group. Jumps were significantly greater off the dominant leg (2.8 cm on average). No other differences between sides were observed. Significant differences in magnitude (p < 0.05) existed between the men and women in jump height, several anthropometric parameters, minimum ankle and hip angles, and vertical ground reaction forces (peak and average). In conclusion, though a person may jump slightly higher on one leg relative to the other, and women may jump slightly differently than men, the magnitude of the difference should be relatively small and due to the multi-factorial nature of jump performance, individual parameters related to performance may not be consistently different.