KEY POINTS:Inhibition of the gut microbial trimethylamine N -oxide (TMAO) pathway after CKD onset both halted disease progression and reversed pathophysiologic CKD features. Inhibition of TMAO formation in established CKD animals improved kidney function, proteinuria, fibrosis, and uremic toxins. In CKD models, a drug that suppressed gut microbial TMAO production yet remains in the intestinal lumen reversed kidney fibrosis and improved GFR. BACKGROUND:Recent studies demonstrate gut microbial trimethylamine N -oxide (TMAO) is clinically and mechanistically linked to CKD development. Prior studies showed selective targeting of gut microbial TMAO production can prevent CKD initiation. A highly relevant clinical question is whether inhibiting TMAO generation can reverse established kidney dysfunction and fibrosis. METHODS:CD1 mice were first randomized (phase 1) to unilateral nephrectomy (UNx) or sham surgery and fed a high-fat diet ±1% choline supplementation to document CKD through measures of multiple indices of kidney function, tissue remodeling/fibrosis, and uremic toxins. Mice were then randomized (phase 2) to continue the same diet±fluoromethylcholine (FMC), a mechanism-based gut microbial TMAO inhibitor, for 8 additional weeks, and the impact on CKD-related phenotypes was assessed. RESULTS:After 12 weeks, choline-supplemented UNx mice exhibited 15-fold higher circulating TMAO levels. In parallel, multiple kidney dysfunction indices progressed, including 48% reduction in measured GFR (mGFR), elevated cystatin C and creatinine, 3.5-fold greater tubulointerstitial fibrosis, and upregulated profibrotic gene expression. After phase 2 (8 weeks±FMC), choline-supplemented (high-TMAO) UNx mice without FMC showed further elevation of circulating TMAO and multiple indices of progressive kidney function decline and fibrosis. Conversely, FMC-treated animals showed virtual elimination in circulating TMAO and significant improvements in all monitored CKD phenotypes, including >50% higher mGFR, and improved (decreased) creatinine, cystatin C, pseudouridine, urinary albumin/creatinine ratio, kidney fibrosis measures, and many uremic toxin levels ( e.g ., indoxyl sulfate and phenylacetylglycine). Beyond halting progression of kidney functional decline and fibrosis, there was frank regression in tubulointerstitial fibrosis (41% reduction) and significant improvement in mGFR (39% increase, end of phase 2 versus phase 1). CONCLUSIONS:Pharmacologic targeting of gut microbial TMAO production after CKD establishment halted disease progression and promoted regression of key CKD pathophysiologic features.

BACKGROUND AND AIMS:The pathways and metabolites that contribute to residual cardiovascular disease risks are unclear. Low-calorie sweeteners are widely used sugar substitutes in processed foods with presumed health benefits. Many low-calorie sweeteners are sugar alcohols that also are produced endogenously, albeit at levels over 1000-fold lower than observed following consumption as a sugar substitute. METHODS:Untargeted metabolomics studies were performed on overnight fasting plasma samples in a discovery cohort (n = 1157) of sequential stable subjects undergoing elective diagnostic cardiac evaluations; subsequent stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were performed on an independent, non-overlapping validation cohort (n = 2149). Complementary isolated human platelet, platelet-rich plasma, whole blood, and animal model studies examined the effect of xylitol on platelet responsiveness and thrombus formation in vivo. Finally, an intervention study was performed to assess the effects of xylitol consumption on platelet function in healthy volunteers (n = 10). RESULTS:In initial untargeted metabolomics studies (discovery cohort), circulating levels of a polyol tentatively assigned as xylitol were associated with incident (3-year) major adverse cardiovascular event (MACE) risk. Subsequent stable isotope dilution LC-MS/MS analyses (validation cohort) specific for xylitol (and not its structural isomers) confirmed its association with incident MACE risk [third vs. first tertile adjusted hazard ratio (95% confidence interval), 1.57 (1.12-2.21), P < .01]. Complementary mechanistic studies showed xylitol-enhanced multiple indices of platelet reactivity and in vivo thrombosis formation at levels observed in fasting plasma. In interventional studies, consumption of a xylitol-sweetened drink markedly raised plasma levels and enhanced multiple functional measures of platelet responsiveness in all subjects. CONCLUSIONS:Xylitol is associated with incident MACE risk. Moreover, xylitol both enhanced platelet reactivity and thrombosis potential in vivo. Further studies examining the cardiovascular safety of xylitol are warranted.

Artificial sweeteners are widely used sugar substitutes, but little is known about their long-term effects on cardiometabolic disease risks. Here we examined the commonly used sugar substitute erythritol and atherothrombotic disease risk. In initial untargeted metabolomics studies in patients undergoing cardiac risk assessment ( n = 1,157; discovery cohort, NCT00590200 ), circulating levels of multiple polyol sweeteners, especially erythritol, were associated with incident (3 year) risk for major adverse cardiovascular events (MACE; includes death or nonfatal myocardial infarction or stroke). Subsequent targeted metabolomics analyses in independent US ( n = 2,149, NCT00590200 ) and European ( n = 833, DRKS00020915 ) validation cohorts of stable patients undergoing elective cardiac evaluation confirmed this association (fourth versus first quartile adjusted hazard ratio (95% confidence interval), 1.80 (1.18–2.77) and 2.21 (1.20–4.07), respectively). At physiological levels, erythritol enhanced platelet reactivity in vitro and thrombosis formation in vivo. Finally, in a prospective pilot intervention study ( NCT04731363 ), erythritol ingestion in healthy volunteers ( n = 8) induced marked and sustained (>2 d) increases in plasma erythritol levels well above thresholds associated with heightened platelet reactivity and thrombosis potential in in vitro and in vivo studies. Our findings reveal that erythritol is both associated with incident MACE risk and fosters enhanced thrombosis. Studies assessing the long-term safety of erythritol are warranted.

Intestinal dysbiosis is prominent in systemic sclerosis (SSc), but it remains unknown how it contributes to microvascular injury and fibrosis that are hallmarks of this disease. Trimethylamine (TMA) is generated by the gut microbiome and in the host converted by flavin-containing monooxygenase (FMO3) into trimethylamine N-oxide (TMAO), which has been implicated in chronic cardiovascular and metabolic diseases. Using cell culture systems and patient biopsies, we now show that TMAO reprograms skin fibroblasts, vascular endothelial cells, and adipocytic progenitor cells into myofibroblasts via the putative TMAO receptor protein R-like endoplasmic reticulum kinase (PERK). Remarkably, FMO3 was detected in skin fibroblasts and its expression stimulated by TGF-β1. Moreover, FMO3 was elevated in SSc skin biopsies and in SSc fibroblasts. A meta-organismal pathway thus might in SSc link gut microbiome to vascular remodeling and fibrosis via stromal cell reprogramming, implicating the FMO3-TMAO-PERK axis in pathogenesis, and as a promising target for therapy.

Background: Daratumumab (DARA) is an anti-CD38 monoclonal antibody approved for treatment of patients with newly diagnosed and previously treated multiple myeloma (MM). Hence, it is possible that patients initiating DARA may continue to use it as backbone of their treatment even if their line of therapy (LOT) or treatment regimen changes, sometimes with interruptions. Aims: This study examines the continuous duration of DARA treatment and dosing frequency across all LOTs using real-world data from MM patients in the U.S. Methods: This is a retrospective observational study conducted using Optum Clinformatics Data Mart database consisting of some Medicare and commercially insured MM patients utilizing DARA, between 11/1/2015-3/31/2021. Duration of DARA use was defined as the time interval between first initiation and discontinuation of DARA spanning multiple LOTs as a time-to-event outcome using Kaplan-Meier method. A gap of >60 days between two consequent DARA claim dates was defined as DARA discontinuation. Dosing frequency was calculated as the average number of DARA doses during defined time periods. Compliance ratios were calculated as observed dosing frequency divided by the dosing frequency for FDA approved regimens (DARA-Lenalidomide-Dexamethasone [DRd] and DARA-Pomalidomide-Dexamethasone [DPd]). All results were calculated for patients using DARA across multiple LOTs regardless of treatment regimen. Results: A total of 2125 patients initiating DARA therapy were included in this analysis with mean age (SD) of 70.9 (9.8) years, 51.3% males, and 64.8% white. The median length of DARA use spanning multiple LOTs, was 16.6 months. During the first year of DARA use, 90.5%, 83.2%, 72.6% and 63.5% of the patients continued DARA at 3, 6, 9, and 12 months, respectively. At years 2 and 3 of DARA use, 33.1% and 14.5% of the patients, respectively, continued DARA treatment (Figure 1). The mean dosing frequency in real world was similar to the dosing frequency on the approved label (Table 1). The compliance ratio was 0.98 over the period of 36 months. Table 1. - Real-world dosing frequency of daratumumab over 36 months of follow-up (N=2125) Follow-up Period (in months) % of original sample remaining Number of Daratumumab doses from RWDMean (SD) Number of Daratumumab doses based on PI Compliance ratio 0-3 90.5% 9.6 (2.4) 11 0.87 0-6 83.2% 15.0 (3.1) 17 0.88 0-9 72.6% 18.5 (3.4) 20 0.92 0-12 63.5% 22.0 (3.8) 23 0.96 0-18 46.5% 28.8 (4.6) 30 0.96 0-24 33.1% 35.4 (6.0) 36 0.98 0-36 14.5% 48.1 (8.6) 49 0.98 Abbreviations: RWD, real world data; SD, standard deviation; PI, prescribing information for DRd and DPd as approved label Image:Summary/Conclusion: Patients initiating DARA remain on the therapy for a median of 16.6 months with one-sixth continuing DARA beyond 3 years. The dosing frequency observed in the real-world was consistent with the approved label.

BACKGROUND: Patients at increased risk for coronary artery disease and adverse prognosis during heart failure exhibit increased levels of circulating trimethylamine N-oxide (TMAO), a metabolite formed in the metabolism of dietary phosphatidylcholine. We investigated the efficacy of dietary withdrawal of TMAO as well as use of a gut microbe-targeted inhibitor of TMAO production, on cardiac function and structure during heart failure. METHODS AND RESULTS: Male C57BLK/6J mice were fed either control diet, a diet containing TMAO (0.12% wt/wt), a diet containing choline (1% wt/wt), or a diet containing choline (1% wt/wt) plus a microbial choline trimethylamine lyase inhibitor, iodomethylcholine (0.06% wt/wt), starting 3 weeks before transverse aortic constriction. At 6 weeks after transverse aortic constriction, a subset of animals in the TMAO group were switched to a control diet for the remainder of the study. Left ventricular structure and function were monitored at 3-week intervals. Withdrawal of TMAO from the diet attenuated adverse ventricular remodeling and improved cardiac function compared with the TMAO group. Similarly, inhibiting gut microbial conversion of choline to TMAO with a choline trimethylamine lyase inhibitor, iodomethylcholine, improved remodeling and cardiac function compared with the choline-fed group. CONCLUSIONS: These experimental findings are clinically relevant, and they demonstrate that TMAO levels are modifiable following long-term exposure periods with either dietary withdrawal of TMAO or gut microbial blockade of TMAO generation. Furthermore, these therapeutic strategies to reduce circulating TMAO levels mitigate the negative effects of dietary choline and TMAO in heart failure.

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including alpha 2A, alpha 2B, and beta 2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

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.

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.

Background Gut microbes play a critical role in the production of trimethylamine N-oxide (TMAO), an atherogenic metabolite that impacts platelet responsiveness and thrombosis potential. Involving both microbe and host enzymatic machinery, TMAO generation utilizes a metaorganismal pathway, beginning with ingestion of trimethylamine (TMA)-containing dietary nutrients such as choline, phosphatidylcholine and carnitine, which are abundant in a Western diet. Gut microbial TMA lyases use these nutrients as substrates to produce TMA, which upon delivery to the liver via the portal circulation, is converted into TMAO by host hepatic flavin monooxygenases (FMOs). Gut microbial production of TMA is rate limiting in the metaorganismal TMAO pathway because hepatic FMO activity is typically in excess. Objectives FMO3 is the major FMO responsible for host generation of TMAO; however, a role for FMO3 in altering platelet responsiveness and thrombosis potential invivo has not yet been explored. Methods The impact of FMO3 suppression (antisense oligonucleotide-targeting) and overexpression (as transgene) on plasma TMAO levels, platelet responsiveness and thrombosis potential was examined using a murine FeCl3-induced carotid artery injury model. Cecal microbial composition was examined using 16S analyses. Results Modulation of FMO3 directly impacts systemic TMAO levels, platelet responsiveness and rate of thrombus formation invivo. Microbial composition analyses reveal taxa whose proportions are associated with both plasma TMAO levels and invivo thrombosis potential. Conclusions The present studies demonstrate that host hepatic FMO3, the terminal step in the metaorganismal TMAO pathway, participates in diet-dependent and gut microbiota-dependent changes in both platelet responsiveness and thrombosis potential invivo.

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.

Objective—Platelets express a functional ubiquitin–proteasome system. Mass spectrometry shows that platelets contain several deubiquitinases, but whether these are functional, modulate the proteome, or affect platelet reactivity are unknown. Approach and Results—Platelet lysates contained ubiquitin–protein deubiquitinase activity hydrolyzing both Lys48 and Lys63 polyubiquitin conjugates that was suppressed by the chemically unrelated deubiquitinase inhibitors PYR41 and PR619. These inhibitors acutely and markedly increased monoubiquitination and polyubiquitination of the proteome of resting platelets. PYR41 (intravenous, 15 minutes) significantly impaired occlusive thrombosis in FeCl3-damaged carotid arteries, and deubiquitinase inhibition reduced platelet adhesion and retention during high shear flow of whole blood through microfluidic chambers coated with collagen. Total internal reflection microscopy showed that adhesion and spreading in the absence of flow were strongly curtailed by these inhibitors with failure of stable process extension and reduced the retraction of formed clots. Deubiquitinase inhibition also sharply reduced homotypic platelet aggregation in response to not only the incomplete agonists ADP and collagen acting through glycoprotein VI but also to the complete agonist thrombin. Suppressed aggregation was accompanied by curtailed procaspase activating compound-1 binding to activated IIb/IIIa and inhibition of P-selectin translocation to the platelet surface. Deubiquitinase inhibition abolished the agonist-induced spike in intracellular calcium, suppressed Akt phosphorylation, and reduced agonist-stimulated phosphatase and tensin homolog phosphatase phosphorylation. Platelets express the proteasome-associated deubiquitinases USP14 and UCHL5, and selective inhibition of these enzymes by b-AP15 reproduced the inhibitory effect of the general deubiquitinase inhibitors on ex vivo platelet function. Conclusions—Remodeling of the ubiquitinated platelet proteome by deubiquitinases promotes agonist-stimulated intracellular signal transduction and platelet responsiveness.

Atherothrombotic diseases are responsible for morethan 25% of all deaths worldwide. Anti¬-platelet drugs are themainstay treatment because of the direct involvement of plateletsin the initiation and propagation of thrombosis. However, thecurrently available anti-platelet drugs, such as antagonists ofplatelet receptors or of effector systems participating in plateletactivation, have their own limitations. A new mode of affectingplatelet reactivity may prove to offer unique advantages in a hostof clinical settings. Proteasome inhibitors are in clinical use totreat hematologic cancers, but also reduce thrombosis. Whether theproteasome participates in platelet activation or function isopaque since little is known of the proteasome in these terminallydifferentiated cells. Therefore, I investigated the role ofproteasome-mediated proteolysis on platelet function (AIM 1). Ifind platelets displayed all three primary proteasome proteaseactivities, which MG132 and bortezomib (Velcade®) inhibited.Proteasome substrates are marked by ubiquitin, and plateletscontained a functional ubiquitination system that modified theproteome by mono- and poly-ubiquitination. Proteasome inhibitionsuppressed platelet aggregation by low thrombin concentrations andristocetin-stimulated agglutination through the GPIb-IX-V complex.Proteasome inhibitor MG132 reduced stimulated spreading and clotretraction. The effects of proteasome inhibitors were not confinedto a single receptor as MG132 and bortezomib suppressed thrombin-,ADP-, and LPS-stimulated microparticle shedding. Systemic MG132strongly suppressed formation of occlusive, platelet-rich thrombiin FeCl3-damaged carotid arteries. Transfusion of platelets treatedex vivo with MG132 and washed prior to transfusion intothrombocytopenic mice also reduced carotid artery thrombosis.Theinhibition of the proteasome quells the ultimate step ofubiquitin-mediated protein degradation pathway. Proteasome-mediateddegradation is the final common step, however, multiple layers ofregulated processes are involved upstream of this degradativemachine that determines whether to target a protein for degradationor not. Platelets express a number of deubiquitinases that reverseprotein ubiquitination, but their potential function in plateletsis unstudied. So, I investigated the role of deubiquitinase enzymesin modulating platelet reactivity (Aim 2). I show platelets expressdeubiquitinase activity and specific inhibitor of theproteasome-associated deubiquitinases (b-AP15) as well as generaldeubiquitinase inhibitors (PYR41 and PR619) increased mono- andpoly-ubiquitination of platelet proteins. Deubiquitinase inhibitionstrongly suppressed aIIbs3 activation, degranulation, plateletaggregation and adhesion/ spreading in response to diverse plateletagonists. This inhibition also blocked downstream signaling fromplatelet receptors by inhibiting agonist-induced Aktphosphorylation and intracellular calcium release. Inhibition ofplatelet deubiquitinase activity strongly suppressed…

Objective— Proteasome inhibitors used in the treatment of hematologic cancers also reduce thrombosis. Whether the proteasome participates in platelet activation or function is unclear because little is known of the proteasome in these terminally differentiated cells. Approach and Results— Platelets displayed all 3 primary proteasome protease activities, which MG132 and bortezomib (Velcade) inhibited. Proteasome substrates are marked by ubiquitin, and platelets contained a functional ubiquitination system that modified the proteome by monoubiquitination and polyubiquitination. Systemic MG132 strongly suppressed the formation of occlusive, platelet-rich thrombi in FeCl 3 -damaged carotid arteries. Transfusion of platelets treated ex vivo with MG132 and washed before transfusion into thrombocytopenic mice also reduced carotid artery thrombosis. Proteasome inhibition reduced platelet aggregation by low thrombin concentrations and ristocetin-stimulated agglutination through the glycoprotein Ib-IX-V complex. This receptor was not appropriately internalized after proteasome inhibition in stimulated platelets, and spreading and clot retraction after MG132 exposure also were decreased. The effects of proteasome inhibitors were not confined to a single receptor as MG132 suppressed thrombin-stimulated, ADP-stimulated, and lipopolysaccharide-stimulated microparticle shedding. Proteasome inhibition increased ubiquitin decoration of cytoplasmic proteins, including the cytoskeletal proteins Filamin A and Talin-1. Mass spectrometry revealed a single MG132-sensitive tryptic cleavage after R 1745 in an extended Filamin A loop, which would separate its actin-binding domain from its carboxy terminal glycoprotein Ibα–binding domain. Conclusions— Platelets contain a ubiquitin/proteasome system that marks cytoskeletal proteins for proteolytic modification to promote productive platelet–platelet and platelet–wall interactions.