Aesim Cho

Matrix metalloproteinases (MMPs) and, in particular, MMP-9 are important for smooth muscle cell (SMC) migration into the intima. In this study, we sought to determine whether MMP-9 is critical for SMC migration and for the formation of a neointima by using mice in which the gene was deleted (MMP-9(-/-) mice). A denuding injury to the arteries of wild-type mice promoted the migration of medial SMCs into the neointima at 6 days, and a large neointimal lesion was observed after 28 days. In wild-type arteries, medial SMC replication was approximately 8% at day 4, 6% at day 6, and 4% at day 8 and had further decreased to 1% at day 14. Intimal cell replication was 65% at 8 days and had decreased to approximately 10% at 14 days after injury. In MMP-9(-/-) arteries, SMC replication was significantly lower at day 8. In addition, SMC migration and arterial lesion growth were significantly impaired in MMP-9(-/-) arteries. SMCs, isolated from MMP-9(-/-) mouse arteries, showed an impairment of migration and replication in vitro. Thus, our present data indicate that MMP-9 is critical for the development of arterial lesions by regulating both SMC migration and proliferation.

Expression of matrix metalloproteinase (MMP)-9 has been linked to the progression of plaque rupture and intimal formation in arterial lesions. In this study, we determined which factors and signaling pathways are involved in regulating the MMP-9 gene. Rat carotid arterial smooth muscle cells treated with tumor necrosis factor (TNF)-alpha showed a marked increase in MMP-9 activity and mRNA level, whereas platelet-derived growth factor (PDGF) showed a slight induction of the MMP-9 mRNA level. TNF-alpha treatment caused an increase in c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular signal-regulated kinase (ERK) activities, whereas PDGF treatment caused an increase in ERKs and p38 MAPK activities without any effect on JNK activity. Treatment with either SB203580 (inhibitor of p38 MAPK) or U0126 (inhibitor of the ERK pathway) downregulated the TNF-alpha-induced MMP-9 expression in a dose-dependent manner. Treatment of cells with TNF-alpha and PDGF together stimulated the MMP-9 expression at a level higher than that observed with either factor alone, suggesting that TNF-alpha and PDGF have a synergistic effect on MMP-9 expression in arterial smooth muscle cells. Furthermore, suboptimal inhibitory concentrations of SB203580 and U0126 together almost completely inhibited the MMP-9 expression. These results suggest that p38 MAPK and ERK pathways contribute to the transcriptional regulation of MMP-9 in arterial smooth muscle cells.

The phosphoinositide 3-kinase [PI(3)K] pathway is a key signaling pathway important for replication of mammalian cells. In this study, we examined the role of PI(3)K in smooth muscle cell (SMC) replication after balloon catheter injury of rat carotid arteries. Protein kinase B (PKB), a downstream target of PI(3)K, was phosphorylated at 30 and 60 minutes after injury and to a lesser degree after 6 hours and 1 and 2 days but not after 7 days. Wortmannin (10 microgram per rat), a PI(3)K inhibitor, given to rats 60 and 5 minutes before and 11 hours after balloon injury, reduced the levels of phosphorylated PKB. SMC replication quantified between 24 to 48 hours was significantly reduced compared with control replication, as were the levels of cyclin D(1). Wortmannin was also administered to rats between days 7 and 8 and between days 7 and 9 after balloon catheter injury. A reduction in levels of phosphorylated PKB was detected, but no decrease in the replication of intimal SMCs was observed in either experiment. These data demonstrate that the PI(3)K signal transduction pathway plays an important role in medial but not intimal SMC replication.

Neointimal lesion formation was induced in sphingosine 1-phosphate (S1P) receptor 2 (S1P2)-null and wild-type mice by ligation of the left carotid artery. After 28 days, large neointimal lesions developed in S1P2-null but not in wild-type arteries. This was accompanied with a significant increase in both medial and intimal smooth muscle cell (SMC) replication between days 4 to 28, with only minimal replication in wild-type arteries. S1P2-null SMCs showed a significant increase in migration when stimulated with S1P alone and together with platelet-derived growth factor, whereas both wild-type and null SMCs migrated equally well to platelet-derived growth factor. S1P increased Rho activation in wild-type but not in S1P2-null SMCs, and inhibition of Rho activity promoted S1P-induced SMC migration. Plasma S1P levels were similar and did not change after surgery. These results suggest that activation of S1P2 normally acts to suppress SMC growth in arteries and that S1P is a regulator of neointimal development.