by Satoshi Kokura, Robert E. Wolf, Toshikazu Yoshikawa, D

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Presentation transcript:

Molecular Mechanisms of Neutrophil-Endothelial Cell Adhesion Induced by Redox Imbalance by Satoshi Kokura, Robert E. Wolf, Toshikazu Yoshikawa, D. Neil Granger, and Tak Yee Aw Circulation Research Volume 84(5):516-524 March 19, 1999 Copyright © American Heart Association, Inc. All rights reserved.

Time course change of GSH and GSSG-to-GSH ratio in HUVECs exposed to 60 minutes of anoxia followed by 4 hours of reoxygenation. Time course change of GSH and GSSG-to-GSH ratio in HUVECs exposed to 60 minutes of anoxia followed by 4 hours of reoxygenation. Each value indicates mean±SEM of 4 experiments. *P<0.05 and **P<0.01 vs control, preanoxic cells (ie, −60 minutes). Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.

Time course of change in GSH (top) and GSSG-to-GSH ratio (bottom) in HUVECs treated with individual or combinations of chemical thiol agents. Time course of change in GSH (top) and GSSG-to-GSH ratio (bottom) in HUVECs treated with individual or combinations of chemical thiol agents. Each value indicates mean±SEM of 4 experiments. *P<0.05 and **P<0.01 vs 0 minutes. Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.

Time course of neutrophil adherence to HUVECs after redox alteration. Time course of neutrophil adherence to HUVECs after redox alteration. A, HUVECs treated with diamide plus BSO. B, HUVECs treated with diamide alone. C, HUVECs treated with DEM plus BSO. 51Cr-labeled neutrophils were added to HUVEC monolayers after removal of media that contained thiol agents, and neutrophil adherence was determined 30 minutes later. Each value indicates mean±SEM of 5 experiments performed in triplicate. *P<0.05, **P<0.01, ***P<0.001 vs untreated controls. Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.

Effect of NAC on neutrophil adherence to HUVECs in phase 1 and phase 2 after diamide plus BSO incubation. Effect of NAC on neutrophil adherence to HUVECs in phase 1 and phase 2 after diamide plus BSO incubation. Responses are shown for neutrophil adhesion at 15 minutes (phase 1) and 240 minutes (phase 2) after redox change. Each value indicates mean±SEM of 5 experiments performed in duplicate. *P<0.001 vs untreated controls; #P<0.01 vs diamide plus BSO treatment. Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.

A and B, Relationship between phase 1 (15 minutes) neutrophil adherence and GSH level or GSSG/GSH ratio, respectively. A and B, Relationship between phase 1 (15 minutes) neutrophil adherence and GSH level or GSSG/GSH ratio, respectively. C and D, Relationship between phase 2 (240 minutes) neutrophil adherence and GSH level or GSSG/GSH ratio, respectively. Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.

Effect of MAbs to P-selectin (PB1 Effect of MAbs to P-selectin (PB1.3, 20 μg/mL), E-selectin (CL3, 20 μg/mL), and ICAM-1 (8.4A6, 20 μg/mL) on neutrophil adherence to HUVECs in phase 1 and phase 2 after diamide plus BSO incubation. Effect of MAbs to P-selectin (PB1.3, 20 μg/mL), E-selectin (CL3, 20 μg/mL), and ICAM-1 (8.4A6, 20 μg/mL) on neutrophil adherence to HUVECs in phase 1 and phase 2 after diamide plus BSO incubation. Responses are shown for neutrophil adhesion at 15 minutes (phase 1) and 240 minutes (phase 2) after redox change. Each value indicates mean±SEM of 4 experiments performed in triplicate. *P<0.001 vs untreated controls (open bars); #P<0.05 vs diamide plus BSO treatment (solid bars). Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.

Surface expression of ECAMs [P-selectin (A), E-selectin (B), and ICAM-1 (C)] in HUVECs incubated with diamide plus BSO (solid bars). Surface expression of ECAMs [P-selectin (A), E-selectin (B), and ICAM-1 (C)] in HUVECs incubated with diamide plus BSO (solid bars). Each value indicates mean±SEM of 4 experiments performed in triplicate. *P<0.01 vs untreated controls (open bars). Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.

Effect of inhibitors of translation (CHX) or transcription (ActD) on neutrophil adherence to HUVECs in phase 1 and phase 2 after diamide plus BSO incubation. Effect of inhibitors of translation (CHX) or transcription (ActD) on neutrophil adherence to HUVECs in phase 1 and phase 2 after diamide plus BSO incubation. Responses are shown for neutrophil adhesion at 15 minutes (phase 1) and 240 minutes (phase 2) after redox change. Each value indicates mean±SEM of 4 experiments performed in triplicate. *P<0.05 vs untreated controls (open bars); #P<0.05 vs diamide plus BSO treatment (solid bars). Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.

Effect of inhibitors of NF-κB or AP-1 on neutrophil adherence to HUVECs in phase 2 after diamide plus BSO incubation. Effect of inhibitors of NF-κB or AP-1 on neutrophil adherence to HUVECs in phase 2 after diamide plus BSO incubation. A, Effect of MG132 (for NF-κB) or 3AB (for AP-1). B, Effect of κB or AP-1 ds-oligonucleotide decoys. Responses are shown for neutrophil adhesion at 240 minutes (phase 2) after redox change. Each value indicates mean±SEM of 3 or 4 experiments performed in triplicate. *P<0.05 vs untreated controls (open bars); #P<0.05 vs diamide plus BSO treatment (solid bars). Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.

Effect of κB or AP-1 ds-oligonucleotides on diamide plus BSO–induced surface expression of ECAMs [P-selectin (A), E-selectin (B), and ICAM-1 (C)]. Effect of κB or AP-1 ds-oligonucleotides on diamide plus BSO–induced surface expression of ECAMs [P-selectin (A), E-selectin (B), and ICAM-1 (C)]. Non-κB and non–AP-1 represent the respective nonbinding oligonucleotides. Each value indicates mean±SEM of 4 experiments performed in triplicate. *P<0.05 vs untreated controls (open bars); #P<0.05 vs diamide plus BSO treatment (solid bars). Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.

EMSA on nuclear extracts from HUVECs incubated with diamide plus BSO in the presence or absence of κB ds-oligonucleotide. EMSA on nuclear extracts from HUVECs incubated with diamide plus BSO in the presence or absence of κB ds-oligonucleotide. The NF-κB-specific adducts are indicated by bold arrow. Satoshi Kokura et al. Circ Res. 1999;84:516-524 Copyright © American Heart Association, Inc. All rights reserved.