BACKGROUND: A close association links Gamma-GlutamylTransferase (GGT) activity to acute thrombotic events that coexisting diabetes increases exponentially. Tissue Factor (TF), the leading factor of acute thrombotic complications, is also coexpressed with GGT in atherosclerotic plaques raising the issue of a direct effect of GGT to TF activation, a possibility never tested insofar. AIM: To assess the effect of an enzymatically inactive human recombinant GGT on TF antigen (ag), TF mRNA and TF pro-coagulant activity (PCA) in human peripheral blood mononuclear cells. Experiments were conducted in both normal (10 mM) and high (HG, 50mM) glucose concentrations, the diabetic hallmark. METHODS: Human peripheral blood mononuclear cells obtained from healthy donors (discontinuous Ficoll/Hystopaque density gradient) were incubated with GGT (0.5ng/µl) either alone or with anti-GGT antibody (2.5µg/ml). Because of the pivotal role played by NFkB, a redox-sensitive transcription factor encoding TF, we also evaluated the effect NF-κB inhibition by BAY-11-7082 (10-5M) and N-acetylcysteine (10-3M), an antioxidant. TF PCA (1-stage clotting assay, arbitrary units), ag (ELISA, pg/mL) and mRNA (real-time PCR, normalized-fold expression compared to housekeeping genes) were the evaluation variables. RESULTS: In normal glucose conditions, GGT increased TF PCA, ag and mRNA, an effect inhibited by anti-GGT, a specific polyclonal antibody. HG amplified GGT-induced TF stimulation (PCA: from 0.4±0.3 to 4.3±3, n=27, p<0.001; mRNA: from 0.05±0.04 to 0.5±0.35, n=8, p<0.001; TFag: from 267±63 to 939±538, n=16, p<0.001 ). BAY-11-7082 (PCA: from 4.3±3 to 0.2±0.1, n=4, p<0.001; TF ag: from 939±538 to 41±38, n=16, p<0.001) and N-acetylcysteine (PCA: from 3.9±2.8 to 0.6±0.2, n=6, p<0.001; TFag: from 939±538 to 188±42, n=16, p<0.001) inhibited GGT-induced expression in normal glucose an effect more pronounced in HG conditions. CONCLUSIONS: GGT stimulates directly TF expression in human peripheral blood mononuclear cells through a NF-κB-mediated mechanism and HG amplifies that effect, potentially contributing to the atherothrombotic risk conferred by higher GGT levels, more markedly so in diabetic patients.
High glucose increases gamma-glutamyltransferase-induced tissue factor expression in human peripheral blood mononuclear cells
CIANCHETTI, SILVANA;CARNICELLI, VITTORIA;FAITA, FRANCESCA;NERI, TOMMASO;ZUCCHI, RICCARDO;CORTI, ALESSANDRO;CELI, ALESSANDRO;PEDRINELLI, ROBERTO
2016-01-01
Abstract
BACKGROUND: A close association links Gamma-GlutamylTransferase (GGT) activity to acute thrombotic events that coexisting diabetes increases exponentially. Tissue Factor (TF), the leading factor of acute thrombotic complications, is also coexpressed with GGT in atherosclerotic plaques raising the issue of a direct effect of GGT to TF activation, a possibility never tested insofar. AIM: To assess the effect of an enzymatically inactive human recombinant GGT on TF antigen (ag), TF mRNA and TF pro-coagulant activity (PCA) in human peripheral blood mononuclear cells. Experiments were conducted in both normal (10 mM) and high (HG, 50mM) glucose concentrations, the diabetic hallmark. METHODS: Human peripheral blood mononuclear cells obtained from healthy donors (discontinuous Ficoll/Hystopaque density gradient) were incubated with GGT (0.5ng/µl) either alone or with anti-GGT antibody (2.5µg/ml). Because of the pivotal role played by NFkB, a redox-sensitive transcription factor encoding TF, we also evaluated the effect NF-κB inhibition by BAY-11-7082 (10-5M) and N-acetylcysteine (10-3M), an antioxidant. TF PCA (1-stage clotting assay, arbitrary units), ag (ELISA, pg/mL) and mRNA (real-time PCR, normalized-fold expression compared to housekeeping genes) were the evaluation variables. RESULTS: In normal glucose conditions, GGT increased TF PCA, ag and mRNA, an effect inhibited by anti-GGT, a specific polyclonal antibody. HG amplified GGT-induced TF stimulation (PCA: from 0.4±0.3 to 4.3±3, n=27, p<0.001; mRNA: from 0.05±0.04 to 0.5±0.35, n=8, p<0.001; TFag: from 267±63 to 939±538, n=16, p<0.001 ). BAY-11-7082 (PCA: from 4.3±3 to 0.2±0.1, n=4, p<0.001; TF ag: from 939±538 to 41±38, n=16, p<0.001) and N-acetylcysteine (PCA: from 3.9±2.8 to 0.6±0.2, n=6, p<0.001; TFag: from 939±538 to 188±42, n=16, p<0.001) inhibited GGT-induced expression in normal glucose an effect more pronounced in HG conditions. CONCLUSIONS: GGT stimulates directly TF expression in human peripheral blood mononuclear cells through a NF-κB-mediated mechanism and HG amplifies that effect, potentially contributing to the atherothrombotic risk conferred by higher GGT levels, more markedly so in diabetic patients.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
