Suppression of the gut microbiome ameliorates age-related arterial dysfunction and oxidative stress in mice

Abstract

Key points: Age-related arterial dysfunction, characterized by oxidative stress- and inflammation-mediated endothelial dysfunction and arterial stiffening, is the primary risk factor for cardiovascular diseases. To investigate whether age-related changes in the gut microbiome may mediate arterial dysfunction, we suppressed gut microbiota in young and old mice with a cocktail of broad-spectrum, poorly-absorbed antibiotics in drinking water for 3-4 weeks. In old mice, antibiotic treatment reversed endothelial dysfunction and arterial stiffening and attenuated vascular oxidative stress and inflammation. To provide insight into age-related changes in gut microbiota that may underlie these observations, we show that ageing altered the abundance of microbial taxa associated with gut dysbiosis and increased plasma levels of the adverse gut-derived metabolite trimethylamine N-oxide. The results of the present study provide the first proof-of-concept evidence that the gut microbiome is an important mediator of age-related arterial dysfunction and therefore may be a promising therapeutic target for preserving arterial function with ageing, thereby reducing the risk of cardiovascular diseases.

Abstract: Oxidative stress-mediated arterial dysfunction (e.g. endothelial dysfunction and large elastic artery stiffening) is the primary mechanism driving age-related cardiovascular diseases. Accumulating evidence suggests the gut microbiome modulates host physiology because dysregulation ('gut dysbiosis') has systemic consequences, including promotion of oxidative stress. The present study aimed to determine whether the gut microbiome modulates arterial function with ageing. We measured arterial function in young and older mice after 3-4 weeks of treatment with broad-spectrum, poorly-absorbed antibiotics to suppress the gut microbiome. To identify potential mechanistic links between the gut microbiome and age-related arterial dysfunction, we sequenced microbiota from young and older mice and measured plasma levels of the adverse gut-derived metabolite trimethylamine N-oxide (TMAO). In old mice, antibiotics reversed endothelial dysfunction [area-under-the-curve carotid artery dilatation to acetylcholine in young: 345 ± 16 AU vs. old control (OC): 220 ± 34 AU, P < 0.01; vs. old antibiotic-treated (OA): 334 ± 15 AU; P < 0.01 vs. OC] and arterial stiffening (aortic pulse wave velocity in young: 3.62 ± 0.15 m s-1 vs. OC: 4.43 ± 0.38 m s-1 ; vs. OA: 3.52 ± 0.35 m s-1 ; P = 0.03). These improvements were accompanied by lower oxidative stress and greater antioxidant enzyme expression. Ageing altered the abundance of gut microbial taxa associated with gut dysbiosis. Lastly, plasma TMAO was higher with ageing (young: 2.6 ± 0.4 μmol L-1 vs. OC: 7.2 ± 2.0 μmol L-1 ; P < 0.0001) and suppressed by antibiotic treatment (OA: 1.2 ± 0.2 μmol L-1 ; P < 0.0001 vs. OC). The results of the present study provide the first evidence for the gut microbiome being an important mediator of age-related arterial dysfunction and oxidative stress and suggest that therapeutic strategies targeting gut microbiome health may hold promise for preserving arterial function and reducing cardiovascular risk with ageing in humans.

Keywords: Ageing; arterial stiffness; endothelial function; gut dysbiosis; inflammation.

© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.

Figures

Figure 1. Antibiotic treatment suppresses the gut microbiome

A, number of DNA copies measured by qPCR in faecal samples collected after 3–4 weeks on the intervention. Samples were run in duplicate and these data were confirmed and averaged across two separate qPCR runs. No batch effects were detected (P > 0.05 across batches, within animals, for all groups); n = 10–14 per group. B, number of DNA copies in faecal samples collected pre‐ and 3–4 weeks post‐antibiotic treatment; n = 4–6 per group. Data are the mean ± SEM. *P < 0.05 vs. YC. †P < 0.05 vs. OC. ‡P < 0.05 vs. pre‐intervention within groups. C and D, average relative abundance of all detected microbial phyla (C) and microbial families within the phylum Proteobacteria (D). *Significant effect (P < 0.05) of treatment (control vs. antibiotics) on ANCOVA.

Figure 2. Antibiotic treatment restores vascular endothelial function in old mice via improved NO bioavailability

A, dose–response endothelium‐dependent dilatation (EDD) to acetylcholine (ACh). B, peak NO‐mediated dilatation to ACh, assessed as the difference between peak EDD in the absence vs. presence of the NO synthase inhibitor l‐NAME. C, dose–response endothelium independent dilatation to the NO donor SNP. Data are the mean ± SEM. *P < 0.05 vs. YC. YC, young control; YA, young antibiotic‐treated; OC, old control; OA, old antibiotic‐treated.

Figure 3. Antibiotic treatment reverses age‐related arterial stiffening

A, aortic pulse wave velocity pre‐ and post‐intervention; n = 9–15 per group. B, representative stress‐strain curve of an aortic ring from OC and OA mice for determination of ex vivo intrinsic mechanical stiffness. C and D, elastic modulus of the elastin (C) and collagen (D) portions of the stress‐strain curve in aortic rings; n = 8–10 per group. E and F, aortic protein expression of elastin (E) and mature‐type collagen‐1 (F) normalized to GAPDH (loading control), with representative western blot images included below; n = 8–10 per group. G, intima media thickness of the aorta, with representative images of whole aortic sections (bottom) and enlargements of the same sections (top) included below the mean data; n = 8–10 per group. Data are the mean ± SEM. *P < 0.05 vs. YC. †P < 0.05 vs. OC. ‡P < 0.05 pre‐ vs. post‐intervention within groups. YC, young control; YA, young antibiotic‐treated; OC, old control; OA, old antibiotic‐treated. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4. Antibiotic treatment normalizes oxidative stress and inflammation in old mice

A, whole‐cell superoxide production in aortic segments. Representative electron paramagnetic resonance spectra are shown below. Protein expression of nitrotyrosine (NT) (B) and ecSOD (C). Representative western blot images for NT (data are sum of the two bands), ecSOD and GAPDH (loading control) are shown below. D–F, concentrations of pro‐inflammatory cytokines in aortic lysates: IL‐6 (D), TNF‐α (E) and IFN‐γ (F). Data are the mean ± SEM; n = 7–11 per group. *P < 0.05 vs. YC. †P < 0.05 vs. OC. YC, young control; YA, young antibiotic‐treated; OC, old control; OA, old antibiotic‐treated.

Figure 5. Ageing alters the mouse gut microbiome

A, principal co‐ordinate analysis plot of 16S rRNA‐based gut microbial profiling with unweighted UniFrac from young (8–10 months) and older (15–24 months) mice fed a standard diet. B, alpha diversity [Faith's phylogenetic diversity (PD)] in young and older mice. Data are box‐and‐whisker plot. *P < 0.05 vs. young. C, average relative abundance (mean ± SE) of bacterial phyla significantly altered by ageing. *P < 0.05 vs. young. D, relative abundance of bacterial phyla across individual samples from older and young mice. E, centred log ratio (CLR) mean differences (F statistic) in differentially abundant bacterial genera between older vs. young mice, as determined by ANCOM analysis. ‘p’, phylum; ‘f’, family; ‘g’, genus. ‘g__’ and the next highest known order are given for operational taxonomic units that do not have a specific genus name. *Statistical significance.

Figure 6. Ageing is associated with increased TMAO

A, plasma concentrations of TMAO were higher in old mice and suppressed by antibiotic treatment. B, both ageing and antibiotic treatment increased protein expression of the TMA‐converting enzyme FMO3 in the liver. Data are the mean ± SEM. *P < 0.05 vs. YC. †P < 0.05 vs. OC. YC, young control; YA, young antibiotic‐treated; OC, old control; OA, old antibiotic‐treated.