Red wine and equivalent oral pharmacological doses of resveratrol

Atherosclerosis 224 (2012) 136e142

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Red wine and equivalent oral pharmacological doses of resveratrol delay vascular aging but do not extend life span in rats Protásio L. da Luz a, b, c, *, Leonardo Tanaka c, Patrícia Chakur Brum d, Paulo Magno Martins Dourado c, Desidério Favarato a, José Eduardo Krieger b, Francisco Rafael M. Laurindo c a

Clinical Division, Heart Institute (Incor), School of Medicine, University of São Paulo, SP, Brazil Genetic and Molecular Cardiology Laboratory, Heart Institute (Incor), School of Medicine, University of São Paulo, SP, Brazil Vascular Biology Laboratory, Heart Institute (Incor), School of Medicine, University of São Paulo, SP, Brazil d School of Physical Education and Sport, University of São Paulo, SP, Brazil b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 December 2011 Received in revised form 7 May 2012 Accepted 4 June 2012 Available online 26 June 2012

Objective: To investigate, in male Wistar rats, the effects of long-term moderate red wine (RW) consumption (equivalent to w0.15 mg% resveratrol RS), or RS in low (L, 0.15 mg%) or high (H, 400 mg%) doses in chow. Background: Both RW and RS exhibit cardioprotection. RS extends lifespan in obese rats. It is unclear whether RW consumption or low-dose RS delay vascular aging and prolong life span in the absence of overt risk factors. Methods: Endpoints were aerobic performance, exercise capacity, aging biomarkers (p53,p16,p21, telomere length and telomerase activity in aortic homogenates), vascular reactivity. Data were compared with controls (C) given regular chow. Results: Expressions of p53 decreased w50% wwith RW and LRS (p < 0.05 vs. C), p16 by w29% with RW (p < 0.05 vs. C) and p21 was unaltered. RW and LRS increased telomere length >6.5-fold vs. C, and telomerase activity increased with LRS and HRS. All treatments increased aerobic capacity (C 32.5 1.2, RW 38.7 1.7, LRS 38.5 1.6, HRS 38.3 1.8 mlO2 min1 kg1), and RW or LRS also improved time of exercise tolerance vs. C (p < 0.05). Endothelium-dependent relaxation improved with all treatments vs. C. Life span, however, was unaltered with each treatment vs. C ¼ 673 30 days, p ¼ NS. Conclusions: RW and LRS can preserve vascular function indexes in normal rats, although not extending life span. These effects were translated into better aerobic performance and exercise capacity. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Aging Senescence Polyphenols Vascular aging Telomeres p53 p16 Red wine Resveratrol

Aging is a major risk factor for atherosclerosis. Senescence leads to several cell alterations that cause severe organ dysfunction in a way dependent on vascular dysfunction [1e3]. However, mechanisms and consequences of cardiovascular aging are not yet precisely understood. At cellular level, telomere and cell cycle inhibitors proteins, such as p53 and p16, play a crucial role in

Abbreviations: CR, calorie restriction; RS, resveratrol; RW, red wine; HRS, high resveratrol; LRS, low resveratrol; HR, heart rate; EDV, end-diastolic volume; ESV, end-sistolic volume; LVEF, left ventricular ejection fraction; E/A, e wave to a wave ratio; IVRT, isovolumic ventricular relaxation time; TERT, telomerase reverse transcriptase; PGC-1a, Peroxisome proliferator-activated recptor- gamma coactivator 1 alpha; PGC-1b, Peroxisome proliferator-activated recptor- gamma coactivator 1 beta. * Corresponding author. Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo e Avenida Dr. Enéas de Carvalho Aguiar, 44 e Cerqueira César e CEP: 05403-000 e São Paulo e SP, Brazil. Tel./ fax: þ55 11 2661 5952. E-mail address: [email protected] (P.L. da Luz). 0021-9150/$ e see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2012.06.007

vascular aging [4,5]. In fact, telomere length has been considered one important biomarker of cardiovascular disease [6] and its dynamic control is associated to life span in humans and experimental models. On the other hand, calorie restriction (CR) is the only intervention that can halt aging by fostering cell replicative capacity in culture and even extending lifespan in several species including Sacharomyces cerevisiae, Caenorhabditis elegans, Drosophyla melanogaster and N. furzeri [7]. These singular effects have been mainly attributed to activation of sirtuins 1 and 2, which are enzymes of the NAD-dependent protein deacetylases that play essential roles as regulators of metabolism, cell division and aging [7,8]. For instance they can down regulate P53 and stabilize DNA, which may increase survival. Substantial evidence indicates the short and long-term moderate red wine (RW) consumption provides cardioprotective effects, including experimental protection against atherosclerosis [9] and, in humans, increased resistance to risk factors and possibly life span augmentation [10].

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Resveratrol (RS) a natural polyphenol found in RW can mimic CR [11]. More recently, Baur et al. [12] showed that RS itself prolonged life in mice fed a high fat diet. RS inhibits Cox-1, decreases platelet aggregation, inhibits angiogenesis, is a vasodilator, is antioxidant and activates sirtuins [8,13]. Although RS has not increased life span in normal mice, it delayed some aging-related factors, acting partially as a caloric restriction mimetic [14]. However, direct comparisons between cardiovascular aging-protective effects of RW and RS, particularly in amounts of RS equivalent to those of moderate RW consumption, have not been explored. Such analysis has potential relevance; RW is a complex substance, readily available for human use, while RS is simpler but not readily consumed. Therefore, we compared the actions of these substances upon indexes of cardiovascular aging and survival in normal rats.

cut into rings (4 mm long). Two rings were promptly applied to evaluate in vitro vasomotor responses and the rest of aorta was used for other measurements: protein expression, telomere length and telomerase activity. Aortic rings were carefully submerged in an organ bath containing oxygenated (95% O2 and 5% CO2) Krebs solution (mM): NaCl 115, KCL 4.7, MgSO4 1.2, KH2PO4 1.5, NaHCO3 25, CaCl2 2.5, glucose 11.1, mM, at 37 C, pH 7.4. Rings were mounted on a force transducer (BIOPAC, USA), using an initial passive tension of 2.0 g. After 60 min of stabilization changing Krebs solution each 20 min, a single dose of phenilephrine 1 uM was applied to induce vasoconstriction followed by concentrationeresponse curve of acetylcholine (ACh: 1010e104 M), an endothelium-dependent dilator agonist.

1. Experimental procedures

3. Molecular parameters

1.1. Animals, diets and survival analysis

3.1. Protein expression

One hundred twenty male Wistar rats were obtained from the animal facilities CEDEME (EPM-UNIFESP) at 10e11 months of age. Animals were maintained in standard conditions with temperature and lightedark cycle controlled. At 12 months old, or else, after approximately one-month of adaptation, animals were assigned into 4 groups: standard food and water (C), 20e25% RW diluted in drinking water (W), low-resveratrol diet (LRS, 0.0015 mg/kg of chow) and high-resveratrol diet (HRS, 4 mg/kg of chow). RW was a Cabernet-Sauvignon 2008, kindly provided by Salton, Rio Grande doSul, Brazil. The average concentration of RS in brazilian cabernet sauvignon wines is 5.0 mg/L. Concentration of total RS in RW was about 0.15 mg% i.e., similar to LRS dosis. RS was purchased from Xian Medicines & Health Products, China. Life span measurement was performed in twenty animals per group maintaining diets until death and for functional and molecular measurements ten rats per group were treated for six months.

Fifty micrograms of proteins from aorta homogenate were separated by SDS-PAGE in 15% polyacrylamide gels and transferred to a nitrocellulose membrane. The membrane was incubated with 5% nonfat milk for 2 h and primary antibodies against p53 (1:1000, Santa Cruz Biotechnology, CA) p21 (1:500, Calbiochem) and p16 (1:1000, Santa Cruz Biotechnology, CA) overnight at 4 C and subsequently with peroxidase-conjugated secondary antibodies for 2 h at room temperature. The antibody complex was measured through enhanced chemiluminescence system (AmeHRSam Corp., USA) detected by autoradiography. Densitometric analysis was performed by Scion Image software (Scion based on NIH image) and b-actin expression was used as a loading control.

2. Physiological parameters 2.1. Aerobic capacity Rats were individually submitted to a progressive exercise test in order to determine exercise tolerance and achieve maximum oxygen consumption (VO2max). Exercise test was performed in a treadmill consisting of a progressive test with incremental charge of 3 m/min every 3 min until exhaustion (Brooks and White, 1978). Expired gas was analyzed by open-circuit indirect calorimetry to obtain oxygen uptake.

3.2. Telomere length Genomic DNA was extracted from aortic homogenate using Qiagen DNA extraction kit and telomere length determined by realtime RT-PCR (Cawthon 2002). Amplification reaction was composed by 35 ng of DNA, SybrMastermix (Invitrogen) and primers for telomere (forward 50 CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT 30 and reverse 50 GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT 30 ) or 36B4 (forward 50 ACT GGT CTA GGA CCC GAG AAG 30 and reverse 50 TCA ATG GTG CCT CTG GAG ATT 30 ) as housekeeping control. Fluorescence detection was performed with Rotor-Gene 6000 (Corbett Research). One sample from C group was used as calibrator and telomere length was calculated by 2DCt method.

2.2. Echocardiography

3.3. Telomerase activity

Echocardiography was performed in xylazine/ketamineanesthetized rats using a 12 MHz transducer (HDI 5000 e Philips Medical Systems Company, Bothell, WA, USA). Left ventricular systolic function was analyzed by planimetry of the end- diastolic and end-systolic areas; end -diastolic and systolic volumes were estimated by two dimensional mode. Ejection fraction was calculated by the Simpson method [15]. The diastolic function was assessed by Doppler mitral inflow e E and A velocity, deceleration time of the E wave, the E/A relationship and LV isovolumetric time of relaxation. The images were recorded for posterior analysis.

Telomeric repeat amplification protocol assay was analyzed using Telo TAGGG Telomerase PCR Elisa plus (Roche) according to manufacters’ instruction. Briefly, 10 ug of protein from aorta were incubated with synthetic oligonucleotides for vascular telomerase adds telomeric repeats. The elongation products were amplified by PCR, denaturated and hybridized to a telomeric repeat-specific probe and probe detection through colorimetric assay was measured in a microplate reader.

2.3. Vascular reactivity

Results were analyzed using GraphPad Prism Software (San Diego) and are presented as mean SEM. Two-way analyses of variance (ANOVA) followed by Bonferroni’s post hoc test was applied for relaxation doseeresponse curve and one-way ANOVA

Rats were euthanized by asphyxia in a carbon dioxide chamber. Thoracic aorta was quickly excised, cleaned of adhering tissue and

3.4. Statistical analyses

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with Dunett’s post hoc for other parameters. Statistical differences were considered significant with p < 0.05. 4. Results 4.1. Physiological parameters: aerobic capacity, cardiac performance and vascular reactivity We investigated if known impairment of age-related physiological functions could be delayed by RW and RS. Six-month consumption of RW and RS in both doses improved aerobic capacity during exercise test. Except for HRS, maximum oxygen uptake paralleled increased exercise tolerance with RW and LRS, as demonstrated by time to exhaustion and running distance during the test (Fig. 1). The improvement on VO2 max was not associated to increased basal cardiac performance in RW or RS-exposed rats. As shown in Table 1, HR, EDV, ESV, LVEF, IVRT and E/A ratio were not statistically different among treated groups. To further analyze vasomotor function in isolated aortic rings, we performed concentrationeeffect curves to acetylcholine. Compared to young healthy rats, relaxation was mildly impaired in middle-aged animals (data not shown). All groups relaxed with acetylcholine, though with RW, LRS or HRS groups showed greater improvement than controls in one or more indexes reflecting extent of relaxation (Fig. 2). However, increase in maximal relaxation occurred only with RW. Endothelium-independent relaxation was similar among different groups (Fig. 2). 4.2. Molecular biomarkers of aging Given that vascular system was responsive to RW and RS, we investigated molecular aging markers in these tissues in rats followed for six months. Expression of tumor-suppressor p53 was decreased in aortic homogenate with RW and LRS. Additionally, RW reduced p16 expression, although p21 was not altered by any treatment (Fig. 3). No changes in these markers occurred in heart and kidney homogenates. Telomere measurement is a well known aging biomarker, its loss associated with repeated cell cycle progression, as well as with other mechanisms and reposition through telomerase activity. There was improved telomere length with RW and LRS, although not with HRS (Fig. 4). Increase in telomerase activity was detected in LRS and HRS, although not with RW (Fig. 4); in fact the increase in HRS group was 49%higher than LRS group. 4.3. Survival Survival was analyzed in 20 rats per group. We did not observe any difference in middle and maximum life span with RW, LRS and HRS compared to control diet, as follows (days): C-673.0 30.4; RW-672.3 24.4; LRS-689.4 26.9; HRS-662.2 26.5. Such lack of effect was not due to differences in body weight, food or liquid ingestion (Table 2). 5. Discussion The main findings of the present study are that RW and LRS both improved vascular function and aerobic capacity, and also down regulated senescence indicators (P53, P16) and preserved telomere length; yet neither RW nor RS prolonged lifespan in these normal rats. Furthermore, specific indexes of mechanical cardiac function were not influenced by either RW or RS. However, significant differences were observed between LRS and HRS dosis as will be discussed ahead.

Fig. 1. Aerobic capacity and response to exercise. Panel A. Vo2 max is significantly increased in RW, low and high RS treated animals compared to controls. Panel B. Red wine and low dosis resveratrol increased exercise tolerance compared to controls; there was only a trend toward increased exercise tolerance with high resveratrol dosis. Panel C. Time to exhaustion was significantly increased by RW and LRS. Number of experiments were: C, 8; RW, 8; LRS, 8; HRS, 8. See abbreviations.

Vascular function improvement, as indicated by greater flow mediated dilation in aortic rings, also was observed with RW and RS. However, endothelial independent dilation was not altered. Most studies indicate that polyphenols from diet, tea or RW improve endothelial arterial dilation in several animal species as well as healthy men and CAD patients by inducing eNOS activity and NO bioavailability [16e18]. Hence, RW polyphenols and RS improved different indexes of vascular function thus making animals more physically fit during their lifespan. The specific role of alcohol was not an objective of this study. On the other hand, increased aerobic capacity as indicated by increased VO2, larger running time and distance achieved on treadmill was observed with both RW and in LRS. Our findings are

P.L. da Luz et al. / Atherosclerosis 224 (2012) 136e142 Table 1 Body weight, chow ingestion and fluid intake during six months treatment. T1 Body weight (g)

Chow ingestion (g day1)

Fluid intake (ml day1)

C RW LRS HRS C RW LRS HRS C RW LRS HRS

528.6 553.2 509.2 579.9 26.8 24.1 26.5 27.7 27.3 23.4 30.0 22.8

T2

10.2 12.6 12.6* 14.0** 1.7 1.4 0.5 1.8 0.8 2.2 2.4 1.2

539.5 549.7 515.3 575.2 27.8 21.7 24.9 24.2 28.4 25.3 29.4 24.8

T3

10.9 16.5 13.4* 12.4 2.8 1.2 0.5 0.7 2.1 1.5 1.5 1.1

523.3 494.3 486.4 535.4 24.3 21.5 27.4 26.5 26.3 28.3 24.3 27.1

11.2 18.6 15.9* 12.2 1.6 1.2 3.7 2.1 1.7 2.3 1.1 1.9

Body weight, Chow ingestion and Fluid intake during six months treatment. Mean and standard error of the two initial months (T1); intermediate (T2); and last two months of treatment. **p < 0.05 vs. C; *p < 0.05 vs. LRS.

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in agreement with Lagouge’s et al. [19] who analyzed the impact of RS upon mitochondrial function and metabolic homeostasis in mice; RS fed mice displayed increased aerobic capacity, induction of genes of oxidative phosphorylation and mitochondrial biogenesis as well as increased resistance to fatigue in muscle fibers; RS also protected against diet induced obesity and insulin resistance; these effects were explained largely by activation o Sirt-1 and PGC-1a, which are key regulators of mitochondrial function. Baur et al. [12] also found that RS improved mitochondrial function and survival in mice on a high-colorie diet. Interestingly, in our study, the better physical performance achieved by RW and LRS consumers was not due to cardiac pump performance itself, since diastolic and systolic cardiac indexes obtained by echocardiography were not different among treated and control animals. Hence, it seems that the increased aerobic capacity was mainly due to vascular performance which could be, at least in part, attributed to enhanced mitochondrial function. This latter effect is probably due to, at least in part, increased telomerase by RW and LRS.

Fig. 2. Vasomotor function in isolated aortic rings. Panel A. Endothelium dependent dilation induced by acetylcholine was significantly improved by RW and low dosis resveratrol. Panel B. Maximum relaxation was significantly increased by RW only. Panel C. EC50 was significantly increased by RW and both dosis of RS. Panel D. Endothelium-independent relaxation was not affected by any treatment. Emax : maximal relaxation. Number of experiments were: C, 6; RW, 7; LRS, 6; HRS, 5; SNP: sodium nitroprusside. See abbreviations.

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Fig. 4. Telomere and telomerase evaluations. Panel A illustrates telomere length augmentation by RW and LRS compared to controls. Number of experiments: C, 6: RW, 6; LRS, 6; HRS, 4. Panel B shows telomerase augmented activity by LRS and HRS but not by RW; interestingly, RW did not impact upon telomerase activity. Number of experiments: C, 6; RW, 5; LRS, 5; HRS, 4. See abbreviations.

Tyner et al. [20] generated mutant mice expressing phenotypes consistent with activated p53 rather than inactivated p53; those animals exhibited enhanced resistance to spontaneous tumors, but on the other hand, displayed signs of early aging including reduced longevity, osteoporosis, generalized organ atrophy and diminished stress tolerance. Also Sahin et al. [21] demonstrated that telomere dysfunction, in a model of Tert -/- mice, activates Fig. 3. Molecular biomarkers of aging in aortic homogenates. Panel A. P53 was clearly reduced by RW as well as by high and low resveratrol dosis compared to controls. Panel B. P16 also was reduced by RW but not by resveratrol. Panel C. P21 was unaffected by any treatment. No changes regarding these biomarkers were observed in kidney or heart preparations (not shown). Number of experiments: C, 3; RW, 4; LRS, 3; HRS, 2. See abbreviations.

Our observations regarding senescence indexes are of considerable interest. Analysis was made in aorta, heart and kidney homogenates; no changes were seen in hearts and kidneys. In the aorta, P53 and p16 expressions were decreased by RW and LRS while p21 remained unchanged. P53 is a tumor suppressor factor which induces cell cycle arrest, apoptosis and senescence [20]. Indeed

Table 2 Echocardiographic variables. C HR (bpm) EF EDV (ml) ESV (ml) IVRT (ms) E/A

270 0.84 0.31 0.051 35.6 2.15

RW

13 0.02 0.03 0.07 2.4 0.48

266 0.84 0.41 0.063 35.0 2.16

LRS

12 0.01 0.05 0.08 2.5 0.29

266 0.84 0.39 0.064 30.6 2.00

HRS

10 0.02 0.03 0.07 1.9 0.24

290 0.85 0.38 0.057 34.0 2.08

17 0.03 0.06 0.04 1.9 0.26

Echocardiographic parameters in C (n ¼ 8), RW [8], LRS [9] and HRS [5] after 6 months treatment. HR e Heart Rate, EF e Ejection Fraction, EDV e End-Diastolic Volume, ESV e End-Systolic Volume, IVRT e Isovolumetric Relaxation Time, E/A e ratio between Early and late Atrial ventricular filling velocity.


p53, suppresses Ppar-g, PGC-1a, PGC-1b, causes growth arrest and mitochondrial dysfunction. Furthermore, in a colon cancer model, RW polyphenols induced expression of several tumor suppressor agents, including p53 and p16 causing reduction in tumor volume [22]. On the other hand, in a non-cancer model as ours, suppression of p53 and p16 by RW and LRS improved vascular reactivity and aerobic capacity while telomere length was preserved. These elements could indicate that mitochondrial function was preserved, energy production maintained and senescence retarded. This interpretation is supported by findings documenting that mutant, antibody inactivated or viral sequestered p53 all extend lifespan of human fibroblasts; also mouse embryo fibroblasts null for p53 rapidly immortalize compared to their normal controls [23]. Thus, our data may be interpreted as indicative that low p53 and p16 represent a signal of replicative stimulus in aortic tissue. RW and LRS effects also may be relevant to apoptosis. RWP increase apoptosis in tumor cells as mentioned above [22]; also, p53 induces apoptosis through the Bcl-2 family in vascular cells [1]. We hypothesize that by reducing p53 expression in aortic tissue, RWP may have prevented apoptosis in endothelial cells and potentially contributed to preserve endothelial function. As far as the role of P1 6 in senescence, there are suggestive evidences to it. For instance, p16 expression paralleled that of senescence eassociated b-galactosidase, which is associated with senescent phenotype [1]; in fact expression of p16 increases in several tissues with advancing age is viewed as a sign of aging [1]. Hence, suppression of these two proteins in the present context may suggest that cell proliferation is not halted and hence could permit tissue renewal, which may be viewed as a positive signal against tissue senescence. In addition, as mentioned above, telomere dysfunction in telomerase reverse transcriptase knockout mice induced severe mitochondrial dysfunction, diffuse organ atrophy and senescent phenotype. In addition, telomere length was increased in RW and LRS treated animals compared to controls. Several evidences indicate that telomere shortening is an index of vascular aging; for instance, progressive telomere shortening was observed in atherosclerosis prone sites of human arteries [24]. Also, Minamino et al. [24] demonstrated that inhibiting telomere function in human aortic endothelial cells induces endothelial dysfunction and senescence; in the other hand, the aa. noted that inhibition of telomere shortening prevented such alterations with senescence. Telomerase activity was only increased by RS not by RW, especially at high dosis; in fact, HRS was associated with 49% increment compared to LRS. Telomerase activity is required for renewal of telomeres, the terminal end-point structure of chromosomes [25]. Compromised telomere structure halts cell proliferation in response to DNA damage [26]. However, while LRS was associated with increased telomere length, reduced p53 and increased aerobic capacity, HRS was not accompanied by such potential benefits. This apparent paradox may be explained by Gorbunova’s et al. [27] report who showed that human fibroblasts that expressed high TERT escaped replicative senescence; however, a subpopulation of such cells exhibited a senescent phenotype with high telomerase activity, higher percentage of B-galactosidase positive cells and growth arrest. Thus, telomerase may have an optimal dose range below and above which telomeric and non-telomeric effects may occur [28]. Therefore, both telomere length preservation and telomerase activity increase by LRS may suggest anti-aging effects. Taken together these observations suggest that both RW and LRS have anti-aging effects. The mechanisms of LRS and RW actions may involve induction of the ubiquitous NAD-dependent protein deacetylases, termed sirtuins and mitochondrial function preservation; indeed sirtuins can extend life span in model organisms and

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are important mediators of the salutary anti-aging effects of caloric restriction [8]. However these beneficial effects were not translated into greater survival among treated animals. This finding contrasts with previous work in hypercholesterolemic mice [13] but agrees with studies in normal mice [14]; indeed Pearson et al. [14] observed that RS induced genes expression patterns in multiple tissues of normal mice that paralleled CR without extending lifespan. Thus differences in species as well as diets may explain these divergent findings. In comparison, aging was delayed and mortality reduced in Rhesus monkeys subjected to caloric restriction for several years compared to a normal diet, without inducing mal-nutrition [29]. Since RS follows the same metabolic pathway as CR, thus leading to DNA stabilization through sirtuins activation [30] it has been postulated that it could prolong life as well. In the present investigation we tested not only RS but RW as well but no survival benefit occurred. We began administration of RW and RS at rat’s mid-age; whether earlier administration would provide different results remains to be seen. In addition a limitation in our study is that we used 12 months old rats; although followed for additional 6 months, senescence index may not have been fully expressed as in older rats. In conclusion, RW and LRS may preserve vascular function in rats in the absence of classical risk factors. Those beneficial effects were translated into better vascular function and aerobic capacity although lifespan was not increased. Financial support Fundação Zerbini, SP, Br; Vulcabrás Azaléia S/A, SP, Br; Vinícola SaltonRS, Br. References [1] Minamino T, Komuro I. Vascular cell senescence: contribution to atherosclerosis. Circ Res 2007;5100:15e26. [2] Celermajer D, Sorensen K, Spiegelhalter D, et al. Aging is associated with endothelial dysfunction in healthy men years before the age-related decline in women. J Am Coll Cardiol 1994;24:471e6. [3] Kletsas D, Pratsinis H, Mariatos G, et al. The proinflammatory phenotype of senescent cells: the p53-Mediated ICAM-1 expression. Ann N Y Acad Sci 2004; 1019:330e2. [4] Campisi J. Senescent cells, tumor suppression and organismal aging: good citizens, bad neighbors. Cell 2005;120:513e22. [5] Holdt LM, Sass K, Gäbel G, et al. Expression of Chr9p21 genes CDKN2B (p15(INK4b)), CDKN2A (p16(INK4a), p14(ARF)) and MTAP in human atherosclerotic plaque. Atherosclerosis 2011;214:264e70. [6] Serrano AL, Andrés V. Telomeres and cardiovascular disease: does size matter? Circ Res 2004;94:575e84. [7] Barger JL, Walford RL, Weindruch R. The retardation of aging by caloric restriction: its significance in the transgenic era. Exp Gerontol 2003;38: 1343e51. [8] Guarante L. Sirtuins, agingand medicine. NEJM 2011;364:2235e44. [9] da Luz PL, Serrano Júnior CV, Chacra AP, et al. The effect of red wine on experimental atherosclerosis: lipid-independent protection. Exp Mol Pathol 1999;65:150e9. [10] Renaud S, de Lorgeril M. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 1992;339:1523e6. [11] Howitz KT, Bitterman KJ, Cohen HY, et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 2003;425:191e6. [12] Baur JA, Pearson KJ, Price NL, et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature 2006;444:337e42. [13] Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev 2006;5:493e506. [14] Pearson KJ, Baur JA, Lewis KN, et al. Resveratrol delays age-reated deterioration and mimics transcriptional aspects of dietary restriction without extending lifespan. Cell Metab 2008;8:157e68. [15] Lang RM, Bierig M, Devereux RB, et al. Recommendations for chamber quantification: a report from the American society of echocardiography’s guidelines and standards committee and the chamber quantification writing group, developed in conjunction with the European association of echocardiography, a branch of the European society of cardiology. J Am Soc Echocardiogr 2005;18:1440e63. [16] Huang PH, Chen YH, Tsai HY, et al. Intake of read wine increase the number and functional capacity of circulation endothelial progenitor cells by

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