Protein Kinase C α Expression Is Required for Heparin Inhibition of Rat Smooth Muscle Cell Proliferation in Vitro and in Vivo

Jean-Marc Herbert,Monika Clowes,Holly J Lea,Marc Pascal,Alexander W Clowes

doi:10.1074/jbc.271.42.25928

Abstract

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.

Highlights

Proliferation of vascular smooth muscle cells (SMC)1 plays an important role in the pathogenesis of atherosclerosis [1] and is an important contributor to the formation of the fibrocellular lesion following surgical intervention such as percutaneous transluminal coronary angioplasty or bypass graft [2]
To understand better the mechanism by which heparin inhibits SMC proliferation, we asked whether heparin modulated the activity of protein kinase C (PKC) in vitro in cultured rat SMC or in vivo in medial SMC of rat carotid arteries subjected to balloon injury
Heparin and heparan sulfate are structurally complex glycosaminoglycans composed of repeating disaccharide units of alternating glucosamine and uronic acid sugars

Summary

Introduction

Proliferation of vascular smooth muscle cells (SMC) plays an important role in the pathogenesis of atherosclerosis [1] and is an important contributor to the formation of the fibrocellular lesion following surgical intervention such as percutaneous transluminal coronary angioplasty or bypass graft [2]. Heparin inhibits the migration and the proliferation of the cells in response to vascular damage [3]. Heparin inhibits proliferation and promotes the induction of a contractile phenotype in cultured SMCs (4 – 6). Heparin has been demonstrated to act selectively on a PKC-dependent pathway [7, 10, 11], and modulation of kinase activity by heparin in vitro has been frequently observed [12,13,14,15], suggesting that heparin may have multiple targets. To understand better the mechanism by which heparin inhibits SMC proliferation, we asked whether heparin modulated the activity of PKC in vitro in cultured rat SMC or in vivo in medial SMC of rat carotid arteries subjected to balloon injury

Methods

Results

Conclusion