Stimulation of PAI-1 expression in endothelial cells by cultured vascular smooth muscle cells.
Regulation of endothelial cell (EC) plasminogen activator inhibitor type-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (TPA) and urokinase-type plasminogen activator (UPA), by various stimuli has been well characterized. We report the upregulation of secreted and intracellular PAI-1 in human umbilical ECs when cocultured with human smooth muscle cells (SMCs) on amniotic membranes or incubated with SMC conditioned medium (CM) under serum-free conditions as determined by enzyme-linked immunosorbent assay. Cocultured human umbilical vein ECs and SMCs, or human umbilical artery ECs and SMCs, displayed a 73% and 68% increase, respectively, in released PAI-1. SMC-derived stimulatory factor release showed tissue specificity, since only human aortic, umbilical vein, and umbilical artery SMCs upregulated PAI-1 synthesis, whereas SMCs from human mammary artery, pulmonary artery, and saphenous vein did not. Stimulation of EC PAI-1 by SMC CM was both time and concentration dependent, with as much as five- and fourfold increases in supernatants and lysates, respectively. PAI-1 synthesis and activity in ECs from other vascular beds were also upregulated by SMC CM. Northern blot analysis paralleled the protein results, showing as much as a 2.7-fold increase in specific EC PAI-1 mRNA expression after incubation with SMC CM for 8 hours. PAI-1 stimulatory activity in SMC CM was completely abolished by boiling or incubation with protamine sulfate and was reduced by transient acidification or heparin-Sepharose pretreatment by 33% or 48%, respectively. The stimulatory factor(s) appeared to have a molecular mass of 23 kD as determined by gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)
Gallicchio
M
M
Department of Diagnostic Haematology, Royal Melbourne Hospital, Victoria, Australia.
Argyriou
S
S
Ianches
G
G
Filonzi
E L
EL
Zoellner
H
H
Hamilton
J A
JA
McGrath
K
K
Wojta
J
J
D002450:Cell Communication; D002478:Cells, Cultured; D002850:Chromatography, Gel; D017077:Culture Media, Conditioned; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000032:analysis; D006801:Humans; D007375:Interleukin-1 / Q000032:analysis; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
D017077:Culture Media, Conditioned; D007375:Interleukin-1; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016222:Fibroblast Growth Factor 2