Aging is the primary risk factor for cardiovascular diseases, primarily due to development of vascular endothelial dysfunction. The gut microbiome is a strong influencer of host physiology, but few studies have investigated how gut microbiome composition changes with primary (healthy) aging in humans, or how such changes may influence endothelial function. PURPOSE To: 1) determine changes in gut microbiome composition and their relation to endothelial function in healthy late middle‐aged to older (MA/O) vs. young (Y) adults; and 2) investigate potential mechanisms of this link. METHODS & RESULTS N=14/group (MA/O: 60‐79 yrs; Y: 18‐29 yrs). Data are mean ± SE. Gut microbiome composition was assessed via fecal 16S rRNA sequencing. α‐diversity, phylogenetic diversity within each sample, was higher in MA/O vs Y adults (Faith’s PD: 22.2 ± 1.8 vs 15.5 ± 1.3, P = 0.02). β‐diversity, difference in overall composition between samples, was also altered with aging (PERMANOVA: P < 0.05; unweighted UniFrac). Both α‐diversity (R = ‐0.60, P = 0.04) and β‐diversity (R = ‐0.58, P = 0.04) were inversely related to age‐related impairments in endothelial function, measured by brachial artery flow‐mediated dilation (MA/O: 4.5 ± 0.4 vs Y: 7.9 ± 1.7%, P < 0.05). Potential Mechanisms: In preliminary analyses (unpaired t‐tests), changes in gut microbiome composition were accompanied by altered relative abundance of gram‐negative bacteria (e.g., Bacteroides [MA/O: 33 ± 4% vs Y: 20 ± 4%, P = 0.02]) and Enterobacteriaceae [MA/O: 1.5 ± 0.1% vs Y: 0.4 ± 0.1%, P = 0.02]), which contain pro‐inflammatory lipopolysaccharide (LPS) in their cell walls. Translocation of LPS into systemic circulation is facilitated by increased intestinal permeability, which we found was higher with aging, as measured by a lactulose‐mannitol (L:M) test (1‐hour serum L:M ratio, MA/O: 0.012 ± 0.002 vs Y: 0.007 ± 0.001, P = 0.04). As such, plasma LPS‐binding protein, a readily detectable marker of LPS in peripheral blood, was higher in MA/O vs Y adults (31.3 ± 4.1 vs 17.7 ± 2.0 ng/mL, P = 0.01). Once in circulation, LPS is recognized as a pathogen‐associated molecular pattern and can trigger an inflammatory response. Consistent with this, circulating levels of the pro‐inflammatory markers IL‐6 [MA/O: 1.56 ± 0.29 vs Y: 0.70 ± 0.13 pg/ml, P = 0.01] and CRP [MA/O: 1.65 ± 0.27 vs Y: 0.78 ± 0.11 pg/ml, P = 0.02]) were increased with aging. In biopsied venous endothelial cells from a subset of subjects (n = 4‐8/group), abundance of phosphorylated (i.e., activated) NF□B was higher in MA/O vs Y adults (0.40 ± 0.05 vs 0.31 ± 0.04 ng/ml, P = 0.24), with no difference in total NFkB, indicating potentially increased vascular inflammation. CONCLUSIONS Our findings represent initial evidence in humans that the gut microbiome changes with healthy aging and may be an important mediator of age‐related endothelial dysfunction, possibly via increasing circulating LPS and vascular inflammation.

Aging is the primary risk factor for the development of cardiovascular diseases (CVD), largely due to increases in systolic blood pressure (SBP) and oxidative stress‐related impairments in vascular endothelial function. Higher intake of dietary fiber is associated with reduced CVD risk. In healthy late middle‐aged to older (MA/O) adults, limited evidence indicates high‐fiber diet interventions improve SBP, but whether a high‐fiber diet improves endothelial function with aging is unknown. Purpose We tested the hypothesis that a high‐fiber diet would, in addition to reducing SBP, improve endothelial function and reduce oxidative stress in healthy MA/O adults. Methods We performed a controlled feeding study in which 14 MA/O adults (8F/6M; age: 69 ± 5 years) consumed an isocaloric high‐fiber diet (30‐35 g/day) for 7 days. Diets also contained 60% total carbohydrates, 15% protein, 25% fat, and 12‐15% sugar. Baseline (pre) measures were performed under habitual diet conditions (26 ± 6 g fiber/day). On days 0 (pre) and 7 (post) of the diet, endothelial cells were collected via venous endovascular biopsy, and endothelial function was assessed by brachial artery flow‐mediated dilation (FMD) following 20 minutes of intravenous infusion of saline (volume control) or the antioxidant ascorbic acid (0.06 g/kg fat‐free mass in 100 mL saline at 5 mL/min; N=12), to determine whether any improvements in endothelial functional following the high‐fiber diet were due to reduced oxidative stress. Results Consumption of a high‐fiber diet reduced SBP (pre vs. post: 120 ± 3 vs. 116 ± 3 mmHg, p<0.01) and improved endothelial function by ~30% (pre vs. post: 4.1 ± 0.5 vs. 5.3 ± 0.7 %, p=0.03). Abundance of nitrotyrosine (NT), a marker of oxidative stress, in endothelial cells from a subset of subjects (N=6) was lower after the high‐fiber diet (pre vs. post: 0.43 ± 0.16 vs. 0.29 ± 0.12 A.U., p=0.08). At baseline, infusion of ascorbic acid increased FMD by 2.2 ± 0.7 %Δ units (p=0.01 vs. saline), indicating tonic suppression of endothelial function by oxidative stress under habitual diet conditions. Ascorbic acid still increased FMD post‐high‐fiber diet but to a lesser extent (+1.2 ± 0.6 %Δ units, p=0.06 vs. saline), and differences in FMD pre vs. post high‐fiber diet were abolished by ascorbic acid (6.3 ± 0.9 vs. 6.6 ± 0.9 %, p=0.68). These data suggest that the high‐fiber diet improved FMD in part by reducing oxidative stress. Conclusions High‐fiber diet consumption improves SBP in healthy MA/O adults with normal baseline levels and induces clinically significant improvements in FMD possibly via reductions in vascular oxidative stress and tonic oxidative stress‐related suppression of endothelial function. These results suggest that a high‐fiber diet may reduce CVD risk, at least in part, by mitigating increases in SBP and oxidative stress‐related impairments in endothelial function with aging.