Gabexate mesilate, a synthetic anticoagulant, inhibits the expression of endothelial leukocyte adhesion molecules in vitro.
OBJECTIVE
Gabexate mesilate, a synthetic protease inhibitor, has been shown to reduce endotoxin-induced pulmonary vascular injury in an animal model of sepsis by inhibiting leukocyte activation. We examined whether gabexate mesilate inhibits tumor necrosis factor-alpha-induced expression of leukocyte adhesion molecules in cultured endothelial cells.


METHODS
Prospective, randomized, controlled study.


METHODS
Research laboratory at a university medical center.


METHODS
Cultured human umbilical vein endothelial cell (HUVECs).


METHODS
HUVECs were stimulated with tumor necrosis factor-alpha or lipopolysaccharide in the presence or absence of gabexate mesilate. Expression of E-selectin and intercellular adhesion molecule-1 was measured by cellular enzyme-linked immunosorbent assay. Messenger RNA levels of E-selectin and intercellular adhesion molecule-1 were determined by reverse transcription-polymerase chain reaction. DNA-binding activity of p65 in the nuclear extracts was evaluated by enzyme-linked immunosorbent assay. Nuclear translocation of nuclear factor-kappaB induced by tumor necrosis factor-alpha was evaluated by immunocytostaining and Western blot analysis. Degradation and phosphorylation of inhibitor of nuclear factor-kappaB (IkappaB) induced by tumor necrosis factor-alpha were evaluated by Western blot analysis.


RESULTS
Gabexate mesilate inhibited the tumor necrosis factor-alpha-induced increases in the endothelial expression of E-selectin and intercellular adhesion molecule-1 by inhibiting the transcription. Tumor necrosis factor-alpha-induced increase in DNA binding of p65 was inhibited by gabexate mesilate through inhibition of the nuclear translocation of p65. Gabexate mesilate inhibited the tumor necrosis factor-alpha-induced degradation of IkappaBalpha, an inhibitor of nuclear factor-kappaB, by inhibiting phosphorylation of IkappaBalpha in HUVECs.


CONCLUSIONS
Gabexate mesilate inhibited the expression of leukocyte adhesion molecules by inhibiting the nuclear factor-kappaB-mediated transcription in HUVECs. Inhibition of nuclear factor-kappaB activation by gabexate mesilate could be explained by inhibition of degradation of IkappaB. Gabexate mesilate might reduce lipopolysaccharide-induced pulmonary vascular injury not only by inhibiting monocytic tumor necrosis factor-alpha production but by inhibiting the expression of endothelial leukocyte adhesion molecules.
Uchiba
Mitsuhiro
M
Department of Laboratory Medicine, Kumamoto University School of Medicine, Kumamoto, Japan.
Okajima
Kenji
K
Kaun
Christoph
C
Binder
Bernd R
BR
Wojta
Johann
J
D000925:Anticoagulants / Q000494:pharmacology*; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D019040:E-Selectin / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D016670:Gabexate / Q000494:pharmacology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins
D000925:Anticoagulants; D015815:Cell Adhesion Molecules; D019040:E-Selectin; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D015842:Serine Proteinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D016670:Gabexate