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

Short-term calorie restriction reverses vascular endothelial dysfunction in old mice by... Introduction The risk of cardiovascular diseases is markedly increased in older adults and this is linked to the development of vascular endothelial dysfunction, most commonly demonstrated as impaired endothelium-dependent dilation (EDD) ( Celermajer et al. , 1994 ; DeSouza et al. , 2000 ; Lakatta & Levy, 2003 ). The latter is mediated by a reduction in the bioavailability of the dilating molecule nitric oxide (NO) and is linked to the development of vascular oxidative stress ( Luscher & Barton, 1997 ; Taddei et al. , 2001 ). Thus, therapeutic strategies that reduce vascular oxidative stress, increase NO bioavailability and reverse age-associated impairments in EDD have important clinical implications for the prevention of cardiovascular diseases in older adults. Calorie restriction, defined as a reduction in energy intake without malnutrition, extends lifespan and is associated with enhanced physiologic function in several species ( Weindruch & Sohal, 1997 ; Masoro, 2005 ). Although little is known about its potential effects on vascular aging, recent observations indicate that life-long calorie restriction preserves EDD with aging in rats ( Ungvari et al. , 2008 ; Csiszar et al. , 2009 ). This was associated with greater protein expression of endothelial NO synthase (eNOS) and evidence for less production of superoxide in large elastic arteries ( Ungvari et al. , 2008 ; Csiszar et al. , 2009 ), suggesting the possibility of enhanced NO bioavailability and reduced vascular oxidative stress. Recent findings indicate that shorter-term calorie restriction may produce some of the same effects on longevity and physiological function in rodents as life-long restriction of energy intake ( Cao et al. , 2001 ; Dhahbi et al. , 2004 ; Goto, 2006 ). However, it is unknown if short-term calorie restriction can improve or reverse vascular endothelial dysfunction associated with aging and, if so, the mechanisms by which this effect is mediated. In the present study, we used a recently established model of age-associated endothelial dysfunction in large arteries ( Lesniewski et al. , 2009 ) to test the hypothesis that short-term calorie restriction initiated late in life restores EDD by improving NO bioavailability as a result of reducing oxidative stress. We also determined the role of reduced arterial superoxide production, as well as the expression and activities of the oxidant enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and antioxidant enzymes in mediating these effects of calorie restriction. Arterial expression of the deacetylase sirituin 1 (SIRT1–silent mating type information regulation 2 homolog) was determined because it activates eNOS ( Mattagajasingh et al. , 2007 ) and is implicated in the physiological effects of calorie restriction ( Cohen et al. , 2004 ; Csiszar et al. , 2009 ). Because life-long calorie restriction can have anti-inflammatory effects ( Spaulding et al. , 1997 ; Ungvari et al. , 2008 ; Csiszar et al. , 2009 ), we also assessed arterial expression of inflammatory proteins. Finally, vasodilation in response to an NO donor (i.e., endothelium-independent dilation) was assessed to determine if improvements in EDD with calorie restriction might be mediated by increases in vascular smooth muscle responsiveness to NO. Results Food intake, body and fat pad mass, and metabolic characteristics Daily food intake during the 8-week experimental period was 10% less in old ad libitum (OAL) vs. young ad libitum (YAL) (4.4 ± 0.1 vs. 4.9 ± 0.1 g, P < 0.05), whereas all calorie restricted (CR) mice ate 4.1, 3.7 and 3.2 g, respectively, in weeks 1, 2 and 3-8. Body weight did not differ among the groups at baseline: YAL 32.8 ± 0.8 g, YCR 32.4 ± 1.2 g, OAL 34.8 ± 1.4 g, old calorie-restricted (OCR) 35.7 ± 0.9 g. Body weight decreased over the feeding period similarly in the calorie-restricted groups (YCR −9.6 ± 1.5 g, OCR -10.0 ± 0.9), whereas there were no significant changes from baseline in the ad libitum-fed groups (YAL +2.7 ± 1.1 g, P = 0.14; OAL −3.1 ± 0.8, P = 0.21). At the end of the treatments, epididymal white adipose tissue mass was lower in the calorie-restricted compared with the ad libitum-fed animals at both ages (YCR 0.15 ± 0.03 vs. YAL 0.71 ± 0.22; OCR 0.11 ± 0.02 vs. OAL 0.25 ± 0.04 g, both P < 0.05). Blood glucose and plasma triglycerides were lower in both CR groups compared with ad libitum-fed controls of the same age, but there was no significant change in plasma insulin, free fatty acids or cholesterol associated with short-term calorie restriction ( Table 1 ). Short-term calorie restriction restored EDD in old mice without influencing endothelium-independent dilation Carotid artery preconstriction to phenylephrine was not different in the ad libitum and CR groups ( P = 0.15). Peak carotid artery dilation in response to acetylcholine was impaired in OAL vs. YAL (74 ± 5% vs. 95 ± 2%, P < 0.05), but was preserved in OCR (96 ± 1%), not differing from YAL or YCR (94 ± 3%) (left panel Fig. 1 ). Neither vasodilatory sensitivity (IC 50 ) to acetylcholine (YAL: 2.1 E −8 ± 9.8 E −9 , YCR: 1.8 E −8 ± 3.8E −9 , OAL: 2.6 E −8 ± 1.43 E −8 , OCR: 2.6 E −8 ± 1.56 E −8 ) nor endothelium-independent dilation to sodium nitroprusside (right panel Fig. 1 ) differed among the groups (all P > 0.5). Short-term calorie restriction-related improvements in EDD in old mice were mediated by increases in NO bioavailability NO inhibition with L-NAME (N G -nitro-L-arginine methyl ester) reduced peak carotid artery dilation to acetylcholine in all groups ( P < 0.005). However, the reduction in carotid artery dilation to acetylcholine after pretreatment with L-NAME compared to acetylcholine alone (NO-mediated dilation, left panel Fig. 2 ) was smaller in OAL vs. YAL ( P < 0.05), and was associated with lower aortic eNOS protein expression ( P < 0.05, right panel Fig. 2 ). In contrast, NO-mediated dilation and eNOS were preserved in OCR compared with YAL and YCR. Superoxide-associated oxidative stress is reduced in short-term calorie restricted old mice and contributes to preserved EDD Nitrotyrosine staining (55-kDa protein band), a cellular marker of oxidant modification of tyrosine residues of proteins, was 80% higher in aorta of OAL vs. YAL ( P < 0.05), whereas staining in OCR was markedly lower than OAL ( P < 0.005), somewhat lower than YAL and similar to YCR (left panel Fig. 3 ). Aortic superoxide production, measured directly by electron paramagnetic resonance, was 150% greater in OAL vs. YAL ( P < 0.05). In contrast, superoxide production in OCR was similar to YAL and not significantly different than YCR (middle panel Fig. 3 ). TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl), a superoxide dismutase (SOD) mimetic, restored carotid artery dilation to acetylcholine in OAL to levels observed in YAL, but had no effect in OCR or the young groups (right panel Fig. 3 ). eNOS inhibition with L-NAME abolished the selective TEMPOL-mediated restoration of the peak vasodilatory response to acetylcholine in OAL such that there were no longer differences among the groups (right panel, Fig. 3 ). Together these observations are consistent with the idea that short-term calorie restriction reduced superoxide bioactivity, which, in turn, increased NO bioavailability and improved EDD. Down-regulation of NADPH oxidase contributes to reduced superoxide–associated improvements in EDD in short-term calorie restricted old mice NADPH oxidase activity and p67phox subunit expression were greater in aorta of OAL vs. YAL ( P < 0.05), whereas OCR demonstrated levels similar to YAL and YCR (left panel Fig. 4 ). Apocynin, an NADPH oxidase inhibitor, restored carotid artery dilation to acetylcholine in OAL to that observed in YAL, but had no effect in OCR or the young groups (middle and right panels Fig. 4 ). There were no differences in dilation among the groups in the presence of apocynin. Short-term calorie restriction increased manganese SOD activity in old mice Protein expression of manganese SOD (MnSOD) in aorta did not differ among the groups ( Fig. 5 ). However, aortic MnSOD activity was lower in OAL vs. YAL ( P < 0.05), whereas the activity in OCR was preserved at the levels of YAL and YCR ( Fig. 5 ). Catalase protein expression and activity were not significantly different among the groups (all P > 0.05, data not shown). Short-term calorie restriction increases SIRT1 expression in aorta of old mice, but has no effect on inflammatory cytokines Protein expression of the deacetylase SIRT1 was lower in aorta of OAL vs. YAL, whereas OCR demonstrated levels not significantly different from YAL and YCR ( Fig. 6 ). SIRT1 protein expression was positively related to peak carotid artery dilation in response to acetylcholine ( r = 0.63, P < 0.001). Expression of the inflammatory cytokines interleukin-1ॆ, interleukin-6, interferon े and tumor necrosis factor ॅ was greater in OAL vs. YAL and in OCR vs. YCR (all P < 0.05, Table 2 ). However, short-term calorie restriction had no significant effect on these inflammatory proteins in either young or old mice. Discussion EDD, NO bioavailability and eNOS Life-long restriction of energy intake preserves EDD in skeletal muscle arterioles and aorta of F344 rats ( Ungvari et al. , 2008 ; Csiszar et al. , 2009 ). Here, we extend these findings by showing that short-term calorie restriction initiated late in life completely reverses the impairment in carotid artery EDD observed in old B6D2F1 mice. Endothelium-independent dilation to sodium nitroprusside was unchanged with energy intake restriction, indicating that the improvements in EDD were independent of changes in vascular smooth muscle sensitivity to NO. Rather our findings show that the improvements in EDD were mediated by increases in NO bioavailability. This is supported by the fact that inhibition of NO production with L-NAME produced a suppression of acetylcholine-induced dilation in energy restricted old mice that was similar to that of young mice, such that EDD was not different in the ad libitum and CR groups in the absence of NO. It is possible that increased expression of eNOS protein contributed to the increase in NO bioavailability associated with short-term calorie restriction by increasing NO production. We found that eNOS in the aorta of the energy intake restricted old mice was approximately 30% greater than in old ad libitum-fed mice, was similar to young mice, and correlated with peak dilation in response to acetylcholine in all animals (r = 0.57, P < 0.05). This is consistent with previous observations that eNOS is greater in large arteries of old rats subjected to life-long calorie restriction than in ad libitum-fed old animals ( Ungvari et al. , 2008 ). Oxidative stress Independent of these increases in eNOS, our data demonstrate that the primary mechanism by which short-term energy intake restriction restores NO bioavailability and EDD in old mice is by reducing oxidative stress. Several lines of evidence support this idea. Nitrotyrosine, a marker of cellular oxidative stress ( Reiter et al. , 2000 ), increases with aging in vascular endothelial cells of humans and is inversely related to EDD ( Donato et al. , 2007 ). In the present study, nitrotyrosine was markedly lower in aorta of the OCR compared with old ad libitum-fed mice, and was similar to levels observed in young mice. This extends to aging arteries previous observations of reduced oxidative modifications in other tissues in response to short- or long-term energy intake restriction ( Gredilla et al. , 2001 ; Bevilacqua et al. , 2005 ). Aortic superoxide production, measured directly with EPR, was markedly elevated in old mice that were ad libitum-fed, but old mice that underwent short-term calorie restriction demonstrated much lower levels, not different than young mice. This finding is consistent with recent results using a fluorescent dye and chemiluminescence methods that suggest reduced superoxide production in arteries of life-long calorically restricted old rats compared with ad libitum-fed controls ( Ungvari et al. , 2008 ; Csiszar et al. , 2009 ). Finally, we found that a SOD mimetic restored EDD in ad libitum-fed, but not calorie-restricted old mice, indicating an absence of superoxide-mediated suppression of EDD in the latter group. This observation directly links the reduction in superoxide to improved vascular endothelial function, extending recent findings in life-long calorically restricted rats ( Csiszar et al. , 2009 ). We further extended these findings by showing that inhibition of NO production with L-NAME abolished the selective improvement in the EDD response to TEMPOL in the ad libitum-fed old mice. Considered together, our data provide evidence that short-term energy intake restriction restores carotid artery EDD in old mice by reducing superoxide-induced oxidative stress, which, in turn, increases NO bioavailability. NADPH oxidase Our results also provide insight into the mechanisms by which short-term calorie restriction reduced vascular oxidative stress in old mice. NADPH oxidase is a major source of superoxide production in arteries ( Zalba et al. , 2000 ) and is increased with aging in humans ( Donato et al. , 2007 ) and, as confirmed here, in ad libitum-fed rodents ( Csiszar et al. , 2007 ; Lesniewski et al. , 2009 ). In the present study, we found that both protein expression and activity of this enzyme were lower in aorta of old energy intake restricted mice and similar to that of young mice. We then connected these changes directly to function by showing that inhibition of NADPH oxidase with apocynin selectively restored EDD in old ad libitum-fed mice, indicating that the preserved EDD in the OCR mice was mediated primarily by reduced NADPH oxidase–mediated superoxide production. Antioxidant enzymes The expression of two important antioxidant enzymes, MnSOD and catalase, was not different in aorta of our groups. However, MnSOD activity was reduced in the old ad libitum-fed animals and this was increased to levels of young mice in the calorie-restricted old animals. That TEMPOL, a SOD mimetic, restored EDD in ad libitum-fed, but not calorie-restricted old mice supports the possibility that the increase in bioactivity of this endogenous antioxidant may have contributed to more effective scavenging of superoxide and reduced oxidative stress in arteries of the old energy restricted mice. The mechanism by which calorie restriction restored MnSOD in our old mice is unclear, but nitration of MnSOD reduces its catalytic activity ( Yamakura et al. , 1998 ; Guo et al. , 2003 ). Thus, reduced nitration of MnSOD could explain the increase in activity observed in the calorie-restricted old animals. Because MnSOD deficiency is associated with oxidative stress and endothelial dysfunction with aging in mice ( Brown et al. , 2007 ), the increase in MnSOD activity could have contributed to the improvement in EDD with calorie restriction in our old mice. SIRT-1 SIRT1 is a histone deacetylase that increases in response to calorie restriction in a variety of tissues ( Cohen et al. , 2004 ). Deacetylation by SIRT1 activates eNOS and increases NO production and EDD ( Mattagajasingh et al. , 2007 ). Protein expression of SIRT-1 is increased in coronary artery endothelial cells incubated with serum from calorie-restricted rats ( Csiszar et al. , 2009 ). In the present study, SIRT1 expression in aorta of old ad libitum-fed mice was approximately 40% lower than in young controls, and short-term energy intake restriction restored the expression to young levels. Moreover, peak EDD was positively related to expression of SIRT-1. These observations are in agreement with previous work in young mice demonstrating increases in aortic expression of SIRT1 after 1 year of calorie restriction and that manipulation of SIRT1 expression was consistently related to NO-mediated EDD ( Zhang et al. , 2008 ). Inflammatory cytokines Life-long calorie restriction may suppress vascular inflammation with aging ( Ungvari et al. , 2008 ; Csiszar et al. , 2009 ), and short-term calorie restriction inhibits signaling of the inflammatory nuclear transcription factor nuclear factor ॔B in the kidney of old rats ( Jung et al. , 2009 ). In the present study, protein expression of several pro-inflammatory cytokines was elevated in aorta of old compared with young ad libitum-fed mice; however, calorie restriction did not affect expression in either age group. These observations suggest that short-term restriction of energy intake may not exert anti-inflammatory actions in arteries of old or young mice and, thus, may not contribute to the improved vascular endothelial function in old animals. Circulating metabolic factors In the present study, circulating glucose and triglyceride concentrations were lower in calorically restricted young and old animals, as reported previously in response to both short-term and life-long energy intake restriction ( Lane et al. , 2000 ; Mahoney et al. , 2006 ). It is possible that these changes contributed to improvements in EDD in the OCR mice. However, the reductions in blood glucose and triglycerides were similar in young calorie-restricted animals without any improvement in EDD. Short-term vs. lifelong calorie restriction Short-term calorie restriction initiated in old animals and life-long restriction of energy intake may represent different physiological states/stressors/stimuli. For example, whereas restriction of energy intake is a feature that is common to both interventions, weight loss occurs only during short-term calorie restriction initiated in old and young animals. We recently demonstrated that in middle-aged and older overweight and obese adult humans, energy intake restriction-based weight loss improves EDD by increasing NO bioavailability ( Pierce et al. , 2008 ). However, the old mice in the present study were of normal weight at baseline, and the body weight at the end of the 8-week feeding period was similar to that observed in old mice maintained on life-long calorie restriction ( Turturro et al. , 1999 ). Thus, the arterial adaptations to short-term calorie restriction in old animals may be the result of energy intake restriction, weight loss or both. Conclusions In conclusion, the results of the present study demonstrate that short-term calorie restriction initiated late in life restores vascular endothelial function in old mice. This is mediated by increased NO bioavailability as a result of reductions in superoxide-dependent oxidative stress and, perhaps, increased eNOS protein. Short-term restriction of energy intake in old mice may reduce oxidative stress by down-regulating NADPH oxidase and increasing MnSOD activity. Finally, short-term calorie restriction in old mice induces an increase in arterial expression of SIRT1, but has no obvious effect on arterial inflammatory cytokines. Experimental procedures Ethical approval All animal procedures conformed to the Guide to the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996) and were approved by the UCB Animal Care and Use Committee. Animals and calorie restriction Young (Y: 5–8 months) and old (O: 28–30 months) male B6D2F1 mice ( n = 40) obtained from the rodent colony of the National Institute on Aging were fed ad libitum (NIH-31 diet) for an acclimation period of 2 weeks. The young and old mice were then divided into two subgroups: one continued on the ad libitum regimen (YAL and OAL) and the other was restricted to 3.2 g (NIH-31 fortified diet) for 6 weeks (YCR and OCR) after a 2-week progressive reduction in food intake (4.1 g week 1 and 3.7 g week 2). The mice were fed between 8.00 and 9.00 am each day. All mice were housed in an animal care facility at the University of Colorado at Boulder on a 12:12 light:dark cycle and had continuous access to water. Age at sacrifice was 7–10 and 30–32 months (mean age: 7.4 ± 0.6 and 8.2 ± 0.7 months for YAL and YCR and 30.4 ± 0.7 and 31.0 ± 0.3 months for OAL and OCR respectively). Vasodilatory responses EDD and endothelium-independent dilation were determined ex vivo in isolated carotid arteries as recently described in detail ( Lesniewski et al. , 2009 ). Mice were anesthetized using isoflurane and euthanized by exsanguination via cardiac puncture. The carotid arteries were carefully excised, cannulated onto glass micropipettes and secured with nylon (11-0) suture in myograph chambers (DMT Inc.) containing buffered physiological saline solution. The arteries were pressurized to 50 mmHg at 37° C and were allowed to equilibrate for 1 h. After submaximal preconstriction with phenylepherine (2 ॖ m ), increases in luminal diameter in response to acetylcholine (ACh:1 × 10 −9 –1 × 10 −4 mol/L) with and without co-administration of the NO synthase inhibitor L-NAME, (0.1 mmol L −1 , 30-min incubation) or the SOD mimetic, TEMPOL, (1 mmol L −1 , 30 min incubation), were determined. EDD also was determined in the presence of the NADPH oxidase inhibitor apocynin (1 mmol L −1 , 30 min incubation). Endothelium-independent dilation was determined by vasodilation in response to sodium nitroprusside (SNP: 1 x 10 −10 –1 × 10 −4 mol L −1 ). Arterial protein expression and enzyme activities Aortas were used as a surrogate large elastic artery to provide sufficient tissue for analysis of protein expression by western blot and enzyme activity as described previously ( Cernadas et al. , 1998 ; Blackwell et al. , 2004 ; Ungvari et al. , 2008 ; Lesniewski et al. , 2009 ). Aortas were excised, cleared of surrounding tissues and frozen in liquid nitrogen before storage at -80°C. For assay, the tissue was pulverized over liquid nitrogen and homogenized in ice-cold RIPA lysis buffer containing protease and phosphatase inhibitors [Protease Inhibitor Cocktail Tablet (Roche, Indianapolis, IN, USA) and 0.01% phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA)]. Fifteen micrograms of protein was loaded on 12% polyacrylamide gels, separated by electrophoresis and transferred onto nitrocellulose membranes for western blot analysis. Antibodies for western analysis included anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cell Signaling, Danvers, MA, USA), anti-p67phox, anti-eNOS (BD Biosciences, San Jose, CA, USA), anti-nitrotyrosine, anti-SIRT1, anti-catalase (Abcam, Cambridge, MA, USA), anti-MnSOD (Stressgen, Ann Arbor, MI, USA). Enzyme activity of MnSOD was determined in aortic lysates (1 ॖg protein) using a SOD activity assay kit in the presence of 1 mmol L −1 potassium cyanide to block copper-zinc SOD activities. Enzyme activity for catalase was measured using a kit (Cayman Chemical, Ann Arbor, MI, USA). NADPH oxidase activity (10 ॖg total protein) was measured using an Amplex red xanthine/xanthine oxidase assay kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer instructions with NADPH (200 ॖmol L −1 per reaction) as the reaction substrate. Pro-inflammatory cytokines interleukin-1 beta, interleukin-6, interferon gamma and tumor necrosis factors alpha were measured using a multiplex SearchLight Chemiluminescent Array Kit (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA) according to manufacturer’s instructions. Metabolic factors At the end of the experimental period, blood was collected through cardiac puncture and plasma was stored in −80C. Blood glucose was measured immediately using a glucose meter (One Touch Ultra; LifeScan, Inc., Milpitas, CA, USA). Plasma insulin was measured using an ELISA kit (Alpco, Salem, NH, USA). Blood lipids (Free Fatty acids, Triglycerides and Cholesterol) were assessed using kits obtained from Wako Diagnostics, Richmond VA. Superoxide production Production of superoxide was measured by EPR spectrometry using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH, Alexis Biochemicals). Stock solutions of CMH were prepared in ice-cold deoxygenated Krebs-HEPES buffer (mmol L −1 : NaCl, 99.01, KCl 4.69, CaCl 2 2.50, MgSO 4 1.20, K 2 HPO 4 1.03, NaHCO 3 25.0, glucose 11.10, Na-HEPES 20.00; pH 7.4) containing 0.1 mmol L −1 diethylenetriamine-penta-acetic acid, 5 ॖmol L −1 sodium diethyldithiocarbamate and pretreated with Chelex (Sigma) to minimize auto-oxidation of the spin probe. Three-millimeter aortic rings were washed once in PSS and again in modified Krebs-HEPES buffer. Rings were then incubated for 60 min at 37°C in 200 ॖL Krebs-HEPES buffer containing 0.5 mmol L −1 CMH and analyzed immediately on an MS300 X-band EPR spectrometer (Magnettech, Berlin, Germany). Instrument settings were: microwave frequency 9.43 Ghz, centerfield 3350 G, sweep 80 G, modulation amplitude 3 G, microwave power 10 mW, and receiver gain 50. Statistics Data are presented as mean ± SEM. For the ex vivo vasodilatory dose responses, group differences were determined by repeated measures anova . For variables in which a significant interaction was found, comparisons between groups at particular doses were made using independent t -tests. For maximum dilation, protein expression and enzyme activities, comparisons between groups were made using anova . Significance was determined using P < 0.05. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Aging Cell Wiley

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

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Wiley
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Journal compilation © 2010 Blackwell Publishing Ltd/The Anatomical Society of Great Britain and Ireland
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1474-9718
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1474-9726
DOI
10.1111/j.1474-9726.2010.00557.x
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20121721
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Abstract

Introduction The risk of cardiovascular diseases is markedly increased in older adults and this is linked to the development of vascular endothelial dysfunction, most commonly demonstrated as impaired endothelium-dependent dilation (EDD) ( Celermajer et al. , 1994 ; DeSouza et al. , 2000 ; Lakatta & Levy, 2003 ). The latter is mediated by a reduction in the bioavailability of the dilating molecule nitric oxide (NO) and is linked to the development of vascular oxidative stress ( Luscher & Barton, 1997 ; Taddei et al. , 2001 ). Thus, therapeutic strategies that reduce vascular oxidative stress, increase NO bioavailability and reverse age-associated impairments in EDD have important clinical implications for the prevention of cardiovascular diseases in older adults. Calorie restriction, defined as a reduction in energy intake without malnutrition, extends lifespan and is associated with enhanced physiologic function in several species ( Weindruch & Sohal, 1997 ; Masoro, 2005 ). Although little is known about its potential effects on vascular aging, recent observations indicate that life-long calorie restriction preserves EDD with aging in rats ( Ungvari et al. , 2008 ; Csiszar et al. , 2009 ). This was associated with greater protein expression of endothelial NO synthase (eNOS) and evidence for less production of superoxide in large elastic arteries ( Ungvari et al. , 2008 ; Csiszar et al. , 2009 ), suggesting the possibility of enhanced NO bioavailability and reduced vascular oxidative stress. Recent findings indicate that shorter-term calorie restriction may produce some of the same effects on longevity and physiological function in rodents as life-long restriction of energy intake ( Cao et al. , 2001 ; Dhahbi et al. , 2004 ; Goto, 2006 ). However, it is unknown if short-term calorie restriction can improve or reverse vascular endothelial dysfunction associated with aging and, if so, the mechanisms by which this effect is mediated. In the present study, we used a recently established model of age-associated endothelial dysfunction in large arteries ( Lesniewski et al. , 2009 ) to test the hypothesis that short-term calorie restriction initiated late in life restores EDD by improving NO bioavailability as a result of reducing oxidative stress. We also determined the role of reduced arterial superoxide production, as well as the expression and activities of the oxidant enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and antioxidant enzymes in mediating these effects of calorie restriction. Arterial expression of the deacetylase sirituin 1 (SIRT1–silent mating type information regulation 2 homolog) was determined because it activates eNOS ( Mattagajasingh et al. , 2007 ) and is implicated in the physiological effects of calorie restriction ( Cohen et al. , 2004 ; Csiszar et al. , 2009 ). Because life-long calorie restriction can have anti-inflammatory effects ( Spaulding et al. , 1997 ; Ungvari et al. , 2008 ; Csiszar et al. , 2009 ), we also assessed arterial expression of inflammatory proteins. Finally, vasodilation in response to an NO donor (i.e., endothelium-independent dilation) was assessed to determine if improvements in EDD with calorie restriction might be mediated by increases in vascular smooth muscle responsiveness to NO. Results Food intake, body and fat pad mass, and metabolic characteristics Daily food intake during the 8-week experimental period was 10% less in old ad libitum (OAL) vs. young ad libitum (YAL) (4.4 ± 0.1 vs. 4.9 ± 0.1 g, P < 0.05), whereas all calorie restricted (CR) mice ate 4.1, 3.7 and 3.2 g, respectively, in weeks 1, 2 and 3-8. Body weight did not differ among the groups at baseline: YAL 32.8 ± 0.8 g, YCR 32.4 ± 1.2 g, OAL 34.8 ± 1.4 g, old calorie-restricted (OCR) 35.7 ± 0.9 g. Body weight decreased over the feeding period similarly in the calorie-restricted groups (YCR −9.6 ± 1.5 g, OCR -10.0 ± 0.9), whereas there were no significant changes from baseline in the ad libitum-fed groups (YAL +2.7 ± 1.1 g, P = 0.14; OAL −3.1 ± 0.8, P = 0.21). At the end of the treatments, epididymal white adipose tissue mass was lower in the calorie-restricted compared with the ad libitum-fed animals at both ages (YCR 0.15 ± 0.03 vs. YAL 0.71 ± 0.22; OCR 0.11 ± 0.02 vs. OAL 0.25 ± 0.04 g, both P < 0.05). Blood glucose and plasma triglycerides were lower in both CR groups compared with ad libitum-fed controls of the same age, but there was no significant change in plasma insulin, free fatty acids or cholesterol associated with short-term calorie restriction ( Table 1 ). Short-term calorie restriction restored EDD in old mice without influencing endothelium-independent dilation Carotid artery preconstriction to phenylephrine was not different in the ad libitum and CR groups ( P = 0.15). Peak carotid artery dilation in response to acetylcholine was impaired in OAL vs. YAL (74 ± 5% vs. 95 ± 2%, P < 0.05), but was preserved in OCR (96 ± 1%), not differing from YAL or YCR (94 ± 3%) (left panel Fig. 1 ). Neither vasodilatory sensitivity (IC 50 ) to acetylcholine (YAL: 2.1 E −8 ± 9.8 E −9 , YCR: 1.8 E −8 ± 3.8E −9 , OAL: 2.6 E −8 ± 1.43 E −8 , OCR: 2.6 E −8 ± 1.56 E −8 ) nor endothelium-independent dilation to sodium nitroprusside (right panel Fig. 1 ) differed among the groups (all P > 0.5). Short-term calorie restriction-related improvements in EDD in old mice were mediated by increases in NO bioavailability NO inhibition with L-NAME (N G -nitro-L-arginine methyl ester) reduced peak carotid artery dilation to acetylcholine in all groups ( P < 0.005). However, the reduction in carotid artery dilation to acetylcholine after pretreatment with L-NAME compared to acetylcholine alone (NO-mediated dilation, left panel Fig. 2 ) was smaller in OAL vs. YAL ( P < 0.05), and was associated with lower aortic eNOS protein expression ( P < 0.05, right panel Fig. 2 ). In contrast, NO-mediated dilation and eNOS were preserved in OCR compared with YAL and YCR. Superoxide-associated oxidative stress is reduced in short-term calorie restricted old mice and contributes to preserved EDD Nitrotyrosine staining (55-kDa protein band), a cellular marker of oxidant modification of tyrosine residues of proteins, was 80% higher in aorta of OAL vs. YAL ( P < 0.05), whereas staining in OCR was markedly lower than OAL ( P < 0.005), somewhat lower than YAL and similar to YCR (left panel Fig. 3 ). Aortic superoxide production, measured directly by electron paramagnetic resonance, was 150% greater in OAL vs. YAL ( P < 0.05). In contrast, superoxide production in OCR was similar to YAL and not significantly different than YCR (middle panel Fig. 3 ). TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl), a superoxide dismutase (SOD) mimetic, restored carotid artery dilation to acetylcholine in OAL to levels observed in YAL, but had no effect in OCR or the young groups (right panel Fig. 3 ). eNOS inhibition with L-NAME abolished the selective TEMPOL-mediated restoration of the peak vasodilatory response to acetylcholine in OAL such that there were no longer differences among the groups (right panel, Fig. 3 ). Together these observations are consistent with the idea that short-term calorie restriction reduced superoxide bioactivity, which, in turn, increased NO bioavailability and improved EDD. Down-regulation of NADPH oxidase contributes to reduced superoxide–associated improvements in EDD in short-term calorie restricted old mice NADPH oxidase activity and p67phox subunit expression were greater in aorta of OAL vs. YAL ( P < 0.05), whereas OCR demonstrated levels similar to YAL and YCR (left panel Fig. 4 ). Apocynin, an NADPH oxidase inhibitor, restored carotid artery dilation to acetylcholine in OAL to that observed in YAL, but had no effect in OCR or the young groups (middle and right panels Fig. 4 ). There were no differences in dilation among the groups in the presence of apocynin. Short-term calorie restriction increased manganese SOD activity in old mice Protein expression of manganese SOD (MnSOD) in aorta did not differ among the groups ( Fig. 5 ). However, aortic MnSOD activity was lower in OAL vs. YAL ( P < 0.05), whereas the activity in OCR was preserved at the levels of YAL and YCR ( Fig. 5 ). Catalase protein expression and activity were not significantly different among the groups (all P > 0.05, data not shown). Short-term calorie restriction increases SIRT1 expression in aorta of old mice, but has no effect on inflammatory cytokines Protein expression of the deacetylase SIRT1 was lower in aorta of OAL vs. YAL, whereas OCR demonstrated levels not significantly different from YAL and YCR ( Fig. 6 ). SIRT1 protein expression was positively related to peak carotid artery dilation in response to acetylcholine ( r = 0.63, P < 0.001). Expression of the inflammatory cytokines interleukin-1ॆ, interleukin-6, interferon े and tumor necrosis factor ॅ was greater in OAL vs. YAL and in OCR vs. YCR (all P < 0.05, Table 2 ). However, short-term calorie restriction had no significant effect on these inflammatory proteins in either young or old mice. Discussion EDD, NO bioavailability and eNOS Life-long restriction of energy intake preserves EDD in skeletal muscle arterioles and aorta of F344 rats ( Ungvari et al. , 2008 ; Csiszar et al. , 2009 ). Here, we extend these findings by showing that short-term calorie restriction initiated late in life completely reverses the impairment in carotid artery EDD observed in old B6D2F1 mice. Endothelium-independent dilation to sodium nitroprusside was unchanged with energy intake restriction, indicating that the improvements in EDD were independent of changes in vascular smooth muscle sensitivity to NO. Rather our findings show that the improvements in EDD were mediated by increases in NO bioavailability. This is supported by the fact that inhibition of NO production with L-NAME produced a suppression of acetylcholine-induced dilation in energy restricted old mice that was similar to that of young mice, such that EDD was not different in the ad libitum and CR groups in the absence of NO. It is possible that increased expression of eNOS protein contributed to the increase in NO bioavailability associated with short-term calorie restriction by increasing NO production. We found that eNOS in the aorta of the energy intake restricted old mice was approximately 30% greater than in old ad libitum-fed mice, was similar to young mice, and correlated with peak dilation in response to acetylcholine in all animals (r = 0.57, P < 0.05). This is consistent with previous observations that eNOS is greater in large arteries of old rats subjected to life-long calorie restriction than in ad libitum-fed old animals ( Ungvari et al. , 2008 ). Oxidative stress Independent of these increases in eNOS, our data demonstrate that the primary mechanism by which short-term energy intake restriction restores NO bioavailability and EDD in old mice is by reducing oxidative stress. Several lines of evidence support this idea. Nitrotyrosine, a marker of cellular oxidative stress ( Reiter et al. , 2000 ), increases with aging in vascular endothelial cells of humans and is inversely related to EDD ( Donato et al. , 2007 ). In the present study, nitrotyrosine was markedly lower in aorta of the OCR compared with old ad libitum-fed mice, and was similar to levels observed in young mice. This extends to aging arteries previous observations of reduced oxidative modifications in other tissues in response to short- or long-term energy intake restriction ( Gredilla et al. , 2001 ; Bevilacqua et al. , 2005 ). Aortic superoxide production, measured directly with EPR, was markedly elevated in old mice that were ad libitum-fed, but old mice that underwent short-term calorie restriction demonstrated much lower levels, not different than young mice. This finding is consistent with recent results using a fluorescent dye and chemiluminescence methods that suggest reduced superoxide production in arteries of life-long calorically restricted old rats compared with ad libitum-fed controls ( Ungvari et al. , 2008 ; Csiszar et al. , 2009 ). Finally, we found that a SOD mimetic restored EDD in ad libitum-fed, but not calorie-restricted old mice, indicating an absence of superoxide-mediated suppression of EDD in the latter group. This observation directly links the reduction in superoxide to improved vascular endothelial function, extending recent findings in life-long calorically restricted rats ( Csiszar et al. , 2009 ). We further extended these findings by showing that inhibition of NO production with L-NAME abolished the selective improvement in the EDD response to TEMPOL in the ad libitum-fed old mice. Considered together, our data provide evidence that short-term energy intake restriction restores carotid artery EDD in old mice by reducing superoxide-induced oxidative stress, which, in turn, increases NO bioavailability. NADPH oxidase Our results also provide insight into the mechanisms by which short-term calorie restriction reduced vascular oxidative stress in old mice. NADPH oxidase is a major source of superoxide production in arteries ( Zalba et al. , 2000 ) and is increased with aging in humans ( Donato et al. , 2007 ) and, as confirmed here, in ad libitum-fed rodents ( Csiszar et al. , 2007 ; Lesniewski et al. , 2009 ). In the present study, we found that both protein expression and activity of this enzyme were lower in aorta of old energy intake restricted mice and similar to that of young mice. We then connected these changes directly to function by showing that inhibition of NADPH oxidase with apocynin selectively restored EDD in old ad libitum-fed mice, indicating that the preserved EDD in the OCR mice was mediated primarily by reduced NADPH oxidase–mediated superoxide production. Antioxidant enzymes The expression of two important antioxidant enzymes, MnSOD and catalase, was not different in aorta of our groups. However, MnSOD activity was reduced in the old ad libitum-fed animals and this was increased to levels of young mice in the calorie-restricted old animals. That TEMPOL, a SOD mimetic, restored EDD in ad libitum-fed, but not calorie-restricted old mice supports the possibility that the increase in bioactivity of this endogenous antioxidant may have contributed to more effective scavenging of superoxide and reduced oxidative stress in arteries of the old energy restricted mice. The mechanism by which calorie restriction restored MnSOD in our old mice is unclear, but nitration of MnSOD reduces its catalytic activity ( Yamakura et al. , 1998 ; Guo et al. , 2003 ). Thus, reduced nitration of MnSOD could explain the increase in activity observed in the calorie-restricted old animals. Because MnSOD deficiency is associated with oxidative stress and endothelial dysfunction with aging in mice ( Brown et al. , 2007 ), the increase in MnSOD activity could have contributed to the improvement in EDD with calorie restriction in our old mice. SIRT-1 SIRT1 is a histone deacetylase that increases in response to calorie restriction in a variety of tissues ( Cohen et al. , 2004 ). Deacetylation by SIRT1 activates eNOS and increases NO production and EDD ( Mattagajasingh et al. , 2007 ). Protein expression of SIRT-1 is increased in coronary artery endothelial cells incubated with serum from calorie-restricted rats ( Csiszar et al. , 2009 ). In the present study, SIRT1 expression in aorta of old ad libitum-fed mice was approximately 40% lower than in young controls, and short-term energy intake restriction restored the expression to young levels. Moreover, peak EDD was positively related to expression of SIRT-1. These observations are in agreement with previous work in young mice demonstrating increases in aortic expression of SIRT1 after 1 year of calorie restriction and that manipulation of SIRT1 expression was consistently related to NO-mediated EDD ( Zhang et al. , 2008 ). Inflammatory cytokines Life-long calorie restriction may suppress vascular inflammation with aging ( Ungvari et al. , 2008 ; Csiszar et al. , 2009 ), and short-term calorie restriction inhibits signaling of the inflammatory nuclear transcription factor nuclear factor ॔B in the kidney of old rats ( Jung et al. , 2009 ). In the present study, protein expression of several pro-inflammatory cytokines was elevated in aorta of old compared with young ad libitum-fed mice; however, calorie restriction did not affect expression in either age group. These observations suggest that short-term restriction of energy intake may not exert anti-inflammatory actions in arteries of old or young mice and, thus, may not contribute to the improved vascular endothelial function in old animals. Circulating metabolic factors In the present study, circulating glucose and triglyceride concentrations were lower in calorically restricted young and old animals, as reported previously in response to both short-term and life-long energy intake restriction ( Lane et al. , 2000 ; Mahoney et al. , 2006 ). It is possible that these changes contributed to improvements in EDD in the OCR mice. However, the reductions in blood glucose and triglycerides were similar in young calorie-restricted animals without any improvement in EDD. Short-term vs. lifelong calorie restriction Short-term calorie restriction initiated in old animals and life-long restriction of energy intake may represent different physiological states/stressors/stimuli. For example, whereas restriction of energy intake is a feature that is common to both interventions, weight loss occurs only during short-term calorie restriction initiated in old and young animals. We recently demonstrated that in middle-aged and older overweight and obese adult humans, energy intake restriction-based weight loss improves EDD by increasing NO bioavailability ( Pierce et al. , 2008 ). However, the old mice in the present study were of normal weight at baseline, and the body weight at the end of the 8-week feeding period was similar to that observed in old mice maintained on life-long calorie restriction ( Turturro et al. , 1999 ). Thus, the arterial adaptations to short-term calorie restriction in old animals may be the result of energy intake restriction, weight loss or both. Conclusions In conclusion, the results of the present study demonstrate that short-term calorie restriction initiated late in life restores vascular endothelial function in old mice. This is mediated by increased NO bioavailability as a result of reductions in superoxide-dependent oxidative stress and, perhaps, increased eNOS protein. Short-term restriction of energy intake in old mice may reduce oxidative stress by down-regulating NADPH oxidase and increasing MnSOD activity. Finally, short-term calorie restriction in old mice induces an increase in arterial expression of SIRT1, but has no obvious effect on arterial inflammatory cytokines. Experimental procedures Ethical approval All animal procedures conformed to the Guide to the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996) and were approved by the UCB Animal Care and Use Committee. Animals and calorie restriction Young (Y: 5–8 months) and old (O: 28–30 months) male B6D2F1 mice ( n = 40) obtained from the rodent colony of the National Institute on Aging were fed ad libitum (NIH-31 diet) for an acclimation period of 2 weeks. The young and old mice were then divided into two subgroups: one continued on the ad libitum regimen (YAL and OAL) and the other was restricted to 3.2 g (NIH-31 fortified diet) for 6 weeks (YCR and OCR) after a 2-week progressive reduction in food intake (4.1 g week 1 and 3.7 g week 2). The mice were fed between 8.00 and 9.00 am each day. All mice were housed in an animal care facility at the University of Colorado at Boulder on a 12:12 light:dark cycle and had continuous access to water. Age at sacrifice was 7–10 and 30–32 months (mean age: 7.4 ± 0.6 and 8.2 ± 0.7 months for YAL and YCR and 30.4 ± 0.7 and 31.0 ± 0.3 months for OAL and OCR respectively). Vasodilatory responses EDD and endothelium-independent dilation were determined ex vivo in isolated carotid arteries as recently described in detail ( Lesniewski et al. , 2009 ). Mice were anesthetized using isoflurane and euthanized by exsanguination via cardiac puncture. The carotid arteries were carefully excised, cannulated onto glass micropipettes and secured with nylon (11-0) suture in myograph chambers (DMT Inc.) containing buffered physiological saline solution. The arteries were pressurized to 50 mmHg at 37° C and were allowed to equilibrate for 1 h. After submaximal preconstriction with phenylepherine (2 ॖ m ), increases in luminal diameter in response to acetylcholine (ACh:1 × 10 −9 –1 × 10 −4 mol/L) with and without co-administration of the NO synthase inhibitor L-NAME, (0.1 mmol L −1 , 30-min incubation) or the SOD mimetic, TEMPOL, (1 mmol L −1 , 30 min incubation), were determined. EDD also was determined in the presence of the NADPH oxidase inhibitor apocynin (1 mmol L −1 , 30 min incubation). Endothelium-independent dilation was determined by vasodilation in response to sodium nitroprusside (SNP: 1 x 10 −10 –1 × 10 −4 mol L −1 ). Arterial protein expression and enzyme activities Aortas were used as a surrogate large elastic artery to provide sufficient tissue for analysis of protein expression by western blot and enzyme activity as described previously ( Cernadas et al. , 1998 ; Blackwell et al. , 2004 ; Ungvari et al. , 2008 ; Lesniewski et al. , 2009 ). Aortas were excised, cleared of surrounding tissues and frozen in liquid nitrogen before storage at -80°C. For assay, the tissue was pulverized over liquid nitrogen and homogenized in ice-cold RIPA lysis buffer containing protease and phosphatase inhibitors [Protease Inhibitor Cocktail Tablet (Roche, Indianapolis, IN, USA) and 0.01% phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA)]. Fifteen micrograms of protein was loaded on 12% polyacrylamide gels, separated by electrophoresis and transferred onto nitrocellulose membranes for western blot analysis. Antibodies for western analysis included anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cell Signaling, Danvers, MA, USA), anti-p67phox, anti-eNOS (BD Biosciences, San Jose, CA, USA), anti-nitrotyrosine, anti-SIRT1, anti-catalase (Abcam, Cambridge, MA, USA), anti-MnSOD (Stressgen, Ann Arbor, MI, USA). Enzyme activity of MnSOD was determined in aortic lysates (1 ॖg protein) using a SOD activity assay kit in the presence of 1 mmol L −1 potassium cyanide to block copper-zinc SOD activities. Enzyme activity for catalase was measured using a kit (Cayman Chemical, Ann Arbor, MI, USA). NADPH oxidase activity (10 ॖg total protein) was measured using an Amplex red xanthine/xanthine oxidase assay kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer instructions with NADPH (200 ॖmol L −1 per reaction) as the reaction substrate. Pro-inflammatory cytokines interleukin-1 beta, interleukin-6, interferon gamma and tumor necrosis factors alpha were measured using a multiplex SearchLight Chemiluminescent Array Kit (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA) according to manufacturer’s instructions. Metabolic factors At the end of the experimental period, blood was collected through cardiac puncture and plasma was stored in −80C. Blood glucose was measured immediately using a glucose meter (One Touch Ultra; LifeScan, Inc., Milpitas, CA, USA). Plasma insulin was measured using an ELISA kit (Alpco, Salem, NH, USA). Blood lipids (Free Fatty acids, Triglycerides and Cholesterol) were assessed using kits obtained from Wako Diagnostics, Richmond VA. Superoxide production Production of superoxide was measured by EPR spectrometry using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH, Alexis Biochemicals). Stock solutions of CMH were prepared in ice-cold deoxygenated Krebs-HEPES buffer (mmol L −1 : NaCl, 99.01, KCl 4.69, CaCl 2 2.50, MgSO 4 1.20, K 2 HPO 4 1.03, NaHCO 3 25.0, glucose 11.10, Na-HEPES 20.00; pH 7.4) containing 0.1 mmol L −1 diethylenetriamine-penta-acetic acid, 5 ॖmol L −1 sodium diethyldithiocarbamate and pretreated with Chelex (Sigma) to minimize auto-oxidation of the spin probe. Three-millimeter aortic rings were washed once in PSS and again in modified Krebs-HEPES buffer. Rings were then incubated for 60 min at 37°C in 200 ॖL Krebs-HEPES buffer containing 0.5 mmol L −1 CMH and analyzed immediately on an MS300 X-band EPR spectrometer (Magnettech, Berlin, Germany). Instrument settings were: microwave frequency 9.43 Ghz, centerfield 3350 G, sweep 80 G, modulation amplitude 3 G, microwave power 10 mW, and receiver gain 50. Statistics Data are presented as mean ± SEM. For the ex vivo vasodilatory dose responses, group differences were determined by repeated measures anova . For variables in which a significant interaction was found, comparisons between groups at particular doses were made using independent t -tests. For maximum dilation, protein expression and enzyme activities, comparisons between groups were made using anova . Significance was determined using P < 0.05.

Journal

Aging Cell – Wiley

Published: Jun 1, 2010

Keywords: aging; endothelium-dependent dilation; superoxide; superoxide dismutase; sirituin-1

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