The autophagy enhancer spermidine reverses arterial aging

Abstract

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

Figure 1. Spermidine supplementation normalizes large elastic artery stiffness and structural proteins

(A) Aortic pulse wave velocity in young and old control (YC and OC) and young and old spermidine supplemented (YS and OS) mice. Representative ECG and Doppler flow signals at right. (B) Advanced glycation end products (AGEs), and (C) protein expression of collagen I in whole aortas. Representative Western blot images below. Protein expression data expressed relative to GAPDH and normalized to YC mean value. Values are means ± SEM (n = 7–8 per group). *P < 0.05 vs. YC. **P < 0.05 vs. OC.

Figure 2. Spermidine supplementation restores NO-mediated EDD in old mice

(A) Dose responses (EDD) to acetylcholine (ACh) with/without the NO inhibitor L-NAME in carotid arteries of young and old control (YC and OC) and young and old spermidine supplemented (YS and OS) mice. (B) NO-dependent dilation (max dilation_ACh - max dilation_ACh+L-NAME). (C) Endothelium-independent dilation to the NO donor sodium nitroprusside (SNP). All data presented on a percent basis to account for differences in arterial diameter among groups. Values are means ± SEM (n = 7–8 per group). *P < 0.05 vs. YC.

Figure 3. Spermidine supplementation reduces arterial oxidative stress in old mice

(A) Nitrotyrosine, a marker of oxidative protein damage, in aorta of young and old control (YC and OC) and young and old spermidine supplemented (YS and OS) mice. Representative Western blot images below. (B) Mean EPR signal for superoxide in aortic rings. Representative EPR signals below. (C) Maximal carotid artery dilation (EDD) to acetylcholine (ACh) in the presence/absence of the superoxide scavenger TEMPOL. Protein expression data expressed relative to GAPDH. Protein expression and EPR data normalized to YC mean value. Values are means ± SEM (n = 7–8 per group). *P < 0.05 vs. YC.

Figure 4. Spermidine supplementation increases markers of autophagy in old mice

(A) Protein expression of the autophagy marker LC3-II in aorta of young and old control (YC and OC) and young and old spermidine supplemented (YS and OS) mice. (B) The p62 adapter protein, a marker of undegraded autophagy substrates. (C) Acetylation of the autophagy-relevant histone H3. (D) Expression of the core autophagy machinery protein Atg3. Data expressed relative to GAPDH and normalized to YC mean value. Representative Western blot images below. Values are means ± SEM (n = 7–8 per group). *P < 0.05 vs. YC. **P < 0.05 vs. OC.

Figure 5. The protective effects of spermidine are time- and autophagy-dependent

Mean EPR signal for superoxide in cultured aortic rings incubated for 1 or 48 h with or without the oxidative stress inducer pyocyanin (10 µM), spermidine (3 mM) or the autophagy inhibitor chloroquine (50 µM). Data expressed relative to control condition for each trial. Values are means ± SEM (n = 6 per group). *P < 0.05 vs. control.