A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors

The quote only partly states this claim.

PAGly showed similar effects to PAGln on platelet calcium and aggregation.

“Similar results were observed with PAGly (Fig. S2E–G).”

May combine two claims.

Parental HEK293 cells showed no PAGln-evoked change in intracellular cAMP, whereas β2-HEK293 cells showed significant PAGln-induced increases in cAMP.

“parental HEK293 cells showed no PAGln-evoked change in intracellular cAMP levels (green line), whereas β2-HEK293 cells showed significant PAGln induced increases in cytosolic cAMP levels (blue line) (Fig. 6B).”

May combine two claims.

PAGln dose-dependently increased cAMP production in β2 ADR-transfected CHO-K1 cells, and this was inhibited by ICI118,551.

“PAGln dose-dependently increased cAMP production in ADRB2 gene transfected Cricetulus griseus, hamster ovary (CHO-K1) cells”

• Also stated in: § Gain of Function Studies Confirm PAGln Can Signal via α2A, α2B and β2 ADRs · plain

  “Both PAGln- and ISO-induced cAMP production in the β2 ADR transfected CHO-K1 cells was inhibited by addition of ICI118,551 (Fig. S7C).”

May combine two claims.

The DMR response induced by PAGln incubation with MEG01 cells was substantially reduced by each of ICI118,551, propranolol, and RX821002.

“The DMR response induced by PAGln incubation with MEG01 cells was substantially reduced by each of the aforementioned inhibitors, while the inhibitors alone did not affect the DMR response (Fig. 5G).”

May combine two claims.

The PAGln-induced DMR response in MEG01 cells was significantly reduced by pretreatment with a combination of PTX, CTX, and YM-254890 or by the global GPCR inhibitor SCH-202676.

“The PAGln-induced DMR response in MEG01 cells was significantly reduced by pretreating the cells with either a combination of all three modulators (PTX, CTX, and YM-254890) or byuse of the global GPCR inhibitor SCH-202676”

May combine two claims.

In mice, both PAGln and PAGly acutely raised by intraperitoneal injection induced heightened platelet thrombus formation within the injured carotid artery and reduced time to cessation of blood flow after FeCl3 injury compared with Phe or saline.

“Notably, both PAGln and PAGly each induced heightened platelet thrombus formation within the injured carotid artery (Fig. 4A), and correspondingly reduced the time to cessation of blood flow following injury (i.e., the occlusion time) when compared to mice treated with the nutrient precursor Phe or normal saline (vehicle) (Fig. 4B).”

May combine two claims.

CTX or the global GPCR inhibitor SCH-202676 inhibited PAGln-evoked cAMP production in MEG01 cells and platelets.

“pretreatment of each with known GPCR modulators showed CTX or the global GPCR inhibitor SCH-202676 inhibited PAGln-evoked cAMP production”

May combine two claims.

In washed human platelets, PAGln dose-dependently enhanced submaximal ADP-stimulated P-selectin surface expression and GP IIb/IIIa activation, with significant increases at PAGln levels of 10 μM or greater.

“In studies with washed human platelets, PAGln also dose-dependently enhance sub-maximal ADP-stimulated P-selectin surface expression (Fig. 3E) and glycoprotein α2β3 (GP IIb/IIIa) activation (Fig. 3F), with significant increases in platelet activation phenotypes noted with PAGln levels of 10 μM or greater.”

May combine two claims.

Pretreatment with PTX or CTX, but not YM-254890, significantly suppressed the PAGln-induced DMR response in MEG01 cells.

“Further, pretreatment of cells with either PTX or CTX, but not YM-254890, significantly suppressed the PAGln-induced DMR response, suggesting Gαi/o and Gαs involvement”

May combine two claims.

siRNA targeting α2A, α2B, or β2 ADRs each significantly reduced PAGln-induced DMR response in MEG01 cells, while control siRNA had no effect.

“Notably, each of the siRNAs against the three ADRs demonstrated a significant reduction in PAGln-induced DMR response in MEG01 cells while control siRNA had no effect (Fig. 5F).”

May combine two claims.

The ΔcutC,ΔporA C. sporogenes mutant, still possessing functional fldH, no longer generated PAA and only formed PPA.

“In contrast, the (ΔcutC,ΔporA)C. sporogenes mutant (still possessing functional fldH) no longer generated PAA and only formed PPA.”

May combine two claims.

Increasing concentrations of PAGln and norepinephrine, but not Phe, produced saturable and specific DMR dose-response curves in MEG01 cells.

“Examination of increasing concentrations of potential binding ligands (PAGln vs Norepi vs Phe ) with MEG01 cells demonstrated saturable and specific DMR dose-response curves, typical for a ligand-receptor interaction process, with PAGln and Norepi, but not Phe (Fig. 5A).”

May combine two claims.

The ΔcutC,ΔfldH C. sporogenes mutant, still possessing functional porA, no longer generated PPA but did generate PAA.

“the ΔcutC,ΔfldH mutant (still possesses functional porA) no longer generated PPA but did generate PAA (Fig. 4C).”

May combine two claims.

In germ-free mice colonized with ΔcutC,ΔfldH C. sporogenes, after arterial injury both the rate of thrombus generation and the time to cessation of blood flow were significantly reduced.

“Following arterial injury, both the rate of thrombus generation and the time to cessation of blood flow within the injured vessel were significantly reduced in the mice colonized with the (ΔcutC,ΔfldH)C. sporogenes mutant, producing higher levels of PAA and consequently, PAGly (Fig. 4D).”

May combine two claims.

In mice, carvedilol reversed the PAGln-induced acceleration of platelet thrombus generation and shortening of time to cessation of blood flow after FeCl3 arterial injury.

“the pro-thrombotic effect of PAGln was reversed with carvedilol (Fig. 6D,E).”

May combine two claims.

In an independent cohort of 4,000 subjects, PAGln was associated with cardiovascular disease and incident major adverse cardiovascular events (myocardial infarction, stroke, or death).

“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).”

May combine two claims.

Specific ADR siRNA knockdown or ADR antagonists block PAGln-induced pro-thrombotic phenotypes.

“In addition, studies with either specific ADR siRNA knockdown or ADR antagonists were both shown to block PAGln induced pro-thrombotic phenotypes”

May combine two claims.

Pathophysiologically relevant levels of PAGln or PAGly each facilitate enhanced rate of thrombus formation and thrombosis potential in arterial injury models.

“Furthermore, numerous in vivo studies reveal pathophysiologically relevant levels of PAGln or PAGly each facilitate both enhanced rate of thrombus formation and thrombosis potential in arterial injury models, including studies ranging from direct administration of either PAGln or PAGly, to microbial transplantation studies that modulate production of PAA, the precursor of PAGln (and PAGly).”

May combine two claims.

Both loss-of-function and gain-of-function studies, employing genetic or pharmacological approaches in multiple cell types, confirm that PAGln can induce cellular responses via α2A, α2B, and β2 ADRs.

“Thus, both loss of function and gain of function studies, employing either genetic or pharmacological approaches in multiple cell types, confirm that PAGln can induce cellular responses via α2A, α2B and β2 ADRs.”

May combine two claims.

The gut microbiota-dependent metabolites PAGln and PAGly significantly impact platelet function, enhancing platelet adhesion to collagen matrices and both platelet stimulus-dependent rise in intracellular calcium and aggregation in response to agonists.

“The above data demonstrate that the gut microbiota-dependent metabolites PAGln and PAGly significantly impact platelet function, enhancing platelet adhesion to collagen matrices and both platelet stimulus-dependent rise in [Ca2+]i and aggregation in response to agonists.”

Classified as probably not a substantial claim.

ADRs possess a flexible agonist pocket.

“Because ADRs possess a flexible agonist pocket (Masureel et al., 2018)”

• cites Masureel et al., 2018 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Both genetic and pharmacological loss-of-function studies confirm that PAGln can induce cellular responses via α2A, α2B, and β2 ADRs.

“Thus, both genetic and pharmacological loss of function studies confirm PAGln can induce cellular responses via α2A, α2B and β2 ADRs.”

Probably not a substantial claim: may not pass S3 (load-bearing).

PAA can be produced by bacterial fermentation of the essential amino acid Phe in culture.

“other studies have shown PAA can be produced by bacterial fermentation of the essential amino acid Phe in culture (Dodd et al., 2017).”

• cites Dodd et al., 2017 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Carvedilol is a widely used β-blocker in clinical practice.

“a widely used β-blocker in clinical practice, carvedilol (Gupta et al., 2004)”

• cites Gupta et al., 2004 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Isoproterenol predominantly binds β2-ADRs that primarily couple to the Gαs subunit in MEG01 cells and platelets.

“Isoproterenol (ISO), which predominantly binds to β2-ADRs that primarily couple to the Gαs subunit in both MEG01 cells (Fig. S5G) and in platelets (Fig. S5H) (Koryakina et al., 2012)”

• cites Koryakina et al., 2012 sourceNot checked yet

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Classified as probably not a substantial claim. May combine two claims.

Pertussis toxin, cholera toxin, and YM-254890 mask activation of Gαi/o, Gαs, and Gαq subunit-mediated signaling pathways, respectively.

“pertussis toxin (PTX), cholera toxin (CTX) and YM-254890, which mask activation of Gαi/o, Gαs and Gαq subunit-mediated signaling pathways, respectively (Campbell and Smrcka, 2018; Milligan and Kostenis, 2006).”

• cites Campbell and Smrcka, 2018Not checked yet

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• cites Milligan and Kostenis, 2006Not checked yet

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Classified as probably not a substantial claim.

SCH-202676 is a global GPCR inhibitor that blocks agonist and antagonist binding to structurally diverse GPCRs coupling to Gαi/o, Gαs, and Gαq proteins.

“SCH-202676, a global GPCR inhibitor that blocks both agonist and antagonist binding to structurally diverse GPCRs that couple to Gαi/o, Gαs and Gαq proteins (Fawzi et al., 2001; Lewandowicz et al., 2006).”

• cites Fawzi et al., 2001 sourceNot checked yet

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• cites Lewandowicz et al., 2006 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

PAGln can be formed by phenylacetic acid conjugation to glutamine by human and rhesus monkey liver enzymes.

“Previous studies with either liver extracts or isolated enzymes have reported PAGln can be formed by phenylacetic acid (PAA) conjugation to the amino acid glutamine (Gln) by both human and rhesus monkey liver enzymes (Webster et al., 1976)”

• cites Webster et al., 1976 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and PAGly fostering platelet responsiveness and thrombosis potential.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing).

PAA conjugation to glycine generating PAGly has been noted.

“In addition to PAGln, PAA conjugation to the amino acid glycine (Gly) generating PAGly has also been noted (Gonzalez-Guardia et al., 2015; Wang et al., 2013).”

• cites Gonzalez-Guardia et al., 2015 sourceNot checked yet

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• cites Wang et al., 2013 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The microbial porA gene plays a role in oxidative metabolism of Phe to PAA by Clostridium sporogenes.

“Recent studies reported a role for the microbial porA gene in oxidative metabolism of Phe to PAA by the human commensal Clostridium sporogenes (Dodd et al., 2017).”

• cites Dodd et al., 2017 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Norepinephrine binds ADRs and collagen binds the glycoprotein VI receptor.

“known receptor binding ligands like norepinephrine (Norepi) and collagen, which bind to ADRs and the glycoprotein VI (GPVI) receptor (Leitinger, 2011; Small et al., 2003), respectively”

• cites Leitinger, 2011 sourceNot checked yet

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• cites Small et al., 2003 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

PPA was a major product of Phe metabolism by clostridia.

“Elsden and colleagues previously reported PPA was a major product of Phe metabolism by clostridia (Elsden et al., 1976)”

• cites Elsden et al., 1976 sourceNot checked yet

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May combine two claims.

PAGln represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

“PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.”

Probably not a substantial claim: may not pass S3 (load-bearing).

PAGln has long been recognized as a synthetic product of PAA and Gln by liver and renal tissues of humans.

“PAGln has long been recognized as a synthetic product of PAA and Gln by liver and renal tissues of humans (Moldave and Meister, 1957)”

• cites Moldave and Meister, 1957Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All subjects gave written informed consent and the Institutional Review Board of the Cleveland Clinic approved all study protocols.

“All subjects gave written informed consent and the Institutional Review Board of the Cleveland Clinic approved all study protocols.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All cell lines used were tested before use to confirm negative status for mycoplasma.

“Before use, all cell lines used were tested to confirm negative status for mycoplasma (CC cell culture facility).”

Classified as probably not a substantial claim.

Adjustments were made for individual traditional cardiac risk factors including age, sex, HDL, LDL, triglycerides, smoking, diabetes mellitus, systolic blood pressure, and high-sensitivity C-reactive protein level.

“Adjustments were made for individual traditional cardiac risk factors including age, sex, HDL, LDL, triglycerides, smoking, diabetes mellitus, systolic blood pressure, and high-sensitivity C-reactive protein level.”

Classified as probably not a substantial claim.

β-Blocker therapy is known to foster numerous clinical benefits in some subjects with CVD, including reduced risks of heart attack, stroke, heart failure and death.

“β-Blocker therapy is known to foster numerous clinical benefits in some subjects with CVD (e.g. reduced risks of heart attack, stroke, heart failure and death (Black et al., 2010; Burnett et al., 2017; Witte et al., 2018)).”

• cites Black et al., 2010 sourceNot checked yet

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• cites Burnett et al., 2017 sourceNot checked yet

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• cites Witte et al., 2018 sourceNot checked yet

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Classified as probably not a substantial claim.

Addition of any agonist over the CTX-induced baseline does not lead to detectable increase in Gαs subunit driven cAMP production, thereby masking the signaling process.

“Addition of any agonist over this baseline value now does not lead to detectable increase in the Gαs subunit driven cAMP production, thereby resulting in masking of the signaling process.”

• AssumptionRests on an assumption: CTX addition results in stimulation of adenylyl cyclase and a sustained increase in cAMP, which was considered as the normalized baseline.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Quality control samples were run with each batch of samples and inter-batch variations expressed as coefficient of variation were less than 10%.

“Quality control samples were run with each batch of samples and inter-batch variations expressed as coefficient of variation (CV) were less than 10%.”

Classified as probably not a substantial claim.

All subjects had extensive clinical and longitudinal outcome data monitored, including adjudicated outcomes over the ensuing 3 to 5 years after enrollment.

“All subjects had extensive clinical and longitudinal outcome data monitored, including adjudicated outcomes over the ensuing 3 to 5 years after enrollment.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Carvedilol has been shown to promote inhibition of platelet function in several trials.

“It is notable that carvedilol has been shown to promote inhibition of platelet function in several trials (Petrikova et al., 2005; Ilardi et al., 2018)”

• cites Petrikova et al., 2005Not checked yet

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• cites Ilardi et al., 2018 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Time to cessation of blood flow through clot formation for all studies was determined by visual inspection by two independent investigators.

“Time to cessation of blood flow through clot formation for all studies was determined by visual inspection by two independent investigators.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Animal care and experimentation were consistent with NIH guidelines and all animal model studies were approved by the Institutional Animal Care and Use Committee at the Cleveland Clinic.

“Animal care and experimentation were consistent with NIH guidelines and all animal model studies were approved by the Institutional Animal Care and Use Committee at the Cleveland Clinic.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Following colonization, the investigator was not blinded from treatment groups to avoid cross contamination.

“Following colonization, the investigator was not blinded from treatment groups to avoid cross contamination.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The limit of detection and limit of quantification for the targeted LC-MS/MS assay were 0.010 and 0.033 μM, respectively.

“The limit of detection (LOD; 3:1 signal to noise cutoff) and limit of quantification (LOQ; 10:1 signal to noise cutoff) were 0.010 and 0.033 μM, respectively.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Activation of the adrenergic system has profound effects on metabolism, and prolonged activation can lead to insulin resistance, alterations in glucose and fatty acid metabolism, and mitochondrial dysfunction.

“Activation of the adrenergic system also has profound effect on metabolism and prolonged activation can lead to insulin resistance, alterations in glucose and fatty acid metabolism and mitochondrial dysfunction (Ciccarelli et al., 2013; Fu et al., 2017).”

• cites Ciccarelli et al., 2013 sourceNot checked yet

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• cites Fu et al., 2017Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

PAGln levels are substantially increased in subjects with end-stage renal disease.

“PAGln levels were noted to be substantially increased in subjects with end-stage renal disease (to more than 500 μM in some subjects (Meyer et al., 2016))”

• cites Meyer et al., 2016 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

ADRs are widely expressed on virtually every cell type and play important roles in myocardial and vascular function and in regulation of body homeostasis in both health and pathologic states.

“ADRs are widely expressed on virtually every cell type, and known to play important roles in myocardial and vascular function, as well as regulation of body homeostasis in both health and pathologic states (Amrani and Bradding, 2017; Hein and Kobilka, 1997; Lefkowitz et al., 2000; Rockman et al., 2002).”

• cites Amrani and Bradding, 2017Not checked yet

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• cites Hein and Kobilka, 1997Not checked yet

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• cites Lefkowitz et al., 2000 sourceNot checked yet

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Classified as probably not a substantial claim. May combine two claims.

The Discovery Cohort (n=1,157) and the independent non-overlapping Validation Cohort (n=4,000) were obtained from sequential consenting subjects enrolled in GeneBank at the Cleveland Clinic.

“The Discovery Cohort (n=1,157) employed for untargeted metabolomics investigations, and the independent non-overlapping Validation Cohort (n=4,000), were obtained from sequential consenting subjects enrolled in the study GeneBank at the Cleveland Clinic.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In the transfection experiments described, equivalent transfection efficiency for each well was monitored using a GFP reporter and was approximately 80%.

“Equivalent transfection efficiency for each well was monitored using a GFP reporter (pmaxGFP, Lonza, CH) and was ~80%.”

• Also stated in: § METHOD DETAILS · plain

  “Equivalent transfection efficiency for each well was monitored using a GFP reporter (pmaxGFP, Lonza, CH) and was ~80%.”

Probably not a substantial claim: may not pass S3 (load-bearing).

PAGln is a non-invasive probe of citric acid cycle intermediates in humans and primates.

“as well as a non-invasive probe of citric acid cycle intermediates in humans and primates (Yang et al., 1996).”

• cites Yang et al., 1996Not checked yet

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