Atherosclerotic cardiovascular disease (ACVD) is a major cause of mortality and morbidity worldwide, and increased low-density lipoproteins (LDLs) play a critical role in development and progression of atherosclerosis. Here, we examined for the first time gut immunomodulatory effects of the microbiota-derived metabolite propionic acid (PA) on intestinal cholesterol metabolism. Using both human and animal model studies, we demonstrate that treatment with PA reduces blood total and LDL cholesterol levels. In apolipoprotein E^−/− (Apoe ^−/−) mice fed a high-fat diet (HFD), PA reduced intestinal cholesterol absorption and aortic atherosclerotic lesion area. Further, PA increased regulatory T-cell numbers and interleukin (IL)-10 levels in the intestinal microenvironment, which in turn suppressed the expression of Niemann-Pick C1-like 1 (Npc1l1), a major intestinal cholesterol transporter. Blockade of IL-10 receptor signalling attenuated the PA-related reduction in total and LDL cholesterol and augmented atherosclerotic lesion severity in the HFD-fed Apoe ^−/− mice. To translate these preclinical findings to humans, we conducted a randomized, double-blinded, placebo-controlled human study (clinical trial no. NCT03590496). Oral supplementation with 500 mg of PA twice daily over the course of 8 weeks significantly reduced LDL [−15.9 mg/dL (−8.1%) vs. −1.6 mg/dL (−0.5%), P = 0.016], total [−19.6 mg/dL (−7.3%) vs. −5.3 mg/dL (−1.7%), P = 0.014] and non-high-density lipoprotein cholesterol levels [PA vs. placebo: −18.9 mg/dL (−9.1%) vs. −0.6 mg/dL (−0.5%), P = 0.002] in subjects with elevated baseline LDL cholesterol levels. Our findings reveal a novel immune-mediated pathway linking the gut microbiota-derived metabolite PA with intestinal Npc1l1 expression and cholesterol homeostasis. The results highlight the gut immune system as a potential therapeutic target to control dyslipidaemia that may introduce a new avenue for prevention of ACVDs. The figure illustrates the proposed model of the cholesterol-lowering and atheroprotective properties of propionate. A high-fat high-cholesterol diet causes a disbalance of immune cells in the small intestinal microenvironment, with reduced regulatory T cell frequencies and interleukin-10 concentrations. Altered regulatory T cell levels are rescued upon exogenous propionate supplementation, with increased local levels. This in turn modulates NPC1L1 expression and membrane density, with a subsequent reduction in cholesterol absorption, ultimately leading to reduced atherogenesis. The illustration was adopted from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Common Attribution 3.0 Generic License. HFD, high-fat diet; IL-10, interleukin-10; NPC1L1, Niemann-Pick C1-like 1; PA, propionic acid; Treg, regulatory T cell.

Using an untargeted metabolomics approach in initial ( N = 99 subjects) and replication cohorts ( N = 1,162), we discovered and structurally identified a plasma metabolite associated with cardiovascular disease (CVD) risks, N 6, N 6, N 6-trimethyl-L-lysine (trimethyllysine, TML). Stable-isotope-dilution tandem mass spectrometry analyses of an independent validation cohort ( N = 2,140) confirmed TML levels are independently associated with incident (3-year) major adverse cardiovascular event risks (hazards ratio [HR], 2.4; 95% CI, 1.7–3.4) and incident (5-year) mortality risk (HR, 2.9; 95% CI, 2.0–4.2). Genome-wide association studies identified several suggestive loci for TML levels, but none reached genome-wide significance; and d9(trimethyl)-TML isotope tracer studies confirmed TML can serve as a nutrient precursor for gut microbiota–dependent generation of trimethylamine (TMA) and the atherogenic metabolite trimethylamine N -oxide (TMAO). Although TML was shown to be abundant in both plant- and animal-derived foods, mouse and human fecal cultures (omnivores and vegans) showed slow conversion of TML to TMA. Furthermore, unlike chronic dietary choline, TML supplementation in mice failed to elevate plasma TMAO or heighten thrombosis potential in vivo. Thus, TML is identified as a strong predictor of incident CVD risks in subjects and to serve as a dietary precursor for gut microbiota–dependent generation of TMAO; however, TML does not appear to be a major microbial source for TMAO generation in vivo.

Background: The gut microbiota-dependent metabolite phenylacetylgutamine (PAGln) is both associated with atherothrombotic heart disease in humans, and mechanistically linked to cardiovascular disease pathogenesis in animal models via modulation of adrenergic receptor signaling. Methods: Here we examined both clinical and mechanistic relationships between PAGln and heart failure (HF). First, we examined associations among plasma levels of PAGln and HF, left ventricular ejection fraction, and N-terminal pro-B-type natriuretic peptide in 2 independent clinical cohorts of subjects undergoing coronary angiography in tertiary referral centers (an initial discovery US Cohort, n=3256; and a validation European Cohort, n=829). Then, the impact of PAGln on cardiovascular phenotypes relevant to HF in cultured cardiomyoblasts, and in vivo were also examined. Results: Circulating PAGln levels were dose-dependently associated with HF presence and indices of severity (reduced ventricular ejection fraction, elevated N-terminal pro-B-type natriuretic peptide) independent of traditional risk factors and renal function in both cohorts. Beyond these clinical associations, mechanistic studies showed both PAGln and its murine counterpart, phenylacetylglycine, directly fostered HF-relevant phenotypes, including decreased cardiomyocyte sarcomere contraction, and B-type natriuretic peptide gene expression in both cultured cardiomyoblasts and murine atrial tissue. Conclusions: The present study reveals the gut microbial metabolite PAGln is clinically and mechanistically linked to HF presence and severity. Modulating the gut microbiome, in general, and PAGln production, in particular, may represent a potential therapeutic target for modulating HF. Registration: URL: https://clinicaltrials.gov/ ; Unique identifier: NCT00590200 and URL: https://drks.de/drks_web/ ; Unique identifier: DRKS00020915.

Precision microbiome modulation as a novel treatment strategy is a rapidly evolving and sought goal. The aim of this study is to determine relationships among systemic gut microbial metabolite levels and incident cardiovascular disease risks to identify gut microbial pathways as possible targets for personalized therapeutic interventions. Stable isotope dilution mass spectrometry methods to quantitatively measure aromatic amino acids and their metabolites were used to examine sequential subjects undergoing elective diagnostic cardiac evaluation in two independent cohorts with longitudinal outcome data [US (n = 4000) and EU (n = 833) cohorts]. It was also used in plasma from humans and mice before vs. after a cocktail of poorly absorbed antibiotics to suppress gut microbiota. Multiple aromatic amino acid-derived metabolites that originate, at least in part, from gut bacteria are associated with incident (3-year) major adverse cardiovascular event (MACE) risks (myocardial infarction, stroke, or death) and all-cause mortality independent of traditional risk factors. Key gut microbiota-derived metabolites associated with incident MACE and poorer survival risks include: (i) phenylacetyl glutamine and phenylacetyl glycine (from phenylalanine); (ii) p-cresol (from tyrosine) yielding p-cresol sulfate and p-cresol glucuronide; (iii) 4-OH-phenyllactic acid (from tyrosine) yielding 4-OH-benzoic acid and 4-OH-hippuric acid; (iv) indole (from tryptophan) yielding indole glucuronide and indoxyl sulfate; (v) indole-3-pyruvic acid (from tryptophan) yielding indole-3-lactic acid and indole-3-acetyl-glutamine, and (vi) 5-OH-indole-3-acetic acid (from tryptophan). Key gut microbiota-generated metabolites derived from aromatic amino acids independently associated with incident adverse cardiovascular outcomes are identified, and thus will help focus future studies on gut-microbial metabolic outputs relevant to host cardiovascular health. Gut microbiota-generated metabolites and pathways derived from aromatic amino acids that independently associate with incident major adverse cardiovascular event (MACE) and death risk are identified using a combination of large-scale clinical association studies (US and European cohorts with longitudinal outcome data), and both human and animal studies involving gut microbial suppression with oral ingestion of a cocktail of poorly absorbed antibiotics.