Fibrinogen-induced endothelin-1 production from endothelial cells.
We previously demonstrated that fibrinogen (Fg) binding to the vascular endothelial intercellular adhesion molecule-1 (ICAM-1) leads to microvascular constriction in vivo and in vitro. Although a role of endothelin-1 (ET-1) in this Fg-induced vasoconstriction was suggested, the mechanism of action was not clear. In the current study, we tested the hypothesis that Fg-induced vasoconstriction results from ET-1 production by vascular endothelial cells (EC) and is mediated by activation of extracellular signal-regulated kinase -1/2 (ERK-1/2). Confluent, rat heart microvascular endothelial cells (RHMECs) were treated with one of the following: Fg (2 or 4 mg/ml), Fg (4 mg/ml) with ERK-1/2 kinase inhibitors (PD-98059 or U-0126), Fg (4 mg/ml) with an antibody against ICAM-1, or medium alone for 45 min. The amount of ET-1 formed and the concentration of released von Willebrand factor (vWF) in the cell culture medium were measured by ELISAs. Fg-induced exocytosis of Weibel-Palade bodies (WPBs) was assessed by immunocytochemistry. Phosphorylation of ERK-1/2 was detected by Western blot analysis. Fg caused a dose-dependent increase in ET-1 formation and release of vWF from the RHMECs. This Fg-induced increase in ET-1 production was inhibited by specific ERK-1/2 kinase inhibitors and by anti-ICAM-1 antibody. Immunocytochemical staining showed that an increase in Fg concentration enhanced exocytosis of WPBs in ECs. A specific endothelin type B receptor blocker, BQ-788, attenuated the enhanced phosphorylation of ERK-1/2 in ECs caused by increased Fg content in the culture medium. The presence of an endothelin converting enzyme inhibitor, SM-19712, slightly decreased Fg-induced phosphorylation of ERK-1/2, but inhibited production of Fg-induced ET-1 production. These results suggest that Fg-induced vasoconstriction may be mediated, in part, by activation of ERK-1/2 signaling and increased production of ET-1 that further increases EC ERK-1/2 signaling. Thus, an increased content of Fg may enhance vasoconstriction through increased production of ET-1.
Sen
Utpal
U
Dept. of Physiology, Univ. of Louisville, School of Medicine, Bldg. A, Rm. 1115, 500 South Preston St., Louisville, KY 40202, USA.
Tyagi
Neetu
N
Patibandla
Phani K
PK
Dean
William L
WL
Tyagi
Suresh C
SC
Roberts
Andrew M
AM
Lominadze
David
D
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002070:Butadienes / Q000494:pharmacology; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1 / Q000378:metabolism*; D000072396:Endothelin-Converting Enzymes; D005089:Exocytosis; D005340:Fibrinogen / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D055806:Microvessels / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D044023:Receptor, Endothelin B / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013453:Sulfonylurea Compounds / Q000494:pharmacology; D014661:Vasoconstriction; D020302:Weibel-Palade Bodies / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
D002070:Butadienes; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1; D005419:Flavonoids; D009570:Nitriles; D009842:Oligopeptides; D010880:Piperidines; D047428:Protein Kinase Inhibitors; D044023:Receptor, Endothelin B; C419789:SM 19712; D013449:Sulfonamides; D013453:Sulfonylurea Compounds; C113580:U 0126; D014841:von Willebrand Factor; D018799:Intercellular Adhesion Molecule-1; C086539:BQ 788; D005340:Fibrinogen; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one