Elevated Circulating Trimethylamine N-Oxide Levels Contribute to Endothelial Dysfunction in Aged Rats through Vascular Inflammation and Oxidative Stress

Tiejun Li, Yanli Chen, Chaojun Gua, Xiaodong Li, Tiejun Li, Yanli Chen, Chaojun Gua, Xiaodong Li

Abstract

Vascular endothelial dysfunction, a characteristic of the aging process, is an important risk factor for cardiovascular disease in aging. Although, vascular inflammation and oxidative stress are major contributors to endothelial dysfunction in aging, the underlying mechanisms during the aging process are not fully understood. Accumulating evidence reveals that gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) is implicated in the pathogenesis of many cardiovascular diseases. We tested the hypothesis that aging increases circulating TMAO levels, which induce vascular inflammation and oxidative stress, resulting in age-associated endothelial dysfunction. Old (22-mo-old) and young (4-mo-old) Fischer-344 rats were treated without (control) or with 1.0% 3,3-Dimethyl-1-butanol (DMB, an inhibitor of trimethylamine formation) in drinking water for 8 weeks. Compared with young control group, old control group had markedly higher plasma TMAO levels, which were reduced by DMB treatment. Endothelium-dependent relaxation of aorta in response to acetylcholine was impaired in old control group compared with young control group as indicated by decreased maximal relaxation (Emax) and reduced area under the curve (AUC). Emax and AUC were both normalized in old rats treated with DMB. No difference in endothelial-independent relaxation in response to sodium nitroprusside was observed among groups. Molecular studies revealed that old control group exhibits increased expression of proinflammatory cytokines and superoxide production, and decreased expression of endothelial nitric-oxide synthase (eNOS) in the aorta, all of which were restored by DMB treatment. These results suggest that aging increases circulating TMAO levels, which may impair eNOS-derived NO bioavailability by increasing vascular inflammation and oxidative stress, contributing to aging-associated endothelial dysfunction.

Keywords: aging; endothelial dysfunction; inflammation; oxidative stress; trimethylamine N-oxide.

Figures

Figure 1
Figure 1
Effects of aging and 3,3-Dimethyl-1-butanol (DMB, an inhibitor of trimethylamine formation) on circulating TMAO Levels. Old rats had higher circulating TMAO levels than young rats. DMB treatment reduced circulating TMAO levels in both young and old rats. Data are expressed as mean ± SE (n = 9–10 for each group). *P < 0.05 vs. Y-CON; †P < 0.05, O+DMB vs. O-CON.
Figure 2
Figure 2
Effects of aging and DMB on endothelial-dependent relaxation. (A) Dose responses of endothelium-intact aortic rings to acetylcholine (ACh); (B) Maximal relaxation (Emax) to ACh; (C) Area under the curve (AUC), and (D) Half maximal effective concentration (LogEC50). Data are expressed as mean ± SE (n = 9–10 for each group). *P < 0.05 vs. Y-CON; †P < 0.05, O+DMB vs. O-CON.
Figure 3
Figure 3
(A) Effects of aging and DMB on endothelial-dependent relaxation to ACh in endothelium-intact aortic rings in the presence of a non-selective nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME); (B) effects of aging and DMB on endothelial-dependent relaxation to ACh in endothelium-denuded aortic rings, and (C) effects of aging and DMB on endothelial-independent relaxation to sodium nitroprusside (SNP) in endothelium-denuded aortic rings. Data are expressed as mean ± SE (n = 6–10 for each group).
Figure 4
Figure 4
Effects of aging and DMB on expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-α (B), interleukin (IL)-1β (C), total eNOS (D), phosphorylated (Ph-) eNOS (E), and the ratio of Ph-eNOS to total eNOS (F) in the aorta. Representative Western blots from each group are shown in (A). Data are expressed as mean ± SE (n = 7 for each group). *P < 0.05 vs. Y-CON; †P < 0.05, O+DMB vs. O-CON.
Figure 5
Figure 5
Effects of aging and DMB on superoxide production. NADPH oxidase-derived superoxide production in the aorta was detected by dihydroethidium (DHE). (A) Representative DHE images from each group; (B) Quantitative comparison of DHE fluorescence among groups. Data are expressed as mean ± SE (n = 7 for each group). *P < 0.05 vs. Y-CON; †P < 0.05, O+DMB vs. O-CON.
Figure 6
Figure 6
Schematic diagram showing possible mechanisms by which elevated TMAO levels induce endothelial dysfunction in aging.

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