Abstract-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 Ϫ/Ϫ 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 Ϸ8% 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 Ϸ10% at 14 days after injury. In MMP-9 Ϫ/Ϫ arteries, SMC replication was significantly lower at day 8. In addition, SMC migration and arterial lesion growth were significantly impaired in MMP-9 Ϫ/Ϫ arteries. SMCs, isolated from MMP-9 Ϫ/Ϫ 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. Key Words: matrix metalloproteinase-9 Ⅲ mouse arterial injury Ⅲ smooth muscle cell replication Ⅲ migration Ⅲ neointimal formation A rterial lesion growth is an important process in the development of atherosclerosis, restenosis, and vascular graft stenosis. Multiple events participate in this process, including smooth muscle cell (SMC) replication and SMC migration. The importance of SMC migration is often neglected because in most mammalian arteries, there is no obvious way to recognize the movement of SMCs within the artery. However, the migration of SMCs can be studied in the arteries of small mammals because the intima is composed of just the endothelial monolayer, and in these circumstances, the migration of SMCs to the intima can be evaluated.The migration of SMCs is thought to be regulated by proteases such as plasminogen/plasmin and matrix metalloproteinases (MMPs). Both classes of enzymes are expressed by SMCs, and their expression is markedly upregulated in injured arteries; these arteries invariably develop a pronounced thickened neointima. 1 Plasminogen activators convert the inactive plasminogen to plasmin, which in turn can activate latent MMPs. 2 Supporting data that plasmin can regulate SMC migration are given in studies in which the deletion of genes encoding for urokinase plasminogen activator or plasminogen resulted in a smaller intimal lesion size in injured mouse arteries. 3,4 There are also convincing data that MMPs are necessary for the migration of SMCs into the intima. The deletion of tissue inhibitor of metalloproteinases (TIMP)-1 in mice leads to the formation of larger intimal lesions in injured arteries, whereas overexpression of TIMP-2 by adenovirus-mediated transfer leads to a temporary reduction in lesion size in injured rat arteries. 5,6 Similar data were obtained when a synthetic MMP inhibitor was administered to rats subjected to arterial i...

Abstract-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)-␣ 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-␣ 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-␣-induced MMP-9 expression in a dose-dependent manner. Treatment of cells with TNF-␣ and PDGF together stimulated the MMP-9 expression at a level higher than that observed with either factor alone, suggesting that TNF-␣ 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. Key Words: mitogen-activated protein kinases Ⅲ p38 mitogen-activated protein kinase Ⅲ extracellular signal-regulated kinase Ⅲ matrix metalloproteinase-9 Ⅲ tumor necrosis factor D egradation of extracellular matrix by matrix metalloproteinases (MMPs) is thought to be important in the progression of atherosclerosis and plaque rupture. MMP-1, MMP-3, and MMP-9 have been identified in human atherosclerotic lesions, and the enhanced expression of MMP-9 at the shoulders of these lesions has been linked to plaque rupture. 1 In experimental animal models, MMPs are also shown to be important for smooth muscle cell migration into the intima. In a rat arterial injury model, after the initial medial smooth muscle cell replication, medial smooth muscle cells migrate and first appear in the intima 4 days after injury. 2 MMP-9 is expressed within 6 hours after injury in rat carotid arteries and continues to be expressed up to 6 days, whereas MMP-2 activity is markedly increased after 4 days of injury. 3 The importance of these MMPs in the migration of smooth muscle cells is illustrated by the finding that the administration of MMP inhibitor almost completely inhibits the number of smooth muscle cells migrating into the intima. 3 Although MMPs play an important role in lesion growth, little is known about the signaling mechanism involved in regulating MMP expression in vascular smooth muscle cells.Several growth factors and cytokines, such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), tumor necrosis factor (TNF)-␣, and interleukin (IL)-1,...

Abstract-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 g 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 ( Key Words: balloon injury Ⅲ smooth muscle cell replication Ⅲ protein kinase B Ⅲ wortmannin B alloon injury to the rat carotid artery initiates smooth muscle cell (SMC) replication in a predictable manner. Our previous data suggest that the replication of intimal and medial cells is controlled by different mechanisms. For example, the extracellular signal-regulated kinase (ERK)1/2 cascade is involved in the early proliferative response of the artery after mechanical injury, inasmuch as PD98095, a mitogen-activated protein kinase kinase (MEK)1 inhibitor, significantly blocked ERK1/2 phosphorylation and SMC replication 48 hours after injury. In contrast, the MEK1 inhibitor was not able to block intimal SMC replication after 7 days. 1 These data have led us to suggest that other signaling pathways may be important for intimal cell replication.Phosphoinositide 3-kinase [PI(3)K] is activated by many growth factors, and there is good evidence that this pathway is involved in the entry of cells into S phase. 2-4 Activation of PI(3)K leads to the generation of phosphatidylinositol 3,4-diphosphate and phosphatidylinositol 3,4,5-triphosphate in the cell membrane. These phospholipids form a binding site for proteins with a pleckstrin homology domain, such as protein kinase B (PKB), and in doing so a cryptic phosphorylation site is exposed. These sites are then phosphorylated by phosphoinositide-dependent kinases (PDKs), thus leading to their activation. Once activated, PKB phosphorylates several substrates, including glycogen synthase kinase-3␤ 5 and glucose transporter 4. 6 PKB is also known to be involved in regulating cell survival and cell replication. [7][8][9][10]

Abstract-Neointimal lesion formation was induced in sphingosine 1-phosphate (S1P) receptor 2 (S1P 2 )-null and wild-type mice by ligation of the left carotid artery. After 28 days, large neointimal lesions developed in S1P 2 -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. S1P 2 -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 S1P 2 -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 S1P 2 normally acts to suppress SMC growth in arteries and that S1P is a regulator of neointimal development. Key Words: sphingosine 1-phosphate receptors Ⅲ smooth muscle cells Ⅲ neointima S phingosine 1-phosphate (S1P) is a bioactive sphingolipid formed by activation of sphingosine kinases. 1 It exerts pleiotropic effects on many cells by regulating cytoskeletal rearrangement, cell survival, cell migration, cell proliferation, angiogenesis, and vascular development. [2][3][4][5] Recently S1P has received attention as a regulator of the cardiovascular system. In part, this is because there are high levels of S1P in plasma, and a recent report showed that they correlate well with the reoccurrence of vascular events. 6 -10 Further platelets release S1P during their activation, and consequently S1P levels are likely to be high at sites of arterial injury. 11,12 S1P acts through 5 G protein-coupled receptors (S1P 1 to S1P 5 ), although arterial smooth muscle cells (SMCs) express only S1P 1 , S1P 2 , and S1P 3 . 4,13 Initially these receptors were called endothelial cell differentiation gene receptors. 14 Activation of S1P receptors induces coupling to a variety of G proteins, which in turn leads to activation of multiple pathways. In SMCs most work has concentrated on S1P 1 and S1P 2 because they have opposing actions. S1P 1 couples to Gi and leads to activation of extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Rac. 13,15,16 Adult SMCs only weakly express S1P 1 , although it is more highly expressed in pup cells, and this has been linked to their increased ability to migrate and proliferate in response to S1P. 17 S1P 1 is also strongly expressed in SMCs from rat intimal lesions as well as in human atherosclerotic lesions. 17,18 These data have been used to suggest that activation of S1P 1 may induce events leading to restenosis and the formation of arterial lesions. S1P 2 is the main receptor expressed by most adult medial SMCs and couples to Gi, Gq, and G12/13, and its activation by S1P is associated with inhibition of SMC migration. 13,19 This is thought to occur via coupling...

The response of mice arteries to injury varies significantly between strains. FVB mice develop large neointimas after injury, whereas very small lesions form in C57BL/6 mice. After injury, platelet interaction with the denuded artery and early smooth muscle (SMC) replication are identical in both strains; however, the migration of SMCs differs significantly. FVB cells readily move into the developing neointima, whereas only the occasional C57BL/6 cells migrate. Injured arteries showed no difference in matrix metalloproteinases (MMP-2 and MMP-9) and plasminogen activator activities. In vitro, sphingosine-1-phosphate (S1P) in combination with platelet-derived growth factor (PDGF) stimulates migration of FVB cells but inhibits migration of C57BL/6 SMCs. Both SMCs migrate equally well to PDGF alone. One explanation is that the SMCs express different S1P receptors. Real-time polymerase chain reaction shows that FVB cells express higher levels of S1P receptor-1 (S1P(1)) compared with C57BL/6 cells, which express higher levels of S1P receptor-2 (S1P(2)). In addition, the migration of C57BL/6 cells can be increased by inhibiting S1P(2), whereas inhibiting S1P(1) expression slows the migration of FVB cells. Taken together these studies suggest that expression of S1P receptors vary within inbred mouse strains and that S1P is critical for SMC migration and lesion formation after injury.

Objective The objective of this study is to define a role for S1PR3 in intimal hyperplasia. Methods and Results A denudation model of the iliac-femoral artery in wild-type and S1PR3-null mice was used to define a role for S1PR3 in the arterial injury response because we found in humans and mice that expression of S1PR3 is higher in these arteries when compared to carotid arteries. At 28 days after surgery, wild-type arteries form significantly larger lesions than S1PR3-null arteries. BrdU labeling experiments demonstrate that upon injury, wild-type arteries exhibit higher medial as well as intimal proliferation than S1PR3-null arteries. Because S1PR3 expression in vitro is low, we expressed S1PR3 in S1PR3-null SMCs using retroviral-mediated gene transfer to study S1PR3 effects on cell functions and signaling. SMCs expressing S1PR3, but not vector-transfected controls, respond to S1P stimulation with activation of Rac, Erk and Akt. SMCs expressing S1PR3 also grow migrate more. Conclusion In humans and mice, S1PR3 expression is higher in iliac-femoral arteries compared to carotid arteries. S1PR3 promotes neointimal hyperplasia upon denudation of iliac-femoral arteries in mice, likely by stimulating cell migration and proliferation through activation of signaling pathways involving Erk, Akt and Rac.

During development of the cellular slime mold Dictyostelium discoideum, cells migrate in response to cAMP to form aggregates, which give rise to fruiting bodies consisting of two major cell types: spores and stalk cells. Multicellularity is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive binding sites are expressed at the initial stage of development. At the aggregation stage, cells acquire EDTA-resistant binding sites, which are mediated by a cell-surface glycoprotein of Mr80,000 (gp80). gp80 is preferentially associated with cell surface filopodia, which are probably involved in the initiation of contact formation between cells. Covaspheres conjugated with gp80 bind specifically to aggregation-stage cells. The binding can be inhibited by precoating cells with an anti-gp80 monoclonal antibody, thus suggesting that gp80 mediates cell-cell binding via homophilic interaction. The structure of gp80 predicted from its cDNA sequence can be divided into three major domains: a membrane anchor, a hinge, and a globular region. An analysis of fusion proteins containing different gp80 segments shows that the cell-binding activity resides in the globular region. In the postaggregation stages, gp80 is replaced by other surface glycoproteins in maintaining cell-cell adhesion. One of them has a Mr of 150,000 (gp150). Anti-gp150 antibodies have no effect on aggregation-stage cells, but they disrupt cell-cell adhesion at subsequent stages. It becomes evident that the complex phenomena of cell adhesion and tissue organization involve the participation of a number of surface glycoproteins.