Protein kinase C alpha expression is required for heparin inhibition of rat smooth muscle cell proliferation in vitro and in vivo.
Heparin is a complex glycosaminoglycan that inhibits vascular smooth muscle cell (SMC) growth in vitro and in vivo. To define the mechanism by which heparin exerts its antiproliferative effects, we asked whether heparin interferes with the activity of intracellular protein kinase C (PKC). The membrane-associated intracellular PKC activity increased following stimulation of cultured rat SMCs with fetal calf serum and was suppressed by heparin in a time- and dose-dependent manner. Heparin acted through a selective inhibition of the PKC-alpha since preincubation of the cells with a 20-mer phosphorothioate PKC-alpha antisense oligodeoxynucleotide (ODN) eliminated the heparin effect. In vivo, following balloon injury of the rat carotid artery, particulate fraction PKC content increased with a time course and to an extent comparable with the observed changes in vitro. Heparin, administered at the time of injury or shortly thereafter, inhibited the activity of the particulate PKC and suppressed the in situ phosphorylation of an 80-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS), a substrate of PKC. The topical application of the phosphorothioate antisense ODN selectively suppressed the expression of the PKC-alpha isoenzyme in vivo but did not affect injury-induced myointimal proliferation. Topical application of the ODN also eliminated the antiproliferative activity of heparin. These results therefore suggest that heparin might block SMC proliferation by interfering with the PKC pathway through a selective direct inhibition of the PKC-alpha isoenzyme.
Herbert
J M
JM
Sanofi Recherche, 195 Route d'Espagne, 31036 Toulouse Cedex, France.
Clowes
M
M
Lea
H J
HJ
Pascal
M
M
Clowes
A W
AW
D000818:Animals; D002339:Carotid Arteries / Q000166:cytology / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation; D006493:Heparin / Q000494:pharmacology*; D019763:Heparin Lyase; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011133:Polysaccharide-Lyases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D017207:Rats, Sprague-Dawley
D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D006493:Heparin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase