Inhibition of type-1 plasminogen activator inhibitor production by antisense oligonucleotides in human vascular endothelial and smooth muscle cells.
We have hypothesized that type-1 plasminogen activator inhibitor (PAI-1) may exacerbate accumulation of extracellular matrix in atheroma by inhibiting local generation of plasmin and intramural proteolysis. Thus, suppression of PAI-1 expression would decrease atherogenesis. To inhibit expression of PAI-1 in cultured human umbilical vein endothelial and aortic smooth muscle cells, a 20-base antisense phosphorothioate oligonucleotide targeting specific sequences in the 3'-untranslated region of the PAI-1 gene was used. Studies with 32P-labeled oligomers verified stability in media. Secretion of PAI-1 protein assayed by enzyme-linked immunosorbent assay declined specifically and dose-dependently in cells exposed to the antisense oligonucleotide treated under basal conditions and after stimulation of PAI-1 expression with transforming growth factor beta (0.5 ng/ml for endothelial cell, 5 ng/ml for smooth muscle cell). Inhibition of expression was confirmed by immunoprecipitation of 35S-labeled PAI-1 and was paralleled by decreased steady-state levels of PAI-1 mRNA (Northern blots). Decreased PAI-1 synthesis was accompanied by augmentation of cell-associated plasmin activity. Thus, the antisense oligonucleotide down-regulated PAI-1 elaboration, an approach that may be useful in limiting obstructive vascular lesions.
Sawa
H
H
Cardiovascular Division, Washington University, St. Louis, Missouri 63110.
Sobel
B E
BE
Fujii
S
S
D001483:Base Sequence; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism
D016376:Oligonucleotides, Antisense; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D005341:Fibrinolysin