Compound 21, a selective angiotensin II type 2 receptor agonist, downregulates lipopolysaccharide-stimulated tissue factor expression in human peripheral blood mononuclear cells
Abstract:Intricate interrelationships connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally active angiotensin II, a proinflammatory agent with direct TF-stimulating properties mediated by the angiotensin II type 1 receptor (AT1R)s. However, angiotensin II also stimulates angiotensin II type 2 receptor (AT2R)s and they may as well contribute to TF expression, a possibility in need of further evaluation. We investigated the effect of C21, a highly… Show more
“…Within the frame of reference summarized in the previous paragraph, our data confirm the inhibitory action of AT2R agonism by C21 on LPS-stimulated TF expression in human PBMCs [ 14 ] and the complete antagonism exerted by PD123,319 reassures about the specificity of the response. The parallel decrease of TFAg and mRNA is consonant with transcriptional modulation of TF protein likely by NFkB downregulation [ 41 ], an interpretation fully supported by recent reports showing attenuated cytokine production by C21 in LPS-stimulated monocytic cells [ 42 ].…”
Section: Discussionsupporting
confidence: 78%
“…[ 11 ] eligible for paracrine and/or intracrine stimulation. In agreement with that concept, AT1R antagonists downregulate activated TF expression [ 12 , 13 ] among others) but the inhibitory effect of selective AT2R stimulation [ 14 ] is suggestive of the existence of AT2Rs counteracting the AT1R-mediated procoagulant phenotype, as reported for other AT2R-mediated functions [ 15 ].…”
BackgroundIntimate links connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally generated Angiotensin (AT) II, the effector arm of the Renin Angiotensin System (RAS). C21, a selective AT2R agonist, downregulates the transcriptional expression of TF in LPS-activated peripheral blood mononuclear cell(PBMC)s implying the existence of ATII type 2 receptor (AT2R)s whose stimulation attenuates inflammation-mediated procoagulant responses. High glucose, by activating key signalling pathways and increasing the cellular content of RAS components, augments TF expression and potentiates the inhibitory effect of AT1R antagonists. It is unknown, however, the impact of that stimulus on AT2R-mediated TF inhibition, an information useful to understand more precisely the role of that signal transduction pathway in the inflammation-mediated coagulation process. TF antigen (ELISA), procoagulant activity (PCA, 1-stage clotting assay) and TF-mRNA (real-time polymerase chain reaction) were assessed in PBMCs activated by LPS, a pro-inflammatory and procoagulant stimulus, exposed to either normal (N) or HG concentrations (5.5 and 50 mM respectively).ResultsHG upregulated TF expression, an effect abolished by BAY 11-7082, a NFκB inhibitor. C21 inhibited LPS-stimulated PCA, TFAg and mRNA to an extent independent of glucose concentration but the response to Olmesartan, an AT1R antagonist, was quite evidently potentiated by HG.ConclusionsHG stimulates LPS-induced TF expression through mechanisms completely dependent upon NFkB activation. Both AT2R-stimulation and AT1R-blockade downregulate inflammation-mediated procoagulant response in PBMCs but HG impacts differently on the two different signal transduction pathways.
“…Within the frame of reference summarized in the previous paragraph, our data confirm the inhibitory action of AT2R agonism by C21 on LPS-stimulated TF expression in human PBMCs [ 14 ] and the complete antagonism exerted by PD123,319 reassures about the specificity of the response. The parallel decrease of TFAg and mRNA is consonant with transcriptional modulation of TF protein likely by NFkB downregulation [ 41 ], an interpretation fully supported by recent reports showing attenuated cytokine production by C21 in LPS-stimulated monocytic cells [ 42 ].…”
Section: Discussionsupporting
confidence: 78%
“…[ 11 ] eligible for paracrine and/or intracrine stimulation. In agreement with that concept, AT1R antagonists downregulate activated TF expression [ 12 , 13 ] among others) but the inhibitory effect of selective AT2R stimulation [ 14 ] is suggestive of the existence of AT2Rs counteracting the AT1R-mediated procoagulant phenotype, as reported for other AT2R-mediated functions [ 15 ].…”
BackgroundIntimate links connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally generated Angiotensin (AT) II, the effector arm of the Renin Angiotensin System (RAS). C21, a selective AT2R agonist, downregulates the transcriptional expression of TF in LPS-activated peripheral blood mononuclear cell(PBMC)s implying the existence of ATII type 2 receptor (AT2R)s whose stimulation attenuates inflammation-mediated procoagulant responses. High glucose, by activating key signalling pathways and increasing the cellular content of RAS components, augments TF expression and potentiates the inhibitory effect of AT1R antagonists. It is unknown, however, the impact of that stimulus on AT2R-mediated TF inhibition, an information useful to understand more precisely the role of that signal transduction pathway in the inflammation-mediated coagulation process. TF antigen (ELISA), procoagulant activity (PCA, 1-stage clotting assay) and TF-mRNA (real-time polymerase chain reaction) were assessed in PBMCs activated by LPS, a pro-inflammatory and procoagulant stimulus, exposed to either normal (N) or HG concentrations (5.5 and 50 mM respectively).ResultsHG upregulated TF expression, an effect abolished by BAY 11-7082, a NFκB inhibitor. C21 inhibited LPS-stimulated PCA, TFAg and mRNA to an extent independent of glucose concentration but the response to Olmesartan, an AT1R antagonist, was quite evidently potentiated by HG.ConclusionsHG stimulates LPS-induced TF expression through mechanisms completely dependent upon NFkB activation. Both AT2R-stimulation and AT1R-blockade downregulate inflammation-mediated procoagulant response in PBMCs but HG impacts differently on the two different signal transduction pathways.
“…3, left panel). As a comparison, PCA in response to the peak hrGGT concentration (1.0 μg/mL) used in those studies was in the range obtained under identical experimental conditions by a well characterized procoagulant agonist such as LPS at a standard concentration of 0.1 μg/mL (from 0.01 ± 0.008 to 1.12 ± 0.2 AU) [13]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…As detailed elsewhere [13], leukocytes were isolated from fresh buffy coats diluted 1:1 with sodium citrate 0.38 % in saline solution, mixed gently with 0.5 volume of 2 % Dextran T500 and left for 40 min for erythrocyte sedimentation. The leukocyte-rich supernatant was recovered and centrifuged for 10 min at 200xg.…”
BackgroundBesides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the principal initiator of the clotting cascade. Thus, GGT might be involved in TF-mediated coagulation processes, an assumption untested insofar.MethodsExperiments were run with either equine, enzymatically active GGT or human recombinant (hr) GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation. TF Procoagulant Activity (PCA, one-stage clotting assay), TF antigen(ELISA) and TFmRNA(real-time PCR) were assessed in unpooled human peripheral blood mononuclear cell(PBMC) suspensions obtained from healthy donors through discontinuous Ficoll/Hystopaque density gradient.ResultsEquine GGT increased PCA, an effect insensitive to GGT inhibition by acivicin suggesting mechanisms independent of its enzymatic activity, a possibility confirmed by the maintained stimulation in response to hrGGT, an enzymatically inactive molecule. Endotoxin(LPS) contamination of GGT preparations was excluded by heat inactivation studies and direct determination(LAL method) of LPS concentrations <0.1 ng/mL practically devoid of procoagulant effect. Inhibition by anti-GGT antibodies corroborated that conclusion. Upregulation by hrGGT of TF antigen and mRNA and its downregulation by BAY-11-7082, a NFkB inhibitor, and N-acetyl-L-cysteine, an antioxidant, was consistent with a NFkB-driven, redox-sensitive transcriptional site of action.ConclusionsGGT upregulates TF expression independent of its enzymatic activity, a cytokine-like behaviour mediated by NFκB activation, a mechanism contributing to promote acute thrombotic events, a possibility in need, however, of further evaluation.
“…[ 30 ] PBMC cultures, to evaluate in vitro LPS-induced cytokine production, may be a better technology than measuring cytokines in the serum. [ 31 32 33 34 35 36 ] Cytokines in serum may be vulnerable to differential concentrations between the site of cytokine release and blood concentrations and the relatively short half-life of cytokines. The use of PBMC cultures complements the collection of peripheral cytokine measures.…”
There is mounting evidence that inflammation is a major factor in the pathophysiology of schizophrenia. Inflammatory status is commonly ascertained by measuring peripheral cytokine concentrations. An issue concerning research on inflammation and schizophrenia relates to assay methodology. Enzyme-linked immunosorbent assay (ELISA) is the most widely used and the gold standard method used to measure cytokine concentrations. ELISA has a number of limitations. Both ELISA and multiplex are limited by not being able to distinguish between bioactive and inactive molecules and the matrix and heterophilic (auto-) antibody interference. Multiplex assays when combined with gene expression analysis and flow cytometry techniques such as fluorescence-activated cell sorting may be useful to detect abnormalities in specific immune pathways. Peripheral blood mononuclear cells cultures, to evaluate in vitro lipopolysaccharide-induced cytokine production, may be a better technology than measuring cytokines in the serum. The purpose of this paper is to shed light on major methodological issues that need to be addressed in order to advance the study of cytokines in schizophrenia. We make a few recommendations on how to address these issues.
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