Introduction

Aging causes two key changes in arteries that significantly increase the risk of cardiovascular diseases (CVD): stiffening of the large elastic arteries (aorta and carotid arteries) and the development of vascular endothelial dysfunction (Lakatta and Levy, 2003; North and Sinclair, 2012). Arterial stiffening results from age-related changes in the arterial wall including increases in collagen deposition, reductions in elastin and cross-linking of these and other structural proteins via formation of advanced glycation end-products (AGEs) (O'Rourke and Hashimoto, 2007). Vascular endothelial dysfunction develops with age primarily due to reduced nitric oxide (NO) bioavailability, as reflected by impaired NOmediated endothelium-dependent dilation (EDD) (Brandes et al., 2005; Lakatta, 2003a).  Although the mechanisms underlying arterial aging are incompletely understood, the characteristics of age-associated vascular dysfunction are consistent with dysregulated cellular protein homeostasis, i.e., oxidative stress and increased molecular damage that ultimately impair cell and tissue function (Koga et al., 2010; Lakatta, 2003a; Seals et al., 2011b). Autophagy, the cellular process of recycling damaged biomolecules, is a major mechanism for protein homeostasis and defense against oxidative stress (Koga et al., 2010; Mizushima and Komatsu, 2011) and may, therefore, play an important role in arterial aging. Indeed, numerous longevity pathways exert their effects through autophagy (Rubinsztein et al., 2011), and recent work from our laboratory suggests that impaired autophagy contributes to arterial aging (LaRocca et al., 2012).

2.3 Vascular endothelial function

EDD and endothelium-independent dilation were determined ex vivo in isolated carotid arteries as previously described (LaRocca et al., 2012; Rippe et al., 2010b). Mice were anesthetized with isoflurane and sacrificed by exsanguination via cardiac puncture. Carotid arteries were dissected free of surrounding tissue, cleaned and cannulated onto glass micropipettes in myograph chambers (DMT, Aarhus, Denmark). Arteries were pressurized to 50 mmHg at 37°C in physiological saline solution and allowed to equilibrate for 1 h. After preconstriction with phenylephrine (2 µM), NO-mediated EDD was determined by measuring increases in luminal diameter in response to acetylcholine (ACh, 1 ×10−9 − 1×10−4 M, Sigma-Aldrich) in the presence or absence of N G-nitro-L-arginine methyl ester (L-NAME; 0.1 mM, 30 min incubation to block NO production, Sigma-Aldrich) or the superoxide dismutase mimetic TEMPOL (1 mM, 60 min incubation to scavenge superoxide, Sigma-Aldrich). Endothelium-independent dilation was determined as dilation in response to the exogenous NO donor sodium nitroprusside (SNP, 1×10−10 − 1×10−4 M, SigmaAldrich), and is used as a measure of vascular smooth muscle sensitivity to NO. Doseresponse data are presented on a percentage basis to account for differences in carotid artery diameter between young and old animals. NO-dependent dilation was determined from maximal EDD with or without L-NAME as: NO-dependent dilation (%) = max dilationACh - max dilation

2.4 Arterial superoxide production

Superoxide production was assessed by electron paramagnetic resonance (EPR) spectroscopy as previously described (Fleenor et al., 2012; LaRocca et al., 2012).

 Freshly dissected and cleaned 2 mm aortic segments were incubated for 60 min at 37°C in KrebsHEPES buffer with the superoxide-specific spin probe 1-hydroxy-3-methoxycarbonly-2,2,5,5-tetramethylpyrrolidine (0.5 mM; Enzo Life Sciences, Farmingdale, NY, USA). EPR signal amplitude was analyzed immediately on an MS300 X-band EPR spectrometer (Magnettech, Berlin, Germany) with the following settings: centerfield, 3350 G; sweep, 80 G; microwave modulation, 3000 mG; microwave attenuation, 7 dB. Data are expressed relative to the mean of the young control group. 

2.5 Arterial protein expression

Measurements of protein expression were performed on cleaned mouse aortas (a representative large elastic artery) to provide sufficient tissue for analysis. Thoracic aortas were excised, cleaned of surrounding tissue and analyzed by standard Western blotting techniques as previously described (LaRocca et al., 2012; Rippe et al., 2010b). Briefly: whole aortas were homogenized in radio-immunoprecipitation assay lysis buffer with protease and phosphatase inhibitors. 10 µg protein was loaded onto 4–12% polyacrylamide gels, separated by electrophoresis and transferred to nitrocellulose membranes (Criterion System; Bio-Rad, Hercules, CA, USA). Membranes were incubated overnight at 4° C with primary antibodies: collagen-I (1:1000 dilution; Abcam, Cambridge, MA, USA), AGEs (1:2000; Abcam), nitrotyrosine (1:500; Abcam), lipid-modified microtubule-associated protein light chain 3 (LC3-II, 1:2000; Cell Signaling, Danvers, MA, USA), p62 adaptor protein (1:2000; MBL International, Woburn, MA, USA), acetylated (Lys9) histone H3 (1:500; Cell Signaling), autophagy protein Atg3 (1:1000; Cell Signaling). 

Proteins were visualized on a digital acquisition system (ChemiDoc-It; UVP, Upland, CA, USA) using horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) and ECL chemiluminescent substrate (Pierce, Rockford, IL, USA). Protein expression is presented normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000; Cell Signaling), and data expressed as a ratio of the mean of the young control group. 

2.6 In vitro tissue culture experiments 

Aortas were excised from young mice and perivascular fat removed from the arteries. Equal length segments of thoracic aorta were incubated in DMEM (with antibiotics, SigmaAldrich) in a humidified incubator at 37°C and 5% CO2 with or without: pyocyanin (10 µM, Sigma-Aldrich) to induce oxidative stress, as previously described in vascular cells and tissue (Gryglewski et al., 1992; Muller, 2002); chloroquine (50 µM), to inhibit autophagy; or spermidine (3 mM). Half of each aortic segment was removed after 1 h and analyzed for superoxide production by EPR as described above. The remaining half of each segment was incubated for 48 h before analysis. 

2.7 Statistical analyses

 Statistical analysis was performed with SPSS 19.0 software. For dose responses, group differences were determined by repeated measures ANOVA. For aortic pulse wave velocity, maximal dilation, superoxide production and protein expression, comparisons between groups were made using appropriate ANOVA. Significance was determined using P < 0.05. 

3. Results

3.1 Arterial stiffness and wall structural factors

Aortic pulse wave velocity was ~20% greater in old compared with young control mice (Fig. 1A) and was associated with increased formation of AGEs in the aorta (Fig. 1B). Spermidine supplementation normalized both aortic pulse wave velocity and AGEs in old mice without affecting young animals. Aortic collagen I tended to increase with age, and spermidine treatment markedly reduced expression in aortas of old mice (Fig. 1C). These results suggest that spermidine supplementation reverses age-associated stiffening of large elastic arteries, and that these improvements are associated with reductions in structural factors in the arterial wall that promote stiffness. 

3.2 Arterial endothelial function

Carotid artery EDD in response to ACh was ~25% lower in old mice (P < 0.05 vs. young controls, Fig. 2A). Impaired EDD in old animals was a result of reduced NO, indicated by a smaller reduction in EDD upon co-incubation with the NO inhibitor L-NAME (Fig. 2A,B). Spermidine supplementation restored NO-mediated EDD in old mice to levels observed in young control mice. Spermidine did not influence EDD in young animals, but increased the NO component of dilation (Fig. 2A,B). Endothelium-independent dilation to the NO donor sodium nitroprusside was similar among the groups, indicating no differences in vascular smooth muscle sensitivity to NO (Fig. 2C). These observations indicate that spermidine supplementation restores EDD in old mice by restoring NO bioavailability.

3.3 Oxidative stress

Aortas from old control animals had markedly greater levels of nitrotyrosine, a protein marker of superoxide-associated oxidative stress (Fig. 3A), and demonstrated increased superoxide production compared with young controls (Fig. 3B). Spermidine supplementation ameliorated the age-associated increases in both aortic nitrotyrosine levels and superoxide production, while also reducing nitrotyrosine in young animals (Fig. 3A,B). Consistent with these observations, co-incubation with the superoxide scavenger TEMPOL restored maximum carotid artery EDD to acetylcholine in old control mice, suggesting excessive superoxide-mediated suppression of EDD with aging. In contrast, TEMPOL had no effect in spermidine supplemented old or young animals, indicating an absence of superoxide-related impairment of EDD in those groups (Fig. 3C). Collectively, these data demonstrate that spermidine supplementation exerts a powerful antioxidant influence on arteries that appears to mediate improvements in arterial endothelial function.

3.4 Autophagy

Expression of the autophagy marker LC3-II was reduced in aorta of old mice, whereas p62, a marker of undegraded autophagy substrates, was increased relative to young controls (Fig. 4A,B). 

Spermidine supplementation restored aortic expression of LC3-II and reduced p62 in old mice, while having no effect in young animals. These effects of spermidine were associated with reduced acetylation of histone H3 and increased expression of the core autophagy machinery protein Atg3 in both young and old mice (Fig. 4C,D). Taken together, these findings suggest that spermidine supplementation is associated with increased activation of autophagy regulatory systems and corresponding enhancement of protein markers of autophagic processes in arteries. 

3.5 Short- vs. long-term effects of spermidine

Pyocyanin induced “aging-like” oxidative stress in arterial segments isolated from young mice, as indicated by an increase in superoxide production similar in magnitude to that which we observed in aortas of old animals (Fig. 5A,B). Co-incubation with spermidine had no short-term effect on pyocyanin-induced superoxide production (Fig. 5A). In contrast, over a period of 48 h, spermidine treatment normalized superoxide production in pyocyanintreated arteries (Fig. 5B). This protective effect of spermidine was abolished upon coincubation with the autophagy inhibitor chloroquine (Fig. 5B). These findings suggest that spermidine does not directly scavenge superoxide, but may reduce oxidative stress in the long-term via autophagy-dependent antioxidant actions. 

Discussion

Age is the most important determinant of CVD risk (Lloyd-Jones et al., 2010) due in large part to stiffening of large elastic arteries and the development of vascular endothelial dysfunction (Lakatta and Levy, 2003). Suboptimal lifestyle and diet exacerbate these processes, contributing significantly to the global CVD burden (Mozaffarian et al., 2011). 

Thus, identifying dietary patterns and selected nutrients that may prevent or reverse arterial aging is an important research objective. Spermidine and other polyamines are naturally occurring biomolecules involved in numerous cellular functions including growth, development, protein/nucleic acid synthesis and cell signaling (Minois and Carmona-Gutierrez, 2011). Higher intake of polyamines in Mediterranean and Asian diets is related to increased longevity and reduced CVD risk (Binh, 2010; Soda, 2010; Tognon et al., 2010), and recent reports indicate that spermidine in particular is a powerful inducer of autophagy, an important longevity-enhancing pathway (Eisenberg et al., 2009). Polyamine levels decline with aging in some tissues (Minois and Carmona-Gutierrez, 2011) and polyamine supplementation has been reported to reduce mortality in aged mice (Soda et al., 2009). Moreover, plasma spermidine levels may be related to longevity in human subjects (Pucciarelli et al., 2012). The results of the present study extend previous observations by providing the first evidence for the therapeutic potential of polyamine supplementation in the treatment of arterial aging. Our findings here also are the first to show that spermidine per se exerts anti- effects that include normalization of arterial function. Finally, our results provide initial insight into the mechanisms by which spermidine may ameliorate arterial aging, namely by reversing superoxide-associated oxidative stress and restoring NO-bioavailability, perhaps, in part, via enhancement of autophagy.

4.1 Large elastic artery stiffness 

4.1 Large elastic artery stiffness 

Stiffening of large elastic arteries is a major cause of CVD in otherwise healthy people (O'Rourke and Hashimoto, 2007). Indeed, aortic pulse wave velocity, the benchmark clinical measure of large elastic artery stiffness, is a strong independent predictor of incident CVD risk in older adults (Mitchell et al., 2010). The present findings are consistent with previous reports from our laboratory (Fleenor et al., 2012; Sindler et al., 2011a) showing that aortic pulse wave velocity increases with aging in mice and is associated with greater expression of collagen I and AGEs, i.e., changes that contribute to increased stiffness of the aortic wall (O'Rourke and Hashimoto, 2007). Here, we show for the first time that spermidine supplementation reverses age-associated increases in aortic pulse wave velocity and normalizes levels of aortic collagen I and AGEs. These findings are consistent with previous reports linking Mediterranean diet to lower aortic pulse wave velocity (Lydakis et al., 2012), as well as limited in vitro observations of the effects of polyamine administration on collagen and AGEs (Gugliucci and Menini, 2003; Santhanam et al., 2008). Thus, the present findings establish pre-clinical support for the beneficial effects of spermidine on ageassociated arterial stiffness, collagen and AGEs, and provide an experimental basis for assessing the potential de-stiffening effects of this nutraceutical in humans.

4.2 Vascular endothelial dysfunction

Vascular endothelial function (EDD) declines progressively with age and is predictive of future CVD risk (Seals et al., 2011a; Widlansky et al., 2003). 

4.3 Oxidative stress

Oxidative stress is a key mechanism underlying the development of both arterial stiffening and vascular endothelial dysfunction with age (Lakatta, 2003b; North and Sinclair, 2012; Seals et al., 2011a). Age-associated vascular oxidative stress reduces NO bioavailability, induces biomolecular damage as a consequence of increased superoxide bioactivity and accelerates the formation of AGEs (Lakatta, 2003b; Seals et al., 2011a). We recently reported that supplementation with the superoxide dismutase mimetic TEMPOL improves aortic pulse wave velocity and EDD in old mice (Fleenor et al., 2012), suggesting a central role for superoxide in arterial oxidative stress-associated vascular dysfunction with aging. The present findings are consistent with these observations, showing both increased levels of oxidative protein damage (nitrotyrosine) and elevated superoxide production, as well as selective rescue of EDD by ex vivo TEMPOL treatment in arteries of old mice. Here, we identify a potential novel treatment for arterial oxidative stress. Spermidine supplementation reversed the age-associated increase in arterial superoxide production and reduced nitrotyrosine in arteries of old mice, while also reducing nitrotyrosine in young mice. Given the key role of superoxide in arterial aging, these data suggest that suppression of oxidative stress may be an important mechanism underlying the beneficial effects of spermidine on both age-associated arterial stiffening and vascular endothelial dysfunction. Previous reports suggest that spermidine supplementation may reduce oxidative stress in mice and flies (Eisenberg et al., 2009; Guo et al., 2011). 

However, the present observations are the first to show the antioxidant effects of spermidine in vascular tissue and to directly link this action with an improvement of physiological function. The potential mechanisms by which novel therapeutic agents such as spermidine reduce oxidative stress and improve vascular function often involve both direct and indirect effects (Dinkova-Kostova and Talalay, 2008; Forstermann, 2008). Previous reports utilizing cellfree systems indicate that at concentrations used in the present study, spermidine does not directly scavenge superoxide (Das and Misra, 2004; Kafy et al., 1986). In agreement with these observations, we found that short-term spermidine treatment in vitro did not prevent the aging-like increase in arterial superoxide production caused by pyocyanin. Together, these observations suggest an important role for autophagy-dependent indirect actions in mediating the long-term protective and antioxidant effects of spermidine.

Conclusions

In summary, supplementation with the polyamine and potential nutraceutical spermidine reverses large elastic artery stiffening, restores NO-mediated endothelial function and reduces oxidative stress, while enhancing autophagy in arteries of old mice. These novel findings provide the necessary preclinical evidence to support future translational studies on the efficacy of spermidine for treating age-associated arterial dysfunction and prevention CVD in older adults.