Gut Microbiome-Derived Metabolite Trimethylamine N-Oxide Induces Aortic Stiffening and Increases Systolic Blood Pressure With Aging in Mice and Humans

Abstract

[Figure: see text].

Keywords: advanced glycation end products; arterial stiffness; gut microbiota; pulse wave velocity.

Figure 1.. Higher plasma concentrations of trimethylamine N-oxide (TMAO) with aging are related to increased arterial stiffness and systolic blood pressure (BP).

A) Plasma TMAO and B) arterial stiffness (assessed as carotid-femoral pulse wave velocity [c-f PWV]) and casual (resting) systolic and diastolic BP in young (N=14-21) and middle-aged to older (MA/O; N=83-101) adults. Data are mean ± S.E.M. *p<0.05 vs. young adults. C-D) Higher plasma concentrations of TMAO are positively related to c-f PWV (C) and casual SBP (D) in unadjusted linear regression models, with 95% confidence intervals (dashed lines).

Figure 2.. Chronic trimethylamine N-oxide (TMAO) supplementation increases plasma TMAO concentrations, aortic stiffness, and systolic blood pressure (BP) in young and old mice.

In young adult (6-12 months of age) and old (21-24 months) mice supplemented without (Control; YC, OC) or with 0.12% TMAO (YT, OT) for 3-6 months: A) Plasma concentrations of TMAO; B-C) in vivo aortic pulse wave velocity (aPWV); and D-E) in vivo tail cuff systolic BP. All data are mean ± S.E.M. N=7-11/group. *p<0.05 vs. Control, within time point/age group. †p<0.05 vs. baseline (0 months on intervention), within group. ‡p<0.05 OT vs. YC.

Figure 3.. Chronic trimethylamine N-oxide (TMAO) supplementation increases aortic intrinsic mechanical stiffness and abundance of advanced glycation end-products.

In young adult mice (12 months at sacrifice) supplemented without (Control/YC) or with 0.12% TMAO (YT) for 6 months: A) Elastic modulus of the high-force collagen-dominant region (left) and the low-force elastin-dominant region (right) of the stress-strain curve, conducted in 1-2mm segments of thoracic aorta. B) Intima-media thickness measured in 7 μM segments of thoracic aorta, with representative images shown to the right. C-D) Protein abundance of mature type-1 collagen assessed by Western immunoblotting in aortic lysates, with representative Western blot images generated from WES electropherograms shown to the right (C), and assessed across vascular layers by immunohistochemistry in 7μm aorta sections, with representative images shown to the right (D). Arrows denote the medial-adventitial border. E) Protein abundance of advanced glycation end-products (AGEs) assessed by Western immunoblotting in aorta lysates (left) and across vascular layers by immunohistochemistry in 7 μM aorta sections (right), with representative blots/images below/to the right of the graphs. F) Protein abundance of elastin assessed by Western immunoblotting in aorta lysates, with representative Western blot images generated from WES electropherograms. All data are mean ± S.E.M. N=8-10/group for stress-strain testing. N=4-7/group for Western blotting and immunohistochemistry. *p<0.05 vs. Control. ‡p<0.10 vs. Control. AU, arbitrary units.

Figure 4.. Acute ex vivo incubation with TMAO increases intrinsic mechanical stiffness via crosslinking of advanced glycation end-products (AGEs) and reactive oxygen species.

A-C) Elastic modulus of the high-force collagen-dominant region of the stress-strain curve (i.e., intrinsic mechanical stiffness) in multiple segments of thoracic aorta from the same young mice incubated for 72 h in control media (Vehicle) or media supplemented with 30 μM TMAO (A; N=18), with or without the addition of either the AGEs crosslink inhibitor alagebrium (B; N=5) or the superoxide dismutase mimetic TEMPOL (C; N=5). D) Abundance of AGEs in aorta rings from 4 additional young mice following 72 h incubation in control media (Vehicle) or media supplemented with 30 μM TMAO ± the superoxide dismutase mimetic TEMPOL. Data are mean ± S.E.M. *p≤0.05 vs. Vehicle (media only). †p<0.05 vs. TMAO (media only).