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

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.

Phenotype-specific inhibition of the vascular smooth muscle cell cycle by high glucose treatment

“…In addition, overexpression of PKCδ [35] or PKCα [36] inhibits SMC proliferation as well. It is interesting to note that activation of PKC also stimulates SMC proliferation [45]. The different PKC isozymes are thought to have distinct functions regarding the phosphorylation of specific substrate proteins [46].…”

Calcium and Protein Kinase C (PKC)-Related Kinase Mediate α1A-Adrenergic Receptor-Stimulated Activation of Phospholipase D in Rat-1 Cells, Independent of PKC

“…Rat-1 cells were transiently transfected in serum-free DMEM with 10 M antisense (AS) or scrambled (SC) oligonucleotides for 2, 4, 6, or 9 days with new pulses every 48 h. AS and SC oligonucleotides were synthesized at the Molecular Resource Center Synthesis Facility (The University of Tennessee, Memphis, TN). These oligonucleotides have been used by other investigators to specifically down-regulate PKC␣ (Herbert et al, 1996), PKC␦ (Pessino et al, 1995; modified for rat isoform), PKC⑀ (Traub et al, 1997; modified for rat isoform), and PKC , or were designed against the translation initiation site (nucleotides 136 -155) of PKN (Mukai and Ono, 1994). The sequences used were as follows: PKC␣ AS, 5Ј-CAGCCATGGTTCCCCCCAAC-3Ј; PKC␣ SC, 5ЈCCAGTCACTCGCACCATCGC-3Ј; PKC␦ AS, 5Ј-ACGGTGCCAT-GATGGA-3Ј; PKC␦ SC, CGAGTAGTTACAGCGG-3Ј; PKC⑀ AS, 5Ј-CATGAGAGCAGATCTGACCT-3Ј; PKC⑀ SC, 5Ј-AACGCATAACTCG CTTGAGG-3Ј; PKC AS, 5Ј-CTGCTGCCGGAGCCCCGA-3Ј; PKC SC, CCGAGAGCCGCCGTCGTC; PKN AS, 5Ј-AGGTTCACTCTGCA CGGCGT-3Ј; and PKN SC, 5Ј-AGTATTCCGGTCGAGCCCTG-3Ј.…”

Rear Polarization of the Microtubule-Organizing Center in Neointimal Smooth Muscle Cells Depends on PKCα, ARPC5, and RHAMM

“…By contrast, PKC␣ did not control the front polarization of the MTOC in medial SMCs, suggesting different regulatory pathways in the two cell types. Others have shown that PKC␣ activity increases between 6 and 48 hours after balloon injury in vivo, preceding migration of SMCs to the intima, 31 and that PKC inhibition attenuates SMC migration. 32 Other PKC isoforms are expressed in SMCs (including the ␤, ␦, , and isoforms) 31 ; however, our experiments with selective PKC inhibitors demonstrated that none of these other isoforms affect rear polarization of neointimal SMCs.…”