Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk.

Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (n > 4,000) independently predicted incident (3 years) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced sub-maximal stimulus-dependent platelet activation from multiple agonists through augmented Ca(2+) release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk.

Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis.

Trimethylamine (TMA) N-oxide (TMAO), a gut-microbiota-dependent metabolite, both enhances atherosclerosis in animal models and is associated with cardiovascular risks in clinical studies. Here, we investigate the impact of targeted inhibition of the first step in TMAO generation, commensal microbial TMA production, on diet-induced atherosclerosis. A structural analog of choline, 3,3-dimethyl-1-butanol (DMB), is shown to non-lethally inhibit TMA formation from cultured microbes, to inhibit distinct microbial TMA lyases, and to both inhibit TMA production from physiologic polymicrobial cultures (e.g., intestinal contents, human feces) and reduce TMAO levels in mice fed a high-choline or L-carnitine diet. DMB inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in apolipoprotein e(-/-) mice without alterations in circulating cholesterol levels. The present studies suggest that targeting gut microbial production of TMA specifically and non-lethal microbial inhibitors in general may serve as a potential therapeutic approach for the treatment of cardiometabolic diseases.

Gut Microbiota-Derived Trimethylamine N-Oxide Contributes to Abdominal Aortic Aneurysm Through Inflammatory and Apoptotic Mechanisms.

BACKGROUND: Large-scale human and mechanistic mouse studies indicate a strong relationship between the microbiome-dependent metabolite trimethylamine N-oxide (TMAO) and several cardiometabolic diseases. This study aims to investigate the role of TMAO in the pathogenesis of abdominal aortic aneurysm (AAA) and target its parent microbes as a potential pharmacological intervention. METHODS: TMAO and choline metabolites were examined in plasma samples, with associated clinical data, from 2 independent patient cohorts (N=2129 total). Mice were fed a high-choline diet and underwent 2 murine AAA models, angiotensin II infusion in low-density lipoprotein receptor-deficient (Ldlr-/-) mice or topical porcine pancreatic elastase in C57BL/6J mice. Gut microbial production of TMAO was inhibited through broad-spectrum antibiotics, targeted inhibition of the gut microbial choline TMA lyase (CutC/D) with fluoromethylcholine, or the use of mice genetically deficient in flavin monooxygenase 3 (Fmo3-/-). Finally, RNA sequencing of in vitro human vascular smooth muscle cells and in vivo mouse aortas was used to investigate how TMAO affects AAA. RESULTS: Elevated TMAO was associated with increased AAA incidence and growth in both patient cohorts studied. Dietary choline supplementation augmented plasma TMAO and aortic diameter in both mouse models of AAA, which was suppressed with poorly absorbed oral broad-spectrum antibiotics. Treatment with fluoromethylcholine ablated TMAO production, attenuated choline-augmented aneurysm initiation, and halted progression of an established aneurysm model. In addition, Fmo3-/- mice had reduced plasma TMAO and aortic diameters and were protected from AAA rupture compared with wild-type mice. RNA sequencing and functional analyses revealed choline supplementation in mice or TMAO treatment of human vascular smooth muscle cells-augmented gene pathways associated with the endoplasmic reticulum stress response, specifically the endoplasmic reticulum stress kinase PERK. CONCLUSIONS: These results define a role for gut microbiota-generated TMAO in AAA formation through upregulation of endoplasmic reticulum stress-related pathways in the aortic wall. In addition, inhibition of microbiome-derived TMAO may serve as a novel therapeutic approach for AAA treatment where none currently exist.

Atlas of gut microbe-derived products from aromatic amino acids and risk of cardiovascular morbidity and mortality.

AIMS: 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. METHODS AND RESULTS: 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). CONCLUSION: 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.

Exploratory Transcriptomic Profiling Reveals the Role of Gut Microbiota in Vascular Dementia.

Stroke is the second most common cause of cognitive impairment and dementia. Vascular dementia (VaD), a cognitive impairment following a stroke, is common and significantly impacts the quality of life. We recently demonstrated via gut microbe transplant studies that the gut microbe-dependent trimethylamine-N-oxide (TMAO) pathway impacts stroke severity, both infarct size and long-term cognitive outcomes. However, the molecular mechanisms that underly the role of the microbiome in VaD have not been explored in depth. To address this issue, we performed a comprehensive RNA-sequencing analysis to identify differentially expressed (DE) genes in the ischemic cerebral cortex of mouse brains at pre-stroke and post-stroke day 1 and day 3. A total of 4016, 3752 and 7861 DE genes were identified at pre-stroke and post-stroke day 1 and day 3, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated pathways of neurodegeneration in multiple diseases, chemokine signaling, calcium signaling, and IL-17 signaling as the key enriched pathways. Inflammatory response genes interleukin-1 beta (Il-1ß), chemokines (C-X-C motif chemokine ligand 10 (Cxcl10), chemokine ligand 2 (Ccl2)), and immune system genes (S100 calcium binding protein 8 (S100a8), lipocalin-2 (Lcn2)) were among the most significantly upregulated genes. Hypocretin neuropeptide precursor (Hcrt), a neuropeptide, and transcription factors such as neuronal PAS domain protein 4 (Npas4), GATA binding protein 3 (Gata3), and paired box 7 (Pax7) were among the most significantly downregulated genes. In conclusion, our results indicate that higher plasma TMAO levels induce differential mRNA expression profiles in the ischemic brain tissue in our pre-clinical stroke model, and the predicted pathways provide the molecular basis for regulating the TMAO-enhanced neuroinflammatory response in the brain.

Site-specific 5-hydroxytryptophan incorporation into apolipoprotein A-I impairs cholesterol efflux activity and high-density lipoprotein biogenesis.

Apolipoprotein A-I (apoA-I) is the major protein constituent of high-density lipoprotein (HDL) and a target of myeloperoxidase-dependent oxidation in the artery wall. In atherosclerotic lesions, apoA-I exhibits marked oxidative modifications at multiple sites, including Trp72 Site-specific mutagenesis studies have suggested, but have not conclusively shown, that oxidative modification of Trp72 of apoA-I impairs many atheroprotective properties of this lipoprotein. Herein, we used genetic code expansion technology with an engineered Saccharomyces cerevisiae tryptophanyl tRNA-synthetase (Trp-RS):suppressor tRNA pair to insert the noncanonical amino acid 5-hydroxytryptophan (5-OHTrp) at position 72 in recombinant human apoA-I and confirmed site-specific incorporation utilizing MS. In functional characterization studies, 5-OHTrp72 apoA-I (compared with WT apoA-I) exhibited reduced ABC subfamily A member 1 (ABCA1)-dependent cholesterol acceptor activity in vitro (41.73 ± 6.57% inhibition; p < 0.01). Additionally, 5-OHTrp72 apoA-I displayed increased activation and stabilization of paraoxonase 1 (PON1) activity (µmol/min/mg) when compared with WT apoA-I and comparable PON1 activation/stabilization compared with reconstituted HDL (WT apoA-I, 1.92 ± 0.04; 5-OHTrp72 apoA-I, 2.35 ± 0.0; and HDL, 2.33 ± 0.1; p < 0.001, p < 0.001, and p < 0.001, respectively). Following injection into apoA-I-deficient mice, 5-OHTrp72 apoA-I reached plasma levels comparable with those of native apoA-I yet exhibited significantly reduced (48%; p < 0.01) lipidation and evidence of HDL biogenesis. Collectively, these findings unequivocally reveal that site-specific oxidative modification of apoA-I via 5-OHTrp at Trp72 impairs cholesterol efflux and the rate-limiting step of HDL biogenesis both in vitro and in vivo.

Targeted Inhibition of Gut Microbial Trimethylamine N-Oxide Production Reduces Renal Tubulointerstitial Fibrosis and Functional Impairment in a Murine Model of Chronic Kidney Disease.

OBJECTIVE: Gut microbial metabolism of dietary choline, a nutrient abundant in a Western diet, produces trimethylamine (TMA) and the atherothrombosis- and fibrosis-promoting metabolite TMA-N-oxide (TMAO). Recent clinical and animal studies reveal that elevated TMAO levels are associated with heightened risks for both cardiovascular disease and incident chronic kidney disease development. Despite this, studies focusing on therapeutically targeting gut microbiota-dependent TMAO production and its impact on preserving renal function are limited. Approach and Results: Herein we examined the impact of pharmacological inhibition of choline diet-induced gut microbiota-dependent production of TMA, and consequently TMAO, on renal tubulointerstitial fibrosis and functional impairment in a model of chronic kidney disease. Initial studies with a gut microbial choline TMA-lyase mechanism-based inhibitor, iodomethylcholine, confirmed both marked suppression of TMA generation, and consequently TMAO levels, and selective targeting of the gut microbial compartment (ie, both accumulation of the drug in intestinal microbes and limited systemic exposure in the host). Dietary supplementation of either choline or TMAO significantly augmented multiple indices of renal functional impairment and fibrosis associated with chronic subcutaneous infusion of isoproterenol. However, the presence of the gut microbiota-targeting inhibitor iodomethylcholine blocked choline diet-induced elevation in TMAO, and both significantly improved decline in renal function, and significantly attenuated multiple indices of tubulointerstitial fibrosis. Iodomethylcholine treatment also reversed many choline diet-induced changes in cecal microbial community composition associated with TMAO and renal functional impairment. CONCLUSIONS: Selective targeting of gut microbiota-dependent TMAO generation may prevent adverse renal structural and functional alterations in subjects at risk for chronic kidney disease.

The Nutritional Supplement L-Alpha Glycerylphosphorylcholine Promotes Atherosclerosis.

L-alpha glycerylphosphorylcholine (GPC), a nutritional supplement, has been demonstrated to improve neurological function. However, a new study suggests that GPC supplementation increases incident stroke risk thus its potential adverse effects warrant further investigation. Here we show that GPC promotes atherosclerosis in hyperlipidemic Apoe-/- mice. GPC can be metabolized to trimethylamine N-oxide, a pro-atherogenic agent, suggesting a potential molecular mechanism underlying the observed atherosclerosis progression. GPC supplementation shifted the gut microbial community structure, characterized by increased abundance of Parabacteroides, Ruminococcus, and Bacteroides and decreased abundance of Akkermansia, Lactobacillus, and Roseburia, as determined by 16S rRNA gene sequencing. These data are consistent with a reduction in fecal and cecal short chain fatty acids in GPC-fed mice. Additionally, we found that GPC supplementation led to an increased relative abundance of choline trimethylamine lyase (cutC)-encoding bacteria via qPCR. Interrogation of host inflammatory signaling showed that GPC supplementation increased expression of the proinflammatory effectors CXCL13 and TIMP-1 and activated NF-κB and MAPK signaling pathways in human coronary artery endothelial cells. Finally, targeted and untargeted metabolomic analysis of murine plasma revealed additional metabolites associated with GPC supplementation and atherosclerosis. In summary, our results show GPC promotes atherosclerosis through multiple mechanisms and that caution should be applied when using GPC as a nutritional supplement.

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

The gut microbe-derived metabolite trimethylamine-N-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.NEW & NOTEWORTHY The gut microbe-dependent metabolite trimethylamine-N-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.

A Systematic Investigation of Structure/Function Requirements for the Apolipoprotein A-I/Lecithin Cholesterol Acyltransferase Interaction Loop of High-density Lipoprotein.

The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) maturation. We previously identified a highly solvent-exposed apoA-I loop domain (Leu(159)-Leu(170)) in nascent HDL, the so-called "solar flare" (SF) region, and proposed that it serves as an LCAT docking site (Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., 3rd, Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol. 14, 861-868). The stability and role of the SF domain of apoA-I in supporting HDL binding and activation of LCAT are debated. Here we show by site-directed mutagenesis that multiple residues within the SF region (Pro(165), Tyr(166), Ser(167), and Asp(168)) of apoA-I are critical for both LCAT binding to HDL and LCAT catalytic efficiency. The critical role for possible hydrogen bond interaction at apoA-I Tyr(166) was further supported using reconstituted HDL generated from apoA-I mutants (Tyr(166) → Glu or Asn), which showed preservation in both LCAT binding affinity and catalytic efficiency. Moreover, the in vivo functional significance of NO2-Tyr(166)-apoA-I, a specific post-translational modification on apoA-I that is abundant within human atherosclerotic plaque, was further investigated by using the recombinant protein generated from E. coli containing a mutated orthogonal tRNA synthetase/tRNACUA pair enabling site-specific insertion of the unnatural amino acid into apoA-I. NO2-Tyr(166)-apoA-I, after subcutaneous injection into hLCAT(Tg/Tg), apoA-I(-/-) mice, showed impaired LCAT activation in vivo, with significant reduction in HDL cholesteryl ester formation. The present results thus identify multiple structural features within the solvent-exposed SF region of apoA-I of nascent HDL essential for optimal LCAT binding and catalytic efficiency.

Eosinophil Peroxidase Catalyzed Protein Carbamylation Participates in Asthma.

The biochemical mechanisms through which eosinophils contribute to asthma pathogenesis are unclear. Here we show eosinophil peroxidase (EPO), an abundant granule protein released by activated eosinophils, contributes to characteristic asthma-related phenotypes through oxidative posttranslational modification (PTM) of proteins in asthmatic airways through a process called carbamylation. Using a combination of studies we now show EPO uses plasma levels of the pseudohalide thiocyanate (SCN-) as substrate to catalyze protein carbamylation, as monitored by PTM of protein lysine residues into Nϵ-carbamyllysine (homocitrulline), and contributes to the pathophysiological sequelae of eosinophil activation. Studies using EPO-deficient mice confirm EPO serves as a major enzymatic source for protein carbamylation during eosinophilic inflammatory models, including aeroallergen challenge. Clinical studies similarly revealed significant enrichment in carbamylation of airway proteins recovered from atopic asthmatics versus healthy controls in response to segmental allergen challenge. Protein-bound homocitrulline is shown to be co-localized with EPO within human asthmatic airways. Moreover, pathophysiologically relevant levels of carbamylated protein either incubated with cultured human airway epithelial cells in vitro, or provided as an aerosolized exposure in non-sensitized mice, induced multiple asthma-associated phenotypes including induction of mucin, Th2 cytokines, IFNγ, TGFß, and epithelial cell apoptosis. Studies with scavenger receptor-A1 null mice reveal reduced IL-13 generation following exposure to aerosolized carbamylated protein, but no changes in other asthma-related phenotypes. In summary, EPO-mediated protein carbamylation is promoted during allergen-induced asthma exacerbation, and can both modulate immune responses and trigger a cascade of many of the inflammatory signals present in asthma.

Gut microbiota-dependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease.

RATIONALE: Trimethylamine-N-oxide (TMAO), a gut microbial-dependent metabolite of dietary choline, phosphatidylcholine (lecithin), and l-carnitine, is elevated in chronic kidney diseases (CKD) and associated with coronary artery disease pathogenesis. OBJECTIVE: To both investigate the clinical prognostic value of TMAO in subjects with versus without CKD, and test the hypothesis that TMAO plays a direct contributory role in the development and progression of renal dysfunction. METHODS AND RESULTS: We first examined the relationship between fasting plasma TMAO and all-cause mortality over 5-year follow-up in 521 stable subjects with CKD (estimated glomerular filtration rate, <60 mL/min per 1.73 m(2)). Median TMAO level among CKD subjects was 7.9 µmol/L (interquartile range, 5.2-12.4 µmol/L), which was markedly higher (P<0.001) than in non-CKD subjects (n=3166). Within CKD subjects, higher (fourth versus first quartile) plasma TMAO level was associated with a 2.8-fold increased mortality risk. After adjustments for traditional risk factors, high-sensitivity C-reactive protein, estimated glomerular filtration rate, elevated TMAO levels remained predictive of 5-year mortality risk (hazard ratio, 1.93; 95% confidence interval, 1.13-3.29; P<0.05). TMAO provided significant incremental prognostic value (net reclassification index, 17.26%; P<0.001 and differences in area under receiver operator characteristic curve, 63.26% versus 65.95%; P=0.036). Among non-CKD subjects, elevated TMAO levels portend poorer prognosis within cohorts of high and low cystatin C. In animal models, elevated dietary choline or TMAO directly led to progressive renal tubulointerstitial fibrosis and dysfunction. CONCLUSIONS: Plasma TMAO levels are both elevated in patients with CKD and portend poorer long-term survival. Chronic dietary exposures that increase TMAO directly contributes to progressive renal fibrosis and dysfunction in animal models.

Transmission of atherosclerosis susceptibility with gut microbial transplantation.

Recent studies indicate both clinical and mechanistic links between atherosclerotic heart disease and intestinal microbial metabolism of certain dietary nutrients producing trimethylamine N-oxide (TMAO). Here we test the hypothesis that gut microbial transplantation can transmit choline diet-induced TMAO production and atherosclerosis susceptibility. First, a strong association was noted between atherosclerotic plaque and plasma TMAO levels in a mouse diversity panel (n = 22 strains, r = 0.38; p = 0.0001). An atherosclerosis-prone and high TMAO-producing strain, C57BL/6J, and an atherosclerosis-resistant and low TMAO-producing strain, NZW/LacJ, were selected as donors for cecal microbial transplantation into apolipoprotein e null mice in which resident intestinal microbes were first suppressed with antibiotics. Trimethylamine (TMA) and TMAO levels were initially higher in recipients on choline diet that received cecal microbes from C57BL/6J inbred mice; however, durability of choline diet-dependent differences in TMA/TMAO levels was not maintained to the end of the study. Mice receiving C57BL/6J cecal microbes demonstrated choline diet-dependent enhancement in atherosclerotic plaque burden as compared with recipients of NZW/LacJ microbes. Microbial DNA analyses in feces and cecum revealed transplantation of donor microbial community features into recipients with differences in taxa proportions between donor strains that were transmissible to recipients and that tended to show coincident proportions with TMAO levels. Proportions of specific taxa were also identified that correlated with plasma TMAO levels in donors and recipients and with atherosclerotic lesion area in recipients. Atherosclerosis susceptibility may be transmitted via transplantation of gut microbiota. Gut microbes may thus represent a novel therapeutic target for modulating atherosclerosis susceptibility.

Effects of native and myeloperoxidase-modified apolipoprotein a-I on reverse cholesterol transport and atherosclerosis in mice.

OBJECTIVE: Preclinical and clinical studies have shown beneficial effects of infusions of apolipoprotein A-I (ApoA-I) on atherosclerosis. ApoA-I is also a target for myeloperoxidase-mediated oxidation, leading in vitro to a loss of its ability to promote ATP-binding cassette transporter A1-dependent macrophage cholesterol efflux. Therefore, we hypothesized that myeloperoxidase-mediated ApoA-I oxidation would impair its promotion of reverse cholesterol transport in vivo and the beneficial effects on atherosclerotic plaques. APPROACH AND RESULTS: ApoA-I(-/-) or apolipoprotein E-deficient mice were subcutaneously injected with native human ApoA-I, oxidized human ApoA-I (myeloperoxidase/hydrogen peroxide/chloride treated), or carrier. Although early postinjection (8 hours) levels of total ApoA-I in plasma were similar for native versus oxidized human ApoA-I, native ApoA-I primarily resided within the high-density lipoprotein fraction, whereas the majority of oxidized human ApoA-I was highly cross-linked and not high-density lipoprotein particle associated, consistent with impaired ATP-binding cassette transporter A1 interaction. In ApoA-I(-/-) mice, ApoA-I oxidation significantly impaired reverse cholesterol transport in vivo. In advanced aortic root atherosclerotic plaques of apolipoprotein E-deficient mice, native ApoA-I injections led to significant decreases in lipid content, macrophage number, and an increase in collagen content; in contrast, oxidized human ApoA-I failed to mediate these changes. The decrease in plaque macrophages with native ApoA-I was accompanied by significant induction of their chemokine receptor CCR7. Furthermore, only native ApoA-I injections led to a significant reduction of inflammatory M1 and increase in anti-inflammatory M2 macrophage markers in the plaques. CONCLUSIONS: Myeloperoxidase-mediated oxidation renders ApoA-I dysfunctional and unable to (1) promote reverse cholesterol transport, (2) mediate beneficial changes in the composition of atherosclerotic plaques, and (3) pacify the inflammatory status of plaque macrophages.

Prognostic value of choline and betaine depends on intestinal microbiota-generated metabolite trimethylamine-N-oxide.

AIMS: Recent metabolomics and animal model studies show trimethylamine-N-oxide (TMAO), an intestinal microbiota-dependent metabolite formed from dietary trimethylamine-containing nutrients such as phosphatidylcholine (PC), choline, and carnitine, is linked to coronary artery disease pathogenesis. Our aim was to examine the prognostic value of systemic choline and betaine levels in stable cardiac patients. METHODS AND RESULTS: We examined the relationship between fasting plasma choline and betaine levels and risk of major adverse cardiac events (MACE = death, myocardial infraction, stroke) in relation to TMAO over 3 years of follow-up in 3903 sequential stable subjects undergoing elective diagnostic coronary angiography. In our study cohort, median (IQR) TMAO, choline, and betaine levels were 3.7 (2.4-6.2)µM, 9.8 (7.9-12.2)µM, and 41.1 (32.5-52.1)µM, respectively. Modest but statistically significant correlations were noted between TMAO and choline (r = 0.33, P < 0.001) and less between TMAO and betaine (r = 0.09, P < 0.001). Higher plasma choline and betaine levels were associated with a 1.9-fold and 1.4-fold increased risk of MACE, respectively (Quartiles 4 vs. 1; P < 0.01, each). Following adjustments for traditional cardiovascular risk factors and high-sensitivity C-reactive protein, elevated choline [1.34 (1.03-1.74), P < 0.05], and betaine levels [1.33 (1.03-1.73), P < 0.05] each predicted increased MACE risk. Neither choline nor betaine predicted MACE risk when TMAO was added to the adjustment model, and choline and betaine predicted future risk for MACE only when TMAO was elevated. CONCLUSION: Elevated plasma levels of choline and betaine are each associated with incident MACE risk independent of traditional risk factors. However, high choline and betaine levels are only associated with higher risk of future MACE with concomitant increase in TMAO.

The cardioprotective protein apolipoprotein A1 promotes potent anti-tumorigenic effects.

Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b(+) F4/80(+) macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.

2H2O-based high-density lipoprotein turnover method for the assessment of dynamic high-density lipoprotein function in mice.

OBJECTIVE: High-density lipoprotein (HDL) promotes reverse cholesterol transport from peripheral tissues to the liver for clearance. Reduced HDL-cholesterol (HDLc) is associated with atherosclerosis; however, as a predictor of cardiovascular disease, HDLc has limitations because it is not a direct marker of HDL functionality. Our objective was to develop a mass spectrometry-based method for the simultaneous measurement of HDLc and ApoAI kinetics in mice, using a single (2)H2O tracer, and use it to examine genetic and drug perturbations on HDL turnover in vivo. APPROACH AND RESULTS: Mice were given (2)H2O in the drinking water, and serial blood samples were collected at different time points. HDLc and ApoAI gradually incorporated (2)H, allowing experimental measurement of fractional catabolic rates and production rates for HDLc and ApoAI. ApoE(-/-) mice displayed increased fractional catabolic rates (P<0.01) and reduced production rates of both HDLc and ApoAI (P<0.05) compared with controls. In human ApoAI transgenic mice, levels and production rates of HDLc and human ApoAI were strikingly higher than in wild-type mice. Myriocin, an inhibitor of sphingolipid synthesis, significantly increased both HDL flux and macrophage-to-feces reverse cholesterol transport, indicating compatibility of this HDL turnover method with the macrophage-specific reverse cholesterol transport assay. CONCLUSIONS: (2)H2O-labeling can be used to measure HDLc and ApoAI flux in vivo, and to assess the role of genetic and pharmacological interventions on HDL turnover in mice. Safety, simplicity, and low cost of the (2)H2O-based HDL turnover approach suggest that this assay can be scaled for human use to study effects of HDL targeted therapies on dynamic HDL function.

Assembling the anaerobic gamma-butyrobetaine to TMA metabolic pathway in Escherichia fergusonii and confirming its role in TMA production from dietary L-carnitine in murine models.

IMPORTANCE: The key atherosclerotic TMAO originates from the initial gut microbial conversion of L-carnitine and other dietary compounds into TMA. Developing therapeutic strategies to block gut microbial TMA production needs a detailed understanding of the different production mechanisms and their relative contributions. Recently, we identified a two-step anaerobic pathway for TMA production from L-carnitine through initial conversion by some microbes into the intermediate γBB which is then metabolized by other microbes into TMA. Investigational studies of this pathway, however, are limited by the lack of single microbes harboring the whole pathway. Here, we engineered E. fergusonii strain to harbor the whole two-step pathway and optimized the expression through cloning a specific chaperone from the original host. Inoculating germ-free mice with this recombinant E. fergusonii is enough to raise serum TMAO to pathophysiological levels upon L-carnitine feeding. This engineered microbe will facilitate future studies investigating the contribution of this pathway to cardiovascular disease.

Two distinct gut microbial pathways contribute to meta-organismal production of phenylacetylglutamine with links to cardiovascular disease.

Recent studies show gut microbiota-dependent metabolism of dietary phenylalanine into phenylacetic acid (PAA) is critical in phenylacetylglutamine (PAGln) production, a metabolite linked to atherosclerotic cardiovascular disease (ASCVD). Accordingly, microbial enzymes involved in this transformation are of interest. Using genetic manipulation in selected microbes and monocolonization experiments in gnotobiotic mice, we identify two distinct gut microbial pathways for PAA formation; one is catalyzed by phenylpyruvate:ferredoxin oxidoreductase (PPFOR) and the other by phenylpyruvate decarboxylase (PPDC). PPFOR and PPDC play key roles in gut bacterial PAA production via oxidative and non-oxidative phenylpyruvate decarboxylation, respectively. Metagenomic analyses revealed a significantly higher abundance of both pathways in gut microbiomes of ASCVD patients compared with controls. The present studies show a role for these two divergent microbial catalytic strategies in the meta-organismal production of PAGln. Given the numerous links between PAGln and ASCVD, these findings will assist future efforts to therapeutically target PAGln formation in vivo.

Gut Microbiota-Generated Phenylacetylglutamine and Heart Failure.

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.