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A cooperation between bradykinin B2 and dopamine D2 receptors regulates neutrophil adhesion to endothelial cells
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Abstracts / Atherosclerosis 263 (2017) e29ee110
Conclusions: Our data demonstrated that blockade of RAGE activation by sRAGE attenuates Ang II-induced endothelial barrier permeability in vitro and in vivo.
SAG130. A COOPERATION BETWEEN BRADYKININ B2 AND DOPAMINE D2 RECEPTORS REGULATES NEUTROPHIL ADHESION TO ENDOTHELIAL CELLS Agnieszka Polit2, Anna LabedzAnna Niewiarowska-Sendo1, Maslowska3, Andrzej Kozik1, Ibeth Guevara-Lora1. 1 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Analytical Biochemistry, Krakow, Poland; 2 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Physical Biochemistry, Krakow, Poland; 3 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Krakow, Poland Aim: Leukocyte adhesion to vascular endothelial cells is a hallmark of inflammation. Biological action of bradykinin (B2R) and dopamine (D2R) receptors in these processes has been widely reported. Both receptors belong to the G-protein-coupled receptor superfamily within which functional and structural interactions were documented. The study was aimed to investigate a potential interaction between B2R and D2R and to demonstrate their cooperation during endothelial dysfunction. Methods: Colocalization of receptors were determined in HEK 293 cells transfected with plasmid vectors encoding B2R and D2R fused with fluorescent proteins with confocal microscopy. Dimer formation B2R-D2R was confirmed with FLIM-FRET technique. Adhesion test was performed to check neutrophil-endothelial cell binding. The expression of adhesion molecules and production of interleukin-8 was analyzed by flow cytometry and ELISA, respectively. Changes in NO production were studied using fluorescent assay. Results: Direct interaction of receptors was demonstrated and their colocalization in the cell membrane was modified by stimulation with B2R and D2R agonists. Neutrophil adhesion to endothelial cells was also regulated by agonist treatment. These effects were accompanied by changes in expression of vascular adhesion molecules, especially E-selectin and ICAM1, and interleukin-8 release. The impact of such interaction on NO concentration was monitored. Conclusions: The results indicate the cooperation between B2R and D2R, causing changes in neutrophil adhesion to endothelial cells. It is supposed that this interaction may be responsible for controlling of NO-mediated signaling pathway during enhanced inflammatory response. These observations contribute to a better understanding of several vascular pathologies, especially associated with inflammatory disorders like atherosclerosis.
SAG131. ENDOTHELIAL MICRORNAS EPIGENETICALLY CONTROL VASCULAR INFLAMMATION AND THROMBOGENICITY IN DIABETES Marco Witkowski1, Alice Weithauser1, Termeh Tabaraie1, Daniel Steffens1, Julian Friebel1, Bernd Stratmann2, Diethelm Tschoepe2, Ulf €tsmedizin, Berlin, e Universita Landmesser1, Ursula Rauch1. 1 Charit Germany; 2 Heart and Diabetes Institute Bad Oeyenhausen, Bad Oeyenhause, Germany Aim: Diabetes mellitus promotes vascular inflammatory processes and is a main risk factor for atherosclerosis and cardiovascular mortality. Under diabetic conditions, expression of tissue factor (TF) is up-regulated leading to sustained vascular inflammation and a pro-thormbotic state. Moreover, impaired glycemic control in diabetes down-regulates endothelial microRNA(miR)s, e.g. miR-126. Here, we sought to investigate the role of microRNA-mediated control of TF expression and procoagulability in diabetes. Methods: Plasma samples of patients with diabetes were analyzed for TF protein and activity as well as expression of microRNA 19a and microRNA126 prior and after optimization of the anti-diabetic treatment. Moreover, human microvascular endothelial cells (HMEC) and monocytes (THP-1)
were transfected with a miR-126 mimic or a control-miR. TF expression and procoagulability were then assessed. Finally, a luciferase assay was performed to confirm the direct binding of the candidate microRNAs to the TF 3'UTR. Results: In the cohort of patients with diabetes high miR-19a and low miR126 levels were associated with markedly reduced TF protein and TFmediated procoagulability. We found plasma miR-19a to strongly correlate with miR-126 (r¼ 0.92, p<0.0001). Both miRs directly bind the TF transcript und thereby downregulate TF-expression and FXa generation in HMEC and THP. With optimization of the anti-diabetic treatment, miR-126 and miR-19a levels increased and the procoagulability was reduced. Conclusions: Circulating miR-126 and miR-19a exhibit anti-thrombotic properties via regulating post-transcriptional TF expression. Reduced expression therefore contribute to vascular inflammation and coagulability in diabetes. Application of both miRs may provide new therapeutic options for patients with diabetes.
SAG132. THE EFFECT OF FACTOR XA AND THROMBIN ON ICAM-1 EXPRESSION ON ENDOTHELIAL PROGENITOR CELLS. THE ROLE OF PAR-1 Styliani Papadaki1, Sofia Sidiropoulou1, Vasileios Chantzichristos1, Minas 1 Alexandros Tselepis1. University of Ioannina, Paschopoulos2, Atherothrombosis Research Centre/Laboratory of Biochemistry, Department of Chemistry, Ioannina, Greece; 2 University of Ioannina, Department of Obstetrics and Gynecology, School of Medicine, Ioannina, Greece Aim: Factor Xa (FXa) and thrombin are important coagulation proteases which also induce cellular pleiotropic effects that are primarily mediated through the protease activated receptors (PARs). PARs are important for thrombin- and FXa-induced cellular activation. We investigated the effect of FXa and thrombin on the membrane expression of adhesion molecule ICAM-1 on CD34+-derived late-outgrowth endothelial cells (OECs) and human umbilical vein endothelial cells (HUVECs). The role of the selective PAR-1 antagonist voraxapar was also investigated. Methods: CD34+ cells were isolated from cord blood mononuclear cells using human CD34 Microbead Kit and appropriately cultured for the formation of OECs. HUVECs were purchased from Lonza. Confluent OECs (passage 4) and HUVECs (passage 3) were incubated for various time intervals up to 24h with 25-100nM FXa or 1-8U/mL thrombin. The effect of FXa- and thrombin-induced ICAM-1 expression (CD54-PE) and the possible inhibitory effect of vorapaxar were evaluated in both cell types using flow cytometry. Results: FXa and thrombin increased ICAM-1 expression dose- and timedependently. The maximum increase of ICAM-1 expression (76.5±5.0% on OECs, 66.7±12.3% on HUVECs) was observed at 50nM FXa after 24h-incubation. The maximum increase of ICAM-1 expression (2.4±0.6 on OECs, 4.5±1.5 times on HUVECs) was observed at 8U/mL thrombin after 24hincubation. Vorapaxar inhibited both FXa- and thrombin-induced ICAM-1 expression on OECs (IC50: 0.3nM and 2.9nM) and HUVECs (IC50: 0.8nM and 1.0nM respectively). Conclusions: FXa and thrombin are mediators of ICAM-1 expression on endothelial progenitor cells, mainly through PAR-1 signalling, playing a key role in inflammation and atherosclerosis.
SAG133. RICE BRAN ENZYMATIC EXTRACT, A SOURCE OF FERULIC ACID, PROTECTS ENDOTHELIAL FUNCTION AND INHIBITS NADPHOX ACTIVITY 2, Juan Parrado3, Maria Jose rez Ternero1, Alba Macia Cristina Pe Maria Alvarez de Sotomayor1, Maria Dolores Motilva2, Herrera1. 1 Department of Pharmacology, School of Pharmacy, University of Seville, Seville, Spain; 2 Food Technology Department, Agrotecnio Center, ria, University of Lleida, Lleida, Escola T ecnica Superior d'Enginyeria Agra Spain; 3 Department of Biochemistry, School of Pharmacy, University of Seville, Seville, Spain Aim: Rice bran is an excellent source of ferulic acid, a well-known antioxidant. Increased NADPHox activity promotes atherogenesis and
Abstracts / Atherosclerosis 263 (2017) e29ee110
Conclusions: Our data demonstrated that blockade of RAGE activation by sRAGE attenuates Ang II-induced endothelial barrier permeability in vitro and in vivo.
SAG130. A COOPERATION BETWEEN BRADYKININ B2 AND DOPAMINE D2 RECEPTORS REGULATES NEUTROPHIL ADHESION TO ENDOTHELIAL CELLS Agnieszka Polit2, Anna LabedzAnna Niewiarowska-Sendo1, Maslowska3, Andrzej Kozik1, Ibeth Guevara-Lora1. 1 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Analytical Biochemistry, Krakow, Poland; 2 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Physical Biochemistry, Krakow, Poland; 3 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Krakow, Poland Aim: Leukocyte adhesion to vascular endothelial cells is a hallmark of inflammation. Biological action of bradykinin (B2R) and dopamine (D2R) receptors in these processes has been widely reported. Both receptors belong to the G-protein-coupled receptor superfamily within which functional and structural interactions were documented. The study was aimed to investigate a potential interaction between B2R and D2R and to demonstrate their cooperation during endothelial dysfunction. Methods: Colocalization of receptors were determined in HEK 293 cells transfected with plasmid vectors encoding B2R and D2R fused with fluorescent proteins with confocal microscopy. Dimer formation B2R-D2R was confirmed with FLIM-FRET technique. Adhesion test was performed to check neutrophil-endothelial cell binding. The expression of adhesion molecules and production of interleukin-8 was analyzed by flow cytometry and ELISA, respectively. Changes in NO production were studied using fluorescent assay. Results: Direct interaction of receptors was demonstrated and their colocalization in the cell membrane was modified by stimulation with B2R and D2R agonists. Neutrophil adhesion to endothelial cells was also regulated by agonist treatment. These effects were accompanied by changes in expression of vascular adhesion molecules, especially E-selectin and ICAM1, and interleukin-8 release. The impact of such interaction on NO concentration was monitored. Conclusions: The results indicate the cooperation between B2R and D2R, causing changes in neutrophil adhesion to endothelial cells. It is supposed that this interaction may be responsible for controlling of NO-mediated signaling pathway during enhanced inflammatory response. These observations contribute to a better understanding of several vascular pathologies, especially associated with inflammatory disorders like atherosclerosis.
SAG131. ENDOTHELIAL MICRORNAS EPIGENETICALLY CONTROL VASCULAR INFLAMMATION AND THROMBOGENICITY IN DIABETES Marco Witkowski1, Alice Weithauser1, Termeh Tabaraie1, Daniel Steffens1, Julian Friebel1, Bernd Stratmann2, Diethelm Tschoepe2, Ulf €tsmedizin, Berlin, e Universita Landmesser1, Ursula Rauch1. 1 Charit Germany; 2 Heart and Diabetes Institute Bad Oeyenhausen, Bad Oeyenhause, Germany Aim: Diabetes mellitus promotes vascular inflammatory processes and is a main risk factor for atherosclerosis and cardiovascular mortality. Under diabetic conditions, expression of tissue factor (TF) is up-regulated leading to sustained vascular inflammation and a pro-thormbotic state. Moreover, impaired glycemic control in diabetes down-regulates endothelial microRNA(miR)s, e.g. miR-126. Here, we sought to investigate the role of microRNA-mediated control of TF expression and procoagulability in diabetes. Methods: Plasma samples of patients with diabetes were analyzed for TF protein and activity as well as expression of microRNA 19a and microRNA126 prior and after optimization of the anti-diabetic treatment. Moreover, human microvascular endothelial cells (HMEC) and monocytes (THP-1)
were transfected with a miR-126 mimic or a control-miR. TF expression and procoagulability were then assessed. Finally, a luciferase assay was performed to confirm the direct binding of the candidate microRNAs to the TF 3'UTR. Results: In the cohort of patients with diabetes high miR-19a and low miR126 levels were associated with markedly reduced TF protein and TFmediated procoagulability. We found plasma miR-19a to strongly correlate with miR-126 (r¼ 0.92, p<0.0001). Both miRs directly bind the TF transcript und thereby downregulate TF-expression and FXa generation in HMEC and THP. With optimization of the anti-diabetic treatment, miR-126 and miR-19a levels increased and the procoagulability was reduced. Conclusions: Circulating miR-126 and miR-19a exhibit anti-thrombotic properties via regulating post-transcriptional TF expression. Reduced expression therefore contribute to vascular inflammation and coagulability in diabetes. Application of both miRs may provide new therapeutic options for patients with diabetes.
SAG132. THE EFFECT OF FACTOR XA AND THROMBIN ON ICAM-1 EXPRESSION ON ENDOTHELIAL PROGENITOR CELLS. THE ROLE OF PAR-1 Styliani Papadaki1, Sofia Sidiropoulou1, Vasileios Chantzichristos1, Minas 1 Alexandros Tselepis1. University of Ioannina, Paschopoulos2, Atherothrombosis Research Centre/Laboratory of Biochemistry, Department of Chemistry, Ioannina, Greece; 2 University of Ioannina, Department of Obstetrics and Gynecology, School of Medicine, Ioannina, Greece Aim: Factor Xa (FXa) and thrombin are important coagulation proteases which also induce cellular pleiotropic effects that are primarily mediated through the protease activated receptors (PARs). PARs are important for thrombin- and FXa-induced cellular activation. We investigated the effect of FXa and thrombin on the membrane expression of adhesion molecule ICAM-1 on CD34+-derived late-outgrowth endothelial cells (OECs) and human umbilical vein endothelial cells (HUVECs). The role of the selective PAR-1 antagonist voraxapar was also investigated. Methods: CD34+ cells were isolated from cord blood mononuclear cells using human CD34 Microbead Kit and appropriately cultured for the formation of OECs. HUVECs were purchased from Lonza. Confluent OECs (passage 4) and HUVECs (passage 3) were incubated for various time intervals up to 24h with 25-100nM FXa or 1-8U/mL thrombin. The effect of FXa- and thrombin-induced ICAM-1 expression (CD54-PE) and the possible inhibitory effect of vorapaxar were evaluated in both cell types using flow cytometry. Results: FXa and thrombin increased ICAM-1 expression dose- and timedependently. The maximum increase of ICAM-1 expression (76.5±5.0% on OECs, 66.7±12.3% on HUVECs) was observed at 50nM FXa after 24h-incubation. The maximum increase of ICAM-1 expression (2.4±0.6 on OECs, 4.5±1.5 times on HUVECs) was observed at 8U/mL thrombin after 24hincubation. Vorapaxar inhibited both FXa- and thrombin-induced ICAM-1 expression on OECs (IC50: 0.3nM and 2.9nM) and HUVECs (IC50: 0.8nM and 1.0nM respectively). Conclusions: FXa and thrombin are mediators of ICAM-1 expression on endothelial progenitor cells, mainly through PAR-1 signalling, playing a key role in inflammation and atherosclerosis.
SAG133. RICE BRAN ENZYMATIC EXTRACT, A SOURCE OF FERULIC ACID, PROTECTS ENDOTHELIAL FUNCTION AND INHIBITS NADPHOX ACTIVITY 2, Juan Parrado3, Maria Jose rez Ternero1, Alba Macia Cristina Pe Maria Alvarez de Sotomayor1, Maria Dolores Motilva2, Herrera1. 1 Department of Pharmacology, School of Pharmacy, University of Seville, Seville, Spain; 2 Food Technology Department, Agrotecnio Center, ria, University of Lleida, Lleida, Escola T ecnica Superior d'Enginyeria Agra Spain; 3 Department of Biochemistry, School of Pharmacy, University of Seville, Seville, Spain Aim: Rice bran is an excellent source of ferulic acid, a well-known antioxidant. Increased NADPHox activity promotes atherogenesis and