Inorganic Nitrite Supplementation Improves Endothelial Function With Aging: Translational Evidence for Suppression of Mitochondria-Derived Oxidative Stress

Abstract

[Figure: see text].

Trial registration: ClinicalTrials.gov NCT02393742.

Keywords: mitochondria; nitric oxide; oxidative stress; reactive oxygen species; sodium nitrite.

Figures

Figure 1.

Brachial artery flow-mediated dilation (FMD) expressed as percent (A and C; treatment x time p<0.05) and absolute (B and D; treatment x time p<0.05) change before and after 12 weeks of placebo (N=21) or sodium nitrite supplementation (N=26). Values are presented as mean±SEM and as individual responses (lower panels). *p<0.05 vs. pre-intervention, within group.

Figure 2.

Nitrotyrosine expression in endothelial cells obtained via endovascular biopsy from study participants before (pre) and after (post) 12 weeks of supplementation with sodium nitrite or placebo (A, N=14–15/group; group x time p=0.28). Total reactive oxygen species (ROS) bioactivity (CellROX signal) (B; treatment x time p<0.05) and mitochondrial ROS bioactivity normalized for mitochondrial volume (MitoSOX relative to MitoTracker signal) (C; treatment x time p<0.05) in cultured human umbilical vein endothelial cells treated with plasma (plasma exposure experiments) from a subset of participants pre and post 12 weeks of supplementation with sodium nitrite or placebo, N=9–11/group. Data are expressed relative to pre-intervention values within group and presented as mean±SEM. *p<0.05 vs. pre-intervention, within group.

Figure 3.

Targeted metabolomics analysis of plasma samples taken before and after chronic supplementation with sodium nitrite or placebo from a subset of subjects (N=8–10/group). 3-Dimensional partial least squares-discriminant analysis depicting the differential response to treatment (expressed as a fold change from baseline within each group) in the sodium nitrite and placebo-treated groups (A). Hierarchical clustering analysis of top 25 metabolites differentially altered by treatment between sodium nitrite and placebo-treated groups (each box represents a fold change from baseline for a given subject) (B).

Figure 4.

Carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine (A) and peak EDD (B) in the absence and presence of the nitric oxide (NO) synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) in young (6 mo) control (YC) and old (27 mo) control (OC) mice and old mice supplemented with sodium nitrite for 8 weeks (ON). Carotid artery endothelium-independent dilation to increasing doses of the NO donor sodium nitroprusside (SNP) (C) and peak dilation to SNP (D). N=9–10/group. All data are mean±SEM. *p<0.05 vs. young control.

Figure 5.

Peak carotid artery endothelium-dependent dilation in the absence and presence of the mitochondrial-targeted antioxidant MitoQ (A) and the absence and presence of the mitochondrial stressor rotenone (B) in young (6 mo) control (YC) and old (27 mo) control (OC) mice and old mice supplemented with sodium nitrite for 8 weeks (ON). Aortic protein abundance of AMP-activated protein kinase (AMPK) (C), peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) (D) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) (E) and mitochondrial cytochrome C oxidase subunit IV (COXIV) (F) normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), with representative images from the same membrane below each panel. N=4–10/group. All data are mean±SEM. *p<0.05 vs. young control or for comparison indicated. #p<0.05 vs. absence of rotenone.