Publications by authors named "Zeneng Wang"

92 Publications

Loop Diuretics Inhibit Renal Excretion of Trimethylamine -Oxide.

JACC Basic Transl Sci 2021 Feb 27;6(2):103-115. Epub 2021 Jan 27.

Center for Microbiome and Human Health, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.

This study demonstrates, for the first time, that renal tubular excretion of trimethylamine -oxide (TMAO) is inhibited by concomitant loop diuretic administration. The observed marked accumulation in the renal parenchyma, and to lesser extent, plasma, implies differential distributions of TMAO across various tissues and/or systems as a consequence of efflux channel control. A better understanding of TMAO renal clearance and its potential interactions with current and future therapies in patients with heart failure are warranted.
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http://dx.doi.org/10.1016/j.jacbts.2020.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907536PMC
February 2021

Plasma trimethylamine N-oxide and its metabolic precursors and risk of mortality, cardiovascular and renal disease in individuals with type 2-diabetes and albuminuria.

PLoS One 2021 3;16(3):e0244402. Epub 2021 Mar 3.

Steno Diabetes Center, Copenhagen, Denmark.

Aims: The trimethylamine N-oxide (TMAO) pathway is related to intestinal microbiota and has been associated to risk of cardiovascular disease (CVD). We investigated associations between four plasma metabolites in the TMAO pathway and risk of all-cause mortality, CVD and deterioration in renal function in individuals with type 2-diabetes (T2D) and albuminuria.

Materials And Methods: Plasma concentrations of TMAO, choline, carnitine, and betaine were measured by liquid chromatography-tandem mass spectrometry at baseline in 311 individuals with T2D and albuminuria. Information on all-cause mortality and fatal/non-fatal CVD during follow-up was obtained from registries. The association of each metabolite, and a weighted sum score of all four metabolites, with the endpoints were examined. Serum creatinine was measured at follow-up visits and the renal endpoint was defined as eGFR-decline of ≥30%. Associations were analysed using proportional hazards models adjusted for traditional risk factors.

Results: Baseline mean(SD) age was 57.2(8.2) years and 75% were males. Follow-up was up to 21.9 years (median (IQR) follow-up 6.8 (6.1-15.5) years for mortality and 6.5 (5.5-8.1) years for CVD events). The individual metabolites and the weighted sum score were not associated with all-cause mortality (n = 106) or CVD (n = 116) (adjusted p≥0.09). Higher choline, carnitine and the weighted sum score of the four metabolites were associated with higher risk of decline in eGFR (n = 106) (adjusted p = 0.001, p = 0.03 and p<0.001, respectively).

Conclusions: In individuals with T2D and albuminuria, higher choline, carnitine and a weighted sum of four metabolites from the TMAO pathway were risk markers for deterioration in renal function during long-term follow-up. Metabolites from the TMAO pathway were not independently related to risk of all-cause mortality or CVD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0244402PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928450PMC
March 2021

Plasma trimethylamine N-oxide (TMAO) levels predict future risk of coronary artery disease in apparently healthy individuals in the EPIC-Norfolk prospective population study.

Am Heart J 2021 Feb 21. Epub 2021 Feb 21.

Center for Microbiome and Human Health, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland, OH; Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland, OH.

Background: Recent studies show a mechanistic link between gut microbiota-dependent formation of the atherosclerosis- and thrombosis-promoting metabolite trimethylamine N-oxide (TMAO) and cardiovascular disease (CVD). The clinical utility of TMAO in apparently healthy subjects for predicting incident CVD risks is unclear.

Methods And Results: In the EPIC-Norfolk community-based study, we examined baseline fasting levels of TMAO and two of its nutrient precursors, choline and betaine, in a case:control design study comparing apparently European healthy middle-aged participants who subsequently develop CVD (Cases, n = 908) vs those who did not (Controls, n = 1,273) over an ensuing average follow-up period of 8 years. In participants who developed CVD vs controls, higher plasma TMAO (3.70 [IQR 2.50-6.41]μM vs 3.25 [IQR 2.19-52,1.15]μM; P < .001) and choline levels (9.09 [IQR 7.87-10.53]μM vs 8.89 [IQR 7.66-10.13]μM; P = .001) were observed. Following adjustments for traditional risk factors, elevated TMAO (adjusted odds ratio (OR) 1.58 [95% confidence interval (CI) 1.21-2.06], P < .001) and choline levels (adjusted OR 1.31 [95%CI 1.00-1.72], P < .05) remained predictive of incident CVD development. The clinical prognostic utility of TMAO remained significant and essentially unchanged regardless of the level of cutoff chosen between 1.5 uM (10%ile) to 10.5 uM (90%ile).

Conclusion: In apparently healthy participants of the community-based middle-aged EPIC-Norfolk population, elevated plasma levels of the gut microbe-dependent metabolite TMAO, and its nutrient precursor choline, predict incident risk for CVD development independent of traditional risk factors.
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http://dx.doi.org/10.1016/j.ahj.2021.01.020DOI Listing
February 2021

Inhibition of microbiota-dependent TMAO production attenuates chronic kidney disease in mice.

Sci Rep 2021 Jan 12;11(1):518. Epub 2021 Jan 12.

Division of Cardiology, Department of Medicine, University of California, 10833 Le Conte Avenue, A2-237 CHS, Los Angeles, CA, 90095-1679, USA.

Patients with chronic kidney disease (CKD) have elevated circulating levels of trimethylamine N-oxide (TMAO), a metabolite derived from gut microbes and associated with cardiovascular diseases. High circulating levels of TMAO and its dietary precursor, choline, predict increased risk for development of CKD in apparently healthy subjects, and studies in mice fed TMAO or choline suggest that TMAO can contribute to kidney impairment and renal fibrosis. Here we examined the interactions between TMAO, kidney disease, and cardiovascular disease in mouse models. We observed that while female hyperlipidemic apoE KO mice fed a 0.2% adenine diet for 14 weeks developed CKD with elevated plasma levels of TMAO, provision of a non-lethal inhibitor of gut microbial trimethylamine (TMA) production, iodomethylcholine (IMC), significantly reduced multiple markers of renal injury (plasma creatinine, cystatin C, FGF23, and TMAO), reduced histopathologic evidence of fibrosis, and markedly attenuated development of microalbuminuria. In addition, while the adenine-induced CKD model significantly increased heart weight, a surrogate marker for myocardial hypertrophy, this was largely prevented by IMC supplementation. Surprisingly, adenine feeding did not increase atherosclerosis and significantly decreased the expression of inflammatory genes in the aorta compared to the control groups, effects unrelated to TMAO levels. Our data demonstrate that inhibition of TMAO production attenuated CKD development and cardiac hypertrophy in mice, suggesting that TMAO reduction may be a novel strategy in treating CKD and its cardiovascular disease complications.
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http://dx.doi.org/10.1038/s41598-020-80063-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804188PMC
January 2021

Bile acids profile, histopathological indices and genetic variants for non-alcoholic fatty liver disease progression.

Metabolism 2021 03 1;116:154457. Epub 2020 Dec 1.

Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH 44195, USA; Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland Clinic, Cleveland, OH 44195, USA; Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA. Electronic address:

Objective: Metabolomic studies suggest plasma levels of bile acids (BAs) are elevated amongst subjects with non-alcoholic fatty liver disease (NAFLD) compared to healthy controls. However, it remains unclear whether or not specific BAs are associated with the clinically relevant transition from nonalcoholic fatty liver (i.e. simple steatosis) to non-alcoholic steatohepatitis (NASH), or enhanced progression of hepatic fibrosis, or genetic determinants of NAFLD/NASH.

Methods: Among sequential subjects (n=102) undergoing diagnostic liver biopsy, we examined the associations of a broad panel of BAs with distinct histopathological features of NAFLD, the presence of NASH, and their associations with genetic variants linked to NAFLD and NASH.

Results: Plasma BA alterations were observed through the entire spectrum of NAFLD, with several glycine conjugated forms of the BAs demonstrating significant associations with higher grades of inflammation and fibrosis. Plasma 7-Keto-DCA levels showed the strongest associations with advanced stages of hepatic fibrosis [odds ratio(95% confidence interval)], 4.2(1.2-16.4), NASH 24.5(4.1-473), and ballooning 18.7(4.8-91.9). Plasma 7-Keto-LCA levels were associated with NASH 9.4(1.5-185) and ballooning 5.9(1.4-28.8). Genetic variants at several NAFLD/NASH loci were nominally associated with increased levels of 7-Keto- and glycine-conjugated forms of BAs, and the NAFLD risk allele at the TRIB1 locus showed strong tendency toward increased plasma levels of GCA (p=0.02) and GUDCA (p=0.009).

Conclusions: Circulating bile acid levels are associated with histopathological and genetic determinants of the transition from simple hepatic steatosis into NASH. Further studies exploring the potential involvement of bile acid metabolism in the development and/or progression of distinct histopathological features of NASH are warranted.
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http://dx.doi.org/10.1016/j.metabol.2020.154457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856026PMC
March 2021

Bacterial second messenger 3',5'-cyclic diguanylate attracts Caenorhabditis elegans and suppresses its immunity.

Commun Biol 2020 Nov 20;3(1):700. Epub 2020 Nov 20.

Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC, 29634, USA.

Cyclic di-nucleotides are important secondary signaling molecules in bacteria that regulate a wide range of processes. In this study, we found that Caenorhabditis elegans can detect and are attracted to multiple signal molecules produced by Vibrio cholerae, specifically the 3',5'-cyclic diguanylate (c-di-GMP), even though this bacterium kills the host at a high rate. C-di-GMP is sensed through C. elegans olfactory AWC neurons, which then evokes a series of signal transduction pathways that lead to reduced activity of two key stress response transcription factors, SKN-1 and HSF-1, and weakened innate immunity. Taken together, our study elucidates the role of c-di-GMP in interkingdom communication. For C. elegans, bacterial c-di-GMP may serve as a cue that they can use to detect food. On the other hand, preexposure to low concentrations of c-di-GMP may impair their immune response, which could facilitate bacterial invasion and survival.
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http://dx.doi.org/10.1038/s42003-020-01436-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679379PMC
November 2020

Effects of Smoking and Smoking Cessation on the Intestinal Microbiota.

J Clin Med 2020 Sep 14;9(9). Epub 2020 Sep 14.

Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792, USA.

We evaluated associations of smoking heaviness markers and the effects of smoking cessation on the intestinal microbiota and cardiovascular disease risk factors in current smokers undertaking a quit attempt. Participants were current smokers enrolled in a prospective randomized clinical trial of smoking cessation therapies with visits at baseline, 2, and 12 weeks. Genomic DNA was extracted from fecal samples followed by 16S rRNA gene sequencing and analysis using the QIIME2 software workflow. Relative abundances of bacterial taxa and alpha- and beta-diversity measures were used for comparisons. The 36 smokers were (mean (standard deviation)) 51.5 (11.1) years old (42% male) and smoked 15.1 (6.4) cigarettes per day for 22.7 (11.9) pack-years. Relative abundances of the phylum Actinobacteria correlated with pack-years (rho = -0.44, = 0.008) and Cyanobacteria correlated with CO levels (rho = 0.39, = 0.021). After 12 weeks, relative abundances of the phylum Bacteroidetes increased ( = 0.048) and Firmicutes decreased ( = 0.036) among abstainers compared to continuing smokers. Increases in alpha-diversity were associated with heart rates (rho = -0.59, = 0.037), systolic blood pressures (rho = -0.58, = 0.043), and C-reactive protein (rho = -0.60, = 0.034). Smoking cessation led to minor changes in the intestinal microbiota. It is unclear if the proven health benefits of smoking cessation lead to salutary changes in the intestinal microbiota.
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http://dx.doi.org/10.3390/jcm9092963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564179PMC
September 2020

Impact of trimethylamine N-oxide (TMAO) metaorganismal pathway on cardiovascular disease.

J Lab Precis Med 2020 Apr 20;5. Epub 2020 Apr 20.

Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.

Host-microbes interaction plays a crucial role in cardiovascular disease (CVD) pathogenesis, mechanistically via metaorganismal pathways. The trimethylamine N-oxide (TMAO) metaorganismal pathway is the most deeply investigated one, which comprises trimethylamine precursors, such as choline, trimethylamine lyase, trimethylamine, host liver FMO3, TMAO, and downstream effectors involving unfolded protein response (UPR), NF-κB and NLRP3 inflammasome. Accumulating data from clinical investigations of CVD patient cohorts and rodent models have supported the critical role of this metaorganismal pathway in the pathogenesis of CVD. We summarize an array of significant animal studies especially for arthrosclerosis with an emphasis on downstream molecular effectors of this metaorganismal pathway. We highlight clinical investigations of the prognostic value of plasma TMAO levels in predicting prospective risk for future major adverse cardiac events (MACE) indicated by composite end points of myocardial infarction (MI), stroke, heart failure (HF), other ischemic cardiovascular events, or death. Further, we discuss the latest advances of preclinical models targeting the gut microbiota trimethylamine lyase of the TMAO metaorganismal pathway for CVD intervention, as well as the catalog of gut microbiota TMA lyase genes and microbes in the human gut as the prerequisite for potential clinical intervention. In-depth characterization of TMAO metaorganismal pathway holds great promise for CVD clinical metagenomics, diagnostics and therapeutics.
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http://dx.doi.org/10.21037/jlpm.2020.01.01DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316184PMC
April 2020

Gut Microbiota-Dependent Trimethylamine N-oxide and Cardiovascular Outcomes in Patients With Prior Myocardial Infarction: A Nested Case Control Study From the PEGASUS-TIMI 54 Trial.

J Am Heart Assoc 2020 05 5;9(10):e015331. Epub 2020 May 5.

TIMI Study Group Division of Cardiovascular Medicine Brigham and Women's Hospital Harvard Medical School Boston MA.

Background Trimethylamine N-oxide (TMAO) may have prothrombotic properties. We examined the association of TMAO quartiles with major adverse cardiovascular events (MACE) and the effect of TMAO on the efficacy of ticagrelor. Methods and Results PEGASUS-TIMI 54 (Prevention of Cardiovascular Events in Patients With Prior Heart Attack Using Ticagrelor Compared to Placebo on a Background of Aspirin - Thrombolysis in Myocardial Infarction 54) randomized patients with prior myocardial infarction to ticagrelor or placebo (median follow-up 33 months). Baseline plasma concentrations of TMAO were measured in a nested case-control study of 597 cases with cardiovascular death, myocardial infarction, or stroke (MACE) and 1206 controls matched for age, sex, and estimated glomerular filtration rate [eGFR]. Odds ratios (OR) were used for the association between TMAO quartiles and MACE, adjusting for baseline clinical characteristics (age, sex, eGFR, region, body mass index, hypertension, hypercholesterolemia, diabetes mellitus, smoking, peripheral artery disease, index event, aspirin dosage and treatment arm), and cardiovascular biomarkers (hs-TnT [high-sensitivity troponin T], hs-CRP [high-sensitivity C-reactive protein], NT-proBNP [N-terminal-pro-B-type natriuretic peptide]). Higher TMAO quartiles were associated with risk of MACE (OR for quartile 4 versus quartile 1, 1.43, 95% CI, 1.06-1.93, trend=0.015). The association was driven by cardiovascular death (OR 2.25, 95% CI, 1.28-3.96, trend=0.003) and stroke (OR 2.68, 95% CI, 1.39-5.17, trend<0.001). After adjustment for clinical factors, the association persisted for cardiovascular death (OR 1.89, 95% CI, 1.03-3.45, trend=0.027) and stroke (OR 2.01, 95% CI, 1.01-4.01, trend=0.022), but was slightly attenuated after adjustment for cardiovascular biomarkers (cardiovascular death: OR 1.74, 95% CI, 0.88-3.45, trend=0.079; and stroke: OR 1.82, 95% CI, 0.88-3.78, trend=0.056). The reduction in MACE with ticagrelor was consistent across TMAO quartiles ( interaction=0.92). Conclusions Among patients with prior myocardial infarction, higher TMAO levels were associated with cardiovascular death and stroke but not with recurrent myocardial infarction. The efficacy of ticagrelor was consistent regardless of TMAO levels. Registration URL: https://www.clini​caltr​ials.gov; Unique identifiers: PEGASUS-TIMI 54, NCT01225562.
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http://dx.doi.org/10.1161/JAHA.119.015331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660879PMC
May 2020

Small molecule inhibition of gut microbial choline trimethylamine lyase activity alters host cholesterol and bile acid metabolism.

Am J Physiol Heart Circ Physiol 2020 06 24;318(6):H1474-H1486. Epub 2020 Apr 24.

Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute Cleveland Clinic, Cleveland, Ohio.

The gut microbe-derived metabolite trimethylamine--oxide (TMAO) has recently been linked to cardiovascular disease (CVD) pathogenesis, prompting the development of therapeutic strategies to reduce TMAO. Previous work has shown that experimental alteration of circulating TMAO levels via dietary alterations or inhibition of the host TMAO producing enzyme flavin containing monooxygenase 3 (FMO3) is associated with reorganization of host cholesterol and bile acid metabolism in mice. In this work, we set out to understand whether recently developed nonlethal gut microbe-targeting small molecule choline trimethylamine (TMA) lyase inhibitors also alter host cholesterol and bile acid metabolism. Treatment of mice with the mechanism-based choline TMA lyase inhibitor, iodomethylcholine (IMC), increased fecal neutral sterol loss in the form of coprostanol, a bacteria metabolite of cholesterol. In parallel, IMC treatment resulted in marked reductions in the intestinal sterol transporter Niemann-pick C1-like 1 (NPC1L1) and reorganization of the gut microbial community, primarily reversing choline supplemented diet-induced changes. IMC also prevented diet-driven hepatic cholesterol accumulation, causing both upregulation of the host hepatic bile acid synthetic enzyme CYP7A1 and altering the expression of hepatic genes critical for bile acid feedback regulation. These studies suggest that the gut microbiota-driven TMAO pathway is closely linked to both microbe and host sterol and bile acid metabolism. Collectively, as gut microbe-targeting choline TMA lyase inhibitors move through the drug discovery pipeline from preclinical models to human studies, it will be important to understand how these drugs impact both microbe and host cholesterol and bile acid metabolism. The gut microbe-dependent metabolite trimethylamine--oxide (TMAO) has been strongly associated with cardiovascular mortality, prompting drug discovery efforts to identify points of therapeutic intervention within the microbe host TMAO pathway. Recently, mechanism-based small molecule inhibitors of the major bacterial trimethylamine (TMA) lyase enzymes have been developed, and these drugs show efficacy as anti-atherothrombotic agents. The novel findings of this study are that small molecule TMA lyase inhibition results in beneficial reorganization of host cholesterol and bile acid metabolism. This study confirms previous observations that the gut microbial TMAO pathway is intimately linked to host cholesterol and bile acid metabolism and provides further rationale for the development of small molecule choline TMA lyase inhibitors for the treatment of cardiometabolic disorders.
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http://dx.doi.org/10.1152/ajpheart.00584.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311702PMC
June 2020

Simultaneous Measurement of Urinary Trimethylamine (TMA) and Trimethylamine -Oxide (TMAO) by Liquid Chromatography-Mass Spectrometry.

Molecules 2020 Apr 17;25(8). Epub 2020 Apr 17.

Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA.

Trimethylamine (TMA) is a gut microbial metabolite-rendered by the enzymatic cleavage of nutrients containing a TMA moiety in their chemical structure. TMA can be oxidized as trimethylamine -oxide (TMAO) catalyzed by hepatic flavin monooxygenases. Circulating TMAO has been demonstrated to portend a pro-inflammatory state, contributing to chronic diseases such as cardiovascular disease and chronic kidney disease. Consequently, TMAO serves as an excellent candidate biomarker for a variety of chronic inflammatory disorders. The highly positive correlation between plasma TMAO and urine TMAO suggests that urine TMAO has the potential to serve as a less invasive biomarker for chronic disease compared to plasma TMAO. In this study, we validated a method to simultaneously measure urine TMA and TMAO concentrations by liquid chromatography-mass spectrometry (LC/MS). Urine TMA and TMAO can be extracted by hexane/butanol under alkaline pH and transferred to the aqueous phase following acidification for LC/MS quantitation. Importantly, during sample processing, none of the nutrients with a chemical structure containing a TMA moiety were spontaneously cleaved to yield TMA. Moreover, we demonstrated that the acidification of urine prevents an increase of TMA after prolonged storage as was observed in non-acidified urine. Finally, here we demonstrated that TMAO can spontaneously degrade to TMA at a very slow rate.
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http://dx.doi.org/10.3390/molecules25081862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222018PMC
April 2020

Gut microbiome and cardiovascular disease.

Curr Opin Cardiol 2020 05;35(3):207-218

Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.

Purpose Of Review: This review aims to highlight the association between gut microbiome and cardiovascular disease (CVD) with emphasis on the possible molecular mechanisms by which how gut microbiome contributes to CVD.

Recent Findings: Increasingly, the roles of gut microbiome in cardiovascular health and disease have gained much attention. Most of the investigations focus on how the gut dysbiosis contributes to CVD risk factors and which gut microbial-derived metabolites mediate such effects.

Summary: In this review, we discuss the molecular mechanisms of gut microbiome contributing to CVD, which include gut microbes translocalization to aortic artery because of gut barrier defect to initiate inflammation and microbial-derived metabolites inducing inflammation-signaling pathway and renal insufficiency. Specifically, we categorize beneficial and deleterious microbial-derived metabolites in cardiovascular health. We also summarize recent findings in the gut microbiome modulation of drug efficacy in treatment of CVD and the microbiome mechanisms by which how physical exercise ameliorates cardiovascular health. Gut microbiome has become an essential component of cardiovascular research and a crucial consideration factor in cardiovascular health and disease.
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http://dx.doi.org/10.1097/HCO.0000000000000720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442318PMC
May 2020

Non-Linear Relationship between Anti-Apolipoprotein A-1 IgGs and Cardiovascular Outcomes in Patients with Acute Coronary Syndromes.

J Clin Med 2019 Jul 9;8(7). Epub 2019 Jul 9.

Division of Cardiology, Cardiology Center, Geneva University Hospital, 1205 Geneva Switzerland.

Autoantibodies against apolipoprotein A-I (anti-apoA-I IgGs) are prevalent in atherosclerosis-related conditions. It remains elusive whether they improve the prognostic accuracy of the Global Registry of Acute Coronary Events (GRACE) score 2.0 (GS) in acute coronary syndromes (ACS). In this prospective multicenter registry, 1713 ACS patients were included and followed for 1 year. The primary endpoint (major adverse cardiovascular events (MACE)) was defined as the composite of myocardial infarction, stroke (including transient ischemic attack), or cardiovascular (CV) death with individual events independently adjudicated. Plasma levels of anti-apoA-I IgGs upon study inclusion were assessed using ELISA. The association between anti-apoA-I IgGs and incident MACE was assessed using Cox models with splines and C-statistics. One-year MACE incidence was 8.4% (144/1713). Anti-apoA-I IgG levels were associated with MACE with a non-linear relationship ( = 0.01), which remained unchanged after adjusting for the GS ( = 0.04). The hazard increased progressively across the two first anti-apoA-I IgG quartiles before decreasing thereafter. Anti-apoA-I IgGs marginally improved the prognostic accuracy of the GS (c-statistics increased from 0.68 to 0.70). In this multicenter study, anti-apoA-I IgGs were predictive of incident MACE in ACS independently of the GS but in a nonlinear manner. The practical implications of these findings remain to be defined.
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http://dx.doi.org/10.3390/jcm8071002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679072PMC
July 2019

Utility of Plasma Concentration of Trimethylamine N-Oxide in Predicting Cardiovascular and Renal Complications in Individuals With Type 1 Diabetes.

Diabetes Care 2019 08 23;42(8):1512-1520. Epub 2019 May 23.

Steno Diabetes Center Copenhagen, Gentofte, Denmark.

Objective: Trimethylamine N-oxide (TMAO) is suggested as an independent gut microbiota-derived risk factor for cardiovascular and renal disease. We investigated associations between plasma TMAO concentrations and cardio-renal outcomes in a prospective study of individuals with type 1 diabetes.

Research Design And Methods: Plasma TMAO was measured at baseline in 1,159 individuals with type 1 diabetes (58% male, mean ± SD age 46 ± 13 years). End points were all-cause and cardiovascular mortality, cardiovascular disease (CVD), and renal events tracked from national registries. Associations between TMAO and end points were tested using Cox regression models.

Results: After 15.0 (6.7-19.3) (median [interquartile range]) years of follow-up, we recorded all-cause and cardiovascular mortality ( = 363 and 120, respectively), combined CVD ( = 406), coronary outcome (myocardial infarction and coronary intervention) ( = 163), stroke ( = 115), hospitalization for heart failure ( = 81), and end-stage renal disease ( = 144). In univariate analyses, higher TMAO concentrations were associated with all end points ( ≤ 0.005). Except for stroke and heart failure, all end points remained significantly associated with higher TMAO concentrations after adjustment for conventional cardiovascular risk factors ( ≤ 0.003). After further adjustment for baseline estimated glomerular filtration rate (eGFR), results became insignificant for all end points. TMAO was inversely associated with baseline eGFR ( = 0.29; < 0.001).

Conclusions: In individuals with type 1 diabetes, higher concentrations of plasma TMAO were associated with mortality, CVD events, and poor renal outcome, independent of conventional risk factors. However, the association became insignificant after further adjustment for baseline eGFR. This could reflect TMAO as a renal function marker or a risk factor for micro- and macrovascular complications mediated through impaired renal function.
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http://dx.doi.org/10.2337/dc19-0048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082641PMC
August 2019

Genetic Deficiency of Flavin-Containing Monooxygenase 3 ( Fmo3) Protects Against Thrombosis but Has Only a Minor Effect on Plasma Lipid Levels-Brief Report.

Arterioscler Thromb Vasc Biol 2019 06;39(6):1045-1054

From the Department of Medicine/Division of Cardiology (D.M.S., Y.M., A.M., A.J.L.), University of California, Los Angeles.

Objective- FMO (flavin-containing monooxygenase) 3 converts bacterial-derived trimethylamine to trimethylamine N-oxide (TMAO), an independent risk factor for cardiovascular disease. We generated FMO3 knockout (FMO3KO) mouse to study its effects on plasma TMAO, lipids, glucose/insulin metabolism, thrombosis, and atherosclerosis. Approach and Results- Previous studies with an antisense oligonucleotide (ASO) knockdown strategy targeting FMO3 in LDLRKO (low-density lipoprotein receptor knockout) mice resulted in major reductions in TMAO levels and atherosclerosis, but also showed effects on plasma lipids, insulin, and glucose. Although FMO3KO mice generated via CRISPR/Cas9 technology bred onto the LDLRKO background did exhibit similar effects on TMAO levels, the effects on lipid metabolism were not as pronounced as with the ASO knockdown model. These differences could result from either off-target effects of the ASO or from a developmental adaptation to the FMO3 deficiency. To distinguish these possibilities, we treated wild-type and FMO3KO mice with control or FMO3 ASOs. FMO3-ASO treatment led to the same extent of lipid-lowering effects in the FMO3KO mice as the wild-type mice, indicating off-target effects. The levels of TMAO in LDLRKO mice fed an atherogenic diet are very low in both wild-type and FMO3KO mice, and no significant effect was observed on atherosclerosis. When FMO3KO and wild-type mice were maintained on a 0.5% choline diet, FMO3KO showed a marked reduction in both TMAO and in vivo thrombosis potential. Conclusions- FMO3KO markedly reduces systemic TMAO levels and thrombosis potential. However, the previously observed large effects of an FMO3 ASO on plasma lipid levels appear to be due partly to off-target effects.
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http://dx.doi.org/10.1161/ATVBAHA.119.312592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531332PMC
June 2019

Genetic Determinants of Circulating Glycine Levels and Risk of Coronary Artery Disease.

J Am Heart Assoc 2019 05;8(10):e011922

1 Department of Preventive Medicine Keck School of Medicine, University of Southern California Los Angeles CA.

Background Recent studies have revealed sexually dimorphic associations between the carbamoyl-phosphate synthase 1 locus, intermediates of the metabolic pathway leading from choline to urea, and risk of coronary artery disease ( CAD ) in women. Based on evidence from the literature, the atheroprotective association with carbamoyl-phosphate synthase 1 could be mediated by the strong genetic effect of this locus on increased circulating glycine levels. Methods and Results We sought to identify additional genetic determinants of circulating glycine levels by carrying out a meta-analysis of genome-wide association study data in up to 30 118 subjects of European ancestry. Mendelian randomization and other analytical approaches were used to determine whether glycine-associated variants were associated with CAD and traditional risk factors. Twelve loci were significantly associated with circulating glycine levels, 7 of which were not previously known to be involved in glycine metabolism ( ACADM , PHGDH , COX 18- ADAMTS 3, PSPH , TRIB 1, PTPRD , and ABO ). Glycine-raising alleles at several loci individually exhibited directionally consistent associations with decreased risk of CAD . However, these effects could not be attributed directly to glycine because of associations with other CAD -related traits. By comparison, genetic models that only included the 2 variants directly involved in glycine degradation and for which there were no other pleiotropic associations were not associated with risk of CAD or blood pressure, lipid levels, and obesity-related traits. Conclusions These results provide additional insight into the genetic architecture of glycine metabolism, but do not yield conclusive evidence for a causal relationship between circulating levels of this amino acid and risk of CAD in humans.
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http://dx.doi.org/10.1161/JAHA.119.011922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585317PMC
May 2019

Trimethyllysine, a trimethylamine N-oxide precursor, provides near- and long-term prognostic value in patients presenting with acute coronary syndromes.

Eur Heart J 2019 08;40(32):2700-2709

Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

Aims: Trimethyllysine (TML) serves as a nutrient precursor of the gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) and is associated with incident cardiovascular (CV) events in stable subjects. We examined the relationship between plasma TML levels and incident CV events in patients presenting with acute coronary syndromes (ACS).

Methods And Results: Plasma levels of TML were quantified in two independent cohorts using mass spectrometry, and its relationship with CV events was investigated. In a Cleveland Cohort (N = 530), comprised of patients presenting to the emergency department with chest pain and suspected ACS, TML was associated with major adverse cardiac events (MACE, myocardial infarction, stroke, need for revascularization, or all-cause mortality) over both 30 days [3rd tertile (T3), adjusted odds ratio (OR) 1.77, 95% confidence interval (CI) 1.04-3.01; P < 0.05] and 6 months (T3, adjusted OR 1.95, 95% CI 1.15-3.32; P < 0.05) of follow-up independent of traditional CV risk factors and indices of renal function. Elevated TML levels were also associated with incident long-term (7-year) all-cause mortality [T3, adjusted hazard ratio (HR) 2.52, 95% CI 1.50-4.24; P < 0.001], and MACE even amongst patients persistently negative for cardiac Troponin T at presentation (e.g. 30-day MACE, T3, adjusted OR 4.49, 95% CI 2.06-9.79; P < 0.001). Trimethyllysine in combination with TMAO showed additive significance for near- and long-term CV events, including patients with 'negative' high-sensitivity Troponin T levels. In a multicentre Swiss Cohort (N = 1683) comprised of ACS patients, similar associations between TML and incident 1-year adverse cardiac risks were observed (e.g. mortality, adjusted T3 HR 2.74, 95% CI 1.28-5.85; P < 0.05; and MACE, adjusted T3 HR 1.55, 95% CI 1.04-2.31; P < 0.05).

Conclusion: Plasma TML levels, alone and together with TMAO, are associated with both near- and long-term CV events in patients with chest pain and ACS.
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http://dx.doi.org/10.1093/eurheartj/ehz259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963132PMC
August 2019

Effects of Lifestyle Intervention on Plasma Trimethylamine N-Oxide in Obese Adults.

Nutrients 2019 Jan 16;11(1). Epub 2019 Jan 16.

Integrative Physiology and Molecular Medicine Laboratory, Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA 70808, USA.

Accumulating evidence linking trimethylamine N-oxide (TMAO) to cardiovascular disease (CVD) risk has prompted interest in developing therapeutic strategies to reduce its production. We compared two lifestyle intervention approaches: hypocaloric versus eucaloric diet, combined with exercise, on TMAO levels in relation to CVD risk factors. Sixteen obese adults (66.1 ± 4.4 years, BMI (body mass index): 35.9 ± 5.3 kg/m², fasting glucose: 106 ± 16 mg/dL, 2-h PPG (postprandial glucose): 168 ± 37 mg/dL) were randomly assigned to 12 weeks of exercise (5 days/week, 80⁻85% HR (maximal heart rate)) plus either a hypocaloric (HYPO) (-500 kcal) or a eucaloric (EU) diet. Outcomes included plasma TMAO, glucose metabolism (oral glucose tolerance test (OGTT) and euglycemic-hyperinsulinemic clamps for glucose disposal rates (GDR)), exercise capacity (VO, maximal oxygen consumption), abdominal adiposity (computed tomography scans), cholesterol, and triglycerides. Results showed that body composition (body weight, subcutaneous adiposity), insulin sensitivity, VO, and cholesterol all improved ( < 0.05). HYPO decreased the percentage change in TMAO compared to an increase after EU (HYPO: -31 ± 0.4% vs. EU: 32 ± 0.6%, = 0.04). Absolute TMAO levels were not impacted (HYPO: = 0.09 or EU: = 0.53 group). The change in TMAO after intervention was inversely correlated with baseline visceral adipose tissue (r = -0.63, = 0.009) and GDR (r = 0.58, = 0.002). A hypocaloric diet and exercise approach appears to be effective in reducing TMAO. Larger trials are needed to support this observation.
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http://dx.doi.org/10.3390/nu11010179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356515PMC
January 2019

Impact of chronic dietary red meat, white meat, or non-meat protein on trimethylamine N-oxide metabolism and renal excretion in healthy men and women.

Eur Heart J 2019 02;40(7):583-594

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Euclid Ave, Cleveland, OH, USA.

Aims: Carnitine and choline are major nutrient precursors for gut microbiota-dependent generation of the atherogenic metabolite, trimethylamine N-oxide (TMAO). We performed randomized-controlled dietary intervention studies to explore the impact of chronic dietary patterns on TMAO levels, metabolism and renal excretion.

Methods And Results: Volunteers (N = 113) were enrolled in a randomized 2-arm (high- or low-saturated fat) crossover design study. Within each arm, three 4-week isocaloric diets (with washout period between each) were evaluated (all meals prepared in metabolic kitchen with 25% calories from protein) to examine the effects of red meat, white meat, or non-meat protein on TMAO metabolism. Trimethylamine N-oxide and other trimethylamine (TMA) related metabolites were quantified at the end of each diet period. A random subset (N = 13) of subjects also participated in heavy isotope tracer studies. Chronic red meat, but not white meat or non-meat ingestion, increased plasma and urine TMAO (each >two-fold; P < 0.0001). Red meat ingestion also significantly reduced fractional renal excretion of TMAO (P < 0.05), but conversely, increased fractional renal excretion of carnitine, and two alternative gut microbiota-generated metabolites of carnitine, γ-butyrobetaine, and crotonobetaine (P < 0.05). Oral isotope challenge revealed red meat or white meat (vs. non-meat) increased TMA and TMAO production from carnitine (P < 0.05 each) but not choline. Dietary-saturated fat failed to impact TMAO or its metabolites.

Conclusion: Chronic dietary red meat increases systemic TMAO levels through: (i) enhanced dietary precursors; (ii) increased microbial TMA/TMAO production from carnitine, but not choline; and (iii) reduced renal TMAO excretion. Discontinuation of dietary red meat reduces plasma TMAO within 4 weeks.
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http://dx.doi.org/10.1093/eurheartj/ehy799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374688PMC
February 2019

l-Carnitine in omnivorous diets induces an atherogenic gut microbial pathway in humans.

J Clin Invest 2019 01 10;129(1):373-387. Epub 2018 Dec 10.

Department of Cellular and Molecular Medicine, Lerner Research Institute.

Background: l-Carnitine, an abundant nutrient in red meat, accelerates atherosclerosis in mice via gut microbiota-dependent formation of trimethylamine (TMA) and trimethylamine N-oxide (TMAO) via a multistep pathway involving an atherogenic intermediate, γ-butyrobetaine (γBB). The contribution of γBB in gut microbiota-dependent l-carnitine metabolism in humans is unknown.

Methods: Omnivores and vegans/vegetarians ingested deuterium-labeled l-carnitine (d3-l-carnitine) or γBB (d9-γBB), and both plasma metabolites and fecal polymicrobial transformations were examined at baseline, following oral antibiotics, or following chronic (≥2 months) l-carnitine supplementation. Human fecal commensals capable of performing each step of the l-carnitine→γBB→TMA transformation were identified.

Results: Studies with oral d3-l-carnitine or d9-γBB before versus after antibiotic exposure revealed gut microbiota contribution to the initial 2 steps in a metaorganismal l-carnitine→γBB→TMA→TMAO pathway in subjects. Moreover, a striking increase in d3-TMAO generation was observed in omnivores over vegans/vegetarians (>20-fold; P = 0.001) following oral d3-l-carnitine ingestion, whereas fasting endogenous plasma l-carnitine and γBB levels were similar in vegans/vegetarians (n = 32) versus omnivores (n = 40). Fecal metabolic transformation studies, and oral isotope tracer studies before versus after chronic l-carnitine supplementation, revealed that omnivores and vegans/vegetarians alike rapidly converted carnitine to γBB, whereas the second gut microbial transformation, γBB→TMA, was diet inducible (l-carnitine, omnivorous). Extensive anaerobic subculturing of human feces identified no single commensal capable of l-carnitine→TMA transformation, multiple community members that converted l-carnitine to γBB, and only 1 Clostridiales bacterium, Emergencia timonensis, that converted γBB to TMA. In coculture, E. timonensis promoted the complete l-carnitine→TMA transformation.

Conclusion: In humans, dietary l-carnitine is converted into the atherosclerosis- and thrombosis-promoting metabolite TMAO via 2 sequential gut microbiota-dependent transformations: (a) initial rapid generation of the atherogenic intermediate γBB, followed by (b) transformation into TMA via low-abundance microbiota in omnivores, and to a markedly lower extent, in vegans/vegetarians. Gut microbiota γBB→TMA/TMAO transformation is induced by omnivorous dietary patterns and chronic l-carnitine exposure.

Trial Registration: ClinicalTrials.gov NCT01731236.

Funding: NIH and Office of Dietary Supplements grants HL103866, HL126827, and DK106000, and the Leducq Foundation.
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http://dx.doi.org/10.1172/JCI94601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307959PMC
January 2019

The utility of biomarkers in diagnosis of aspirin exacerbated respiratory disease.

Respir Res 2018 Oct 30;19(1):210. Epub 2018 Oct 30.

Department of Internal Medicine, Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland.

Background: Aspirin-exacerbated respiratory disease (AERD) is a distinct eosinophilic phenotype of severe asthma with accompanying chronic rhinosinusitis, nasal polyposis, and hypersensitivity to aspirin. Urinary 3-bromotyrosine (uBrTyr) is a noninvasive marker of eosinophil-catalyzed protein oxidation. The lack of in vitro diagnostic test makes the diagnosis of AERD difficult. We aimed to determine uBrTyr levels in patients with AERD (n = 240) and aspirin-tolerant asthma (ATA) (n = 226) and to assess whether its addition to urinary leukotriene E (uLTE) levels and blood eosinophilia can improve the prediction of AERD diagnosis.

Methods: Clinical data, spirometry and blood eosinophilis were evaluated. UBrTyr and uLTE levels were measured in urine by HPLC and ELISA, respectively.

Results: Both groups of asthmatics (AERD, n = 240; ATA, n = 226) had significantly higher uBrTyr, uLTE levels, and blood eosinophils than healthy controls (HC) (n = 71) (p < 0.05). ULTE levels and blood eosinophils were significantly higher in AERD as compared to ATA (p = 0.004, p < 0.0001, respectively). whereas uBrTyr levels were not significantly different between both asthma phenotypes (p = 0.34). Asthmatics with high levels of uBrTyr (> 0.101 ng/mg Cr), uLTE levels (> 800 pg/mg Cr) and blood eosinophils (> 300 cells/ul) were 7 times more likely to have AERD.. However, uBrTyr did not increase the benefit for predicting AERD when uLTE and blood eosinophils were already taken into account (p = 0.57).

Conclusion: UBrTyr levels are elevated both in AERD and ATA as compared to HC, but they could not differentiate between these asthma phenotypes suggesting a similar eosinophilic activation. The addition of uBrTyr to elevated uLTE4 levels and blood eosinophils did not statistically enhance the prediction of AERD diagnosis.
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http://dx.doi.org/10.1186/s12931-018-0909-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208044PMC
October 2018

Microbial Transplantation With Human Gut Commensals Containing CutC Is Sufficient to Transmit Enhanced Platelet Reactivity and Thrombosis Potential.

Circ Res 2018 10;123(10):1164-1176

Center for Microbiome and Human Health, Lerner Research Institute (S.M.S., W.Z., K.A.R., Z.W., J.K., J.A.D., W.H.W.T., S.L.H.), Cleveland Clinic, OH.

Rationale: Gut microbes influence cardiovascular disease and thrombosis risks through the production of trimethylamine N-oxide (TMAO). Microbiota-dependent generation of trimethylamine (TMA)-the precursor to TMAO-is rate limiting in the metaorganismal TMAO pathway in most humans and is catalyzed by several distinct microbial choline TMA-lyases, including the proteins encoded by the cutC/D (choline utilization C/D) genes in multiple human commensals.

Objective: Direct demonstration that the gut microbial cutC gene is sufficient to transmit enhanced platelet reactivity and thrombosis potential in a host via TMA/TMAO generation has not yet been reported.

Methods And Results: Herein, we use gnotobiotic mice and a series of microbial colonization studies to show that microbial cutC-dependent TMA/TMAO production is sufficient to transmit heightened platelet reactivity and thrombosis potential in a host. Specifically, we examine in vivo thrombosis potential employing germ-free mice colonized with either high TMA-producing stable human fecal polymcrobial communities or a defined CutC-deficient background microbial community coupled with a CutC-expressing human commensal±genetic disruption of its cutC gene (ie, Clostridium sporogenes Δ cutC).

Conclusions: Collectively, these studies point to the microbial choline TMA-lyase pathway as a rational molecular target for the treatment of atherothrombotic heart disease.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223262PMC
October 2018

Loss of HDAC6 alters gut microbiota and worsens obesity.

FASEB J 2019 01 13;33(1):1098-1109. Epub 2018 Aug 13.

Department of Medicine New York University School of Medicine (NYUSM), New York, New York, USA.

Alterations in gut microbiota are known to affect intestinal inflammation and obesity. Antibiotic treatment can affect weight gain by elimination of histone deacetylase (HDAC) inhibitor-producing microbes, which are anti-inflammatory by augmenting regulatory T (T) cells. We asked whether mice that lack HDAC6 and have potent suppressive T cells are protected from microbiota-induced accelerated weight gain. We crossed wild-type and HDAC6-deficient mice and subjected the offspring to perinatal penicillin, inducing weight gain via microbiota disturbance. We observed that male HDAC6-deficient mice were not protected and developed profoundly accelerated weight gain. The antibiotic-exposed HDAC6-deficient mice showed a mixed immune phenotype with increased CD4 and CD8 T-cell activation yet maintained the enhanced T cell-suppressive function phenotype characteristic of HDAC6-deficient mice. 16S rRNA sequencing of mouse fecal samples reveals that their microbiota diverged with time, with HDAC6 deletion altering microbiome composition. On a high-fat diet, HDAC6-deficient mice were depleted in representatives of the S24-7 family and Lactobacillus but enriched with Bacteroides and Parabacteroides; these changes are associated with obesity. Our findings further our understanding of the influence of HDACs on microbiome composition and are important for the development of HDAC6 inhibitors in the treatment of human diseases.-Lieber, A. D., Beier, U. H., Xiao, H., Wilkins, B. J., Jiao, J., Li, X. S., Schugar, R. C., Strauch, C. M., Wang, Z., Brown, J. M., Hazen, S. L., Bokulich, N. A., Ruggles, K. V., Akimova, T., Hancock, W. W., Blaser, M. J. Loss of HDAC6 alters gut microbiota and worsens obesity.
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http://dx.doi.org/10.1096/fj.201701586RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355060PMC
January 2019

Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential.

Nat Med 2018 09 6;24(9):1407-1417. Epub 2018 Aug 6.

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.

Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite that enhances both platelet responsiveness and in vivo thrombosis potential in animal models, and TMAO plasma levels predict incident atherothrombotic event risks in human clinical studies. TMAO is formed by gut microbe-dependent metabolism of trimethylamine (TMA) moiety-containing nutrients, which are abundant in a Western diet. Here, using a mechanism-based inhibitor approach targeting a major microbial TMA-generating enzyme pair, CutC and CutD (CutC/D), we developed inhibitors that are potent, time-dependent, and irreversible and that do not affect commensal viability. In animal models, a single oral dose of a CutC/D inhibitor significantly reduced plasma TMAO levels for up to 3 d and rescued diet-induced enhanced platelet responsiveness and thrombus formation, without observable toxicity or increased bleeding risk. The inhibitor selectively accumulated within intestinal microbes to millimolar levels, a concentration over 1-million-fold higher than needed for a therapeutic effect. These studies reveal that mechanism-based inhibition of gut microbial TMA and TMAO production reduces thrombosis potential, a critical adverse complication in heart disease. They also offer a generalizable approach for the selective nonlethal targeting of gut microbial enzymes linked to host disease limiting systemic exposure of the inhibitor in the host.
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http://dx.doi.org/10.1038/s41591-018-0128-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129214PMC
September 2018

Gut Microbiota-Dependent Trimethylamine N-Oxide Predicts Risk of Cardiovascular Events in Patients With Stroke and Is Related to Proinflammatory Monocytes.

Arterioscler Thromb Vasc Biol 2018 09;38(9):2225-2235

From the Department of Cardiology (A.H., D.S., F.Z., N.K., U.L.).

Objective- Gut microbiota-dependent metabolites, in particular trimethylamine N-oxide (TMAO), have recently been reported to promote atherosclerosis and thrombosis. Here, we examined for the first time the relation of TMAO and the risk of incident cardiovascular events in patients with recent first-ever ischemic stroke in 2 independent prospective cohorts. Moreover, the link between TMAO and proinflammatory monocytes as a potential contributing factor for cardiovascular risk in stroke patients was studied. Approach and Results- In a first study (n=78), higher TMAO plasma levels were linked with an increased risk of incident cardiovascular events including myocardial infarction, recurrent stroke, and cardiovascular death (fourth quartile versus first quartile; hazard ratio, 2.31; 95% CI, 1.25-4.23; P<0.01). In the second independent validation cohort (n=593), high TMAO levels again heralded marked increased risk of adverse cardiovascular events (fourth quartile versus first quartile; hazard ratio, 5.0; 95% CI, 1.7-14.8; P<0.01), and also after adjustments for cardiovascular risk factors including hypertension, diabetes mellitus, LDL (low-density lipoprotein) cholesterol, and estimated glomerular filtration rate (hazard ratio, 3.3; 95% CI, 1.2-10.9; P=0.04). A significant correlation was also found between TMAO levels and percentage of proinflammatory intermediate CD14CD16 monocytes ( r=0.70; P<0.01). Moreover, in mice fed a diet enriched with choline to increase TMAO synthesis, levels of proinflammatory murine Ly6C monocytes were higher than in the chow-fed control group (choline: 9.2±0.5×10 per mL versus control: 6.5±0.5×10 per mL; P<0.01). This increase was abolished in mice with depleted gut microbiota (choline+antibiotics: 5.4±0.7×10 per mL; P<0.001 versus choline). Conclusions- The present study demonstrates for the first time a graded relation between TMAO levels and the risk of subsequent cardiovascular events in patients with recent prior ischemic stroke. Our data support the notion that TMAO-related increase of proinflammatory monocytes may add to elevated cardiovascular risk of patients with increased TMAO levels.
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http://dx.doi.org/10.1161/ATVBAHA.118.311023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202215PMC
September 2018

Elevated levels of plasma symmetric dimethylarginine and increased arginase activity as potential indicators of cardiovascular comorbidity in rheumatoid arthritis.

Arthritis Res Ther 2018 06 8;20(1):123. Epub 2018 Jun 8.

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.

Background: Rheumatoid arthritis (RA) patients are at high risk of developing cardiovascular disease (CVD). In RA, chronic inflammation may lead to endothelial dysfunction, an early indicator of CVD, owing to diminished nitric oxide (NO) production. Because L-arginine is the sole precursor of NO, we hypothesized that levels of L-arginine metabolic products reflecting NO metabolism are altered in patients with RA.

Methods: Plasma samples from patients with RA (n = 119) and age- and sex-matched control subjects (n = 238) were used for this study. Using LC-MS/MS, we measured plasma levels of free L-arginine, L-ornithine, L-citrulline, L-N-monomethyl arginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA). We compared global arginine bioavailability ratio (GABR) (i.e., ratio of L-arginine to L-ornithine + L-citrulline) and arginine methylation index (ArgMI) (i.e., ADMA + SDMA/MMA) in patients with RA vs. control subjects. Plasma arginase activity was measured using a sensitive arginase assay kit. The relationship of L-arginine metabolites and arginase activity to CVD risk factors was evaluated using Pearson's chi-square test.

Results: Compared with healthy control subjects, the RA cohort showed significantly lower levels of plasma L-arginine (46.11 ± 17.29 vs. 74.2 ± 22.53 μmol/L, p < 0.001) and GABR (0.36 ± 0.16 vs. 0.73 ± 0.24, p < 0.001), elevated levels of ADMA (0.76 ± 0.12 vs. 0.62 ± 0.12 μmol/L, p < 0.001), SDMA (0.54 ± 0.14 vs. 0.47 ± 0.13 μmol/L, p < 0.001), and ArgMI (6.51 ± 1.86 vs. 5.54 ± 1.51, p < 0.001). We found an approximately fourfold increase in arginase activity (33.8 ± 1.1 vs. 8.4 ± 0.8 U/L, p < 0.001), as well as elevated levels of arginase-mediated L-arginine catalytic product L-ornithine (108.64 ± 30.26 vs. 69.3 ± 20.71 μmol/L, p < 0.001), whereas a nitric oxide synthase (NOS) catalytic product, the L-citrulline level, was diminished in RA (30.32 ± 9.93 vs. 36.17 ± 11.64 μmol/L, p < 0.001). Patients with RA with existing CVD had higher arginase activity than patients with RA without CVD (p = 0.048).

Conclusions: Global L-arginine bioavailability was diminished, whereas plasma arginase activity, ADMA, and SDMA levels were elevated, in patients with RA compared with healthy control subjects. Plasma SDMA was associated with hypertension and hyperlipidemia in patients with RA. This dysregulated L-arginine metabolism may function as a potential indicator of CVD risk in patients with RA.
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http://dx.doi.org/10.1186/s13075-018-1616-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994036PMC
June 2018

Gut microbiota derived metabolites in cardiovascular health and disease.

Protein Cell 2018 05 3;9(5):416-431. Epub 2018 May 3.

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, 44195, USA.

Trillions of microbes inhabit the human gut, not only providing nutrients and energy to the host from the ingested food, but also producing metabolic bioactive signaling molecules to maintain health and elicit disease, such as cardiovascular disease (CVD). CVD is the leading cause of mortality worldwide. In this review, we presented gut microbiota derived metabolites involved in cardiovascular health and disease, including trimethylamine-N-oxide (TMAO), uremic toxins, short chain fatty acids (SCFAs), phytoestrogens, anthocyanins, bile acids and lipopolysaccharide. These gut microbiota derived metabolites play critical roles in maintaining a healthy cardiovascular function, and if dysregulated, potentially causally linked to CVD. A better understanding of the function and dynamics of gut microbiota derived metabolites holds great promise toward mechanistic predicative CVD biomarker discoveries and precise interventions.
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http://dx.doi.org/10.1007/s13238-018-0549-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960473PMC
May 2018

Effect of Vegan Fecal Microbiota Transplantation on Carnitine- and Choline-Derived Trimethylamine-N-Oxide Production and Vascular Inflammation in Patients With Metabolic Syndrome.

J Am Heart Assoc 2018 03 26;7(7). Epub 2018 Mar 26.

Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, The Netherlands

Background: Intestinal microbiota have been found to be linked to cardiovascular disease via conversion of the dietary compounds choline and carnitine to the atherogenic metabolite TMAO (trimethylamine-N-oxide). Specifically, a vegan diet was associated with decreased plasma TMAO levels and nearly absent TMAO production on carnitine challenge.

Methods And Results: We performed a double-blind randomized controlled pilot study in which 20 male metabolic syndrome patients were randomized to single lean vegan-donor or autologous fecal microbiota transplantation. At baseline and 2 weeks thereafter, we determined the ability to produce TMAO from d-choline and d-carnitine (eg, labeled and unlabeled TMAO in plasma and 24-hour urine after oral ingestion of 250 mg of both isotope-labeled precursor nutrients), and fecal samples were collected for analysis of microbiota composition. F-fluorodeoxyglucose positron emission tomography/computed tomography scans of the abdominal aorta, as well as ex vivo peripheral blood mononuclear cell cytokine production assays, were performed. At baseline, fecal microbiota composition differed significantly between vegans and metabolic syndrome patients. With vegan-donor fecal microbiota transplantation, intestinal microbiota composition in metabolic syndrome patients, as monitored by global fecal microbial community structure, changed toward a vegan profile in some of the patients; however, no functional effects from vegan-donor fecal microbiota transplantation were seen on TMAO production, abdominal aortic F-fluorodeoxyglucose uptake, or ex vivo cytokine production from peripheral blood mononuclear cells.

Conclusions: Single lean vegan-donor fecal microbiota transplantation in metabolic syndrome patients resulted in detectable changes in intestinal microbiota composition but failed to elicit changes in TMAO production capacity or parameters related to vascular inflammation.

Clinical Trial Registration: URL: http://www.trialregister.nl. Unique identifier: NTR 4338.
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http://dx.doi.org/10.1161/JAHA.117.008342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5907601PMC
March 2018

Untargeted metabolomics identifies trimethyllysine, a TMAO-producing nutrient precursor, as a predictor of incident cardiovascular disease risk.

JCI Insight 2018 03 22;3(6). Epub 2018 Mar 22.

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.

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, N6,N6,N6-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.
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http://dx.doi.org/10.1172/jci.insight.99096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5926943PMC
March 2018

Effect of long-term dietary sphingomyelin supplementation on atherosclerosis in mice.

PLoS One 2017 14;12(12):e0189523. Epub 2017 Dec 14.

School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, Australia.

Sphingomyelin (SM) levels in the circulation correlate positively with atherosclerosis burden. SM is a ubiquitous component of human diets, but it is unclear if dietary SM increases circulating SM levels. Dietary choline increases atherosclerosis by raising circulating trimethylamine N-oxide (TMAO) levels in mice and humans. As SM has a choline head group, we ask in this study if dietary SM accelerates atherosclerotic lesion development by increasing circulating SM and TMAO levels. Three studies were performed: (Study 1) C57BL/6 mice were maintained on a high fat diet with or without SM supplementation for 4 weeks prior to quantification of serum TMAO and SM levels; (Study 2) atherosclerosis was studied in apoE-/- mice after 16 weeks of a high fat diet without or with SM supplementation and (Study 3) apoE-/- mice were maintained on a chow diet for 19 weeks without or with SM supplementation and antibiotic treatment prior to quantification of atherosclerotic lesions and serum TMAO and SM levels. SM consumption did not increase circulating SM levels or atherosclerosis in high fat-fed apoE-/- mice. Serum TMAO levels in C57BL/6 mice were low and had no effect atherosclerosis lesion development. Dietary SM supplementation significantly reduced atherosclerotic lesion area in the aortic arch of chow-fed apoE-/- mice. This study establishes that dietary SM does not affect circulating SM levels or increase atherosclerosis in high fat-fed apoE-/- mice, but it is anti-atherogenic in chow-fed apoE-/- mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189523PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730175PMC
December 2017