Effects of lipopolysaccharide on the expression of fibrinolytic factors in an established cell line from human endothelial cells.
Human endothelial cells express antithrombotic properties by producing prostacyclin, heparan sulphate and plasminogen activator (PA). Bacterial extract, such as lipopolysaccharide (LPS), damaged the blood vessels and destroyed the balance between the antithrombotic and thrombotic functions of endothelial cells. The fibrinolytic system is involved in antithrombotic functions. The TKM-33 cell line was established from human endothelial cells. In order to determine whether TKM-33 is a good fibrinolytic system endothelial cell expression model, the expression of fibrinolytic factors in TKM-33 cells treated with or without LPS was studied. The endothelial cells which had not been treated with LPS produced and secreted a large amount of urokinase-type PA (u-PA), and small amounts of tissue-type PA (t-PA) and PA inhibitor-1 (PAI-1), which were identified immunohistochemically and by electrophoretic enzymography. Diisopropylfluorophosphate-treated 125I-u-PA bound specifically to acid-treated monolayered endothelial cells with a Kd of 2.83 +/- 0.61 nM, and Bmax of (0.11 +/- 0.01) x 10(6) sites/cell. u-PAR expression was detected in endothelial cells by Northern blot analysis. Thus, endothelial cells was shown to express u-PAR which binds u-PA specifically. In the binding assay, the stimulation of endothelial cells with 0.1, 1.0 and 10 micrograms/ml of LPS altered the Kd values to 6.04 +/- 0.71, 7.03 +/- 1.55 and 7.38 +/- 1.03 nM, respectively. However the Bmax values did not change significantly. Although LPS treatment increased u-PAR expression in endothelial cells in a dose-dependent manner, the expression of u-PA and t-PA mRNAs was not altered significantly. LPS stimulation (10 micrograms/ml) increased the expression of PAI-1 mRNA, significantly. The PA activity recovered from the cell surface fraction was not affected by LPS stimulation, but the PAI-1 activity was increased. These findings suggest that the established endothelial cell line, TKM-33, possesses the characteristics of endothelial cells and they express u-PAR on their cell surface, which is occupied by intrinsic u-PA secreted from the cells, and that treatment of endothelial cells with LPS changes the cell surface characteristics and inhibited the u-PAR expression thus promoting the prothrombotic function concomitantly with increased PAI-1 activity.
Matsumoto
H
H
Department of Physiology, Kinki University School of Medicine, Osakasayama, Japan.
Ueshima
S
S
Fukao
H
H
Mitsui
Y
Y
Matsuo
O
O
D001667:Binding, Competitive; D015152:Blotting, Northern; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology*; D010960:Plasminogen Activators / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D010960:Plasminogen Activators