Sphingosine 1-phosphate: a regulator of arterial lesions.

Sphingosine-1 phosphate (S1P) is a bioactive sphingolipid that is critical in the development of blood vessels, and in the adult regulates vascular functions including vascular tone, endothelial integrity, and angiogenesis. Further, S1P may regulate arterial lesions in disease and after injury by controlling leukocyte recruitment and smooth muscle cell functions.

Modulation of smooth muscle proliferation in rat carotid artery by platelet-derived mediators and fibroblast growth factor-2.

Endothelial denuding injury to the rat carotid artery stimulates smooth muscle cell proliferation in the tunica media. Fibroblast growth factor-2 (FGF2) is responsible for a significant portion of this proliferation but other factors may contribute, particularly those released from adherent platelets. We therefore tested the effects of a range of platelet-derived factors. After filament injury, which minimises FGF2 release, the proliferation rate in thrombocytopaenic rats was decreased by 74% (P < 0.02). After balloon injury, antibody neutralisation of platelet-derived growth factor (PDGF) caused a 27% decrease in proliferation (P < 0.05), while inhibition of histamine H(1) receptors caused a 53% increase (P < 0.05). When filament injury was performed 1 h after FGF2 injection, the proliferation rate increased from 2.3+/-0.7 to 32.8+/-2.7% (P < 0.001), while filament injury alone caused a proliferation rate of only 18.3+/-2.9% (P < 0.01 versus filament plus FGF2). These data suggest that platelet-derived factors interact with FGF2 that is adsorbed to the vessel wall in the control of smooth muscle cell proliferation, and that the net effect of platelets is to stimulate smooth muscle cell proliferation. PDGF, but no other platelet agonist tested, contributes to that stimulation.

Mitogen-activated protein kinases mediate matrix metalloproteinase-9 expression in vascular smooth muscle cells.

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.

Phosphatidylinositol 3-kinase signaling is important for smooth muscle cell replication after arterial injury.

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.

Proliferation of intimal smooth muscle cells. Attenuation of basic fibroblast growth factor 2-stimulated proliferation is associated with increased expression of cell cycle inhibitors.

Basic fibroblast growth factor (FGF2) is a potent mitogen for medial smooth muscle cells and is necessary for their proliferation after balloon catheter injury; however, intimal smooth muscle cells do not require FGF2 for their proliferation, and they respond only weakly to exogenous FGF2. The present study examined the activation of extracellular signal-regulated kinase (ERK) signaling as well as the expression and activity of cell cycle proteins in FGF2-stimulated intimal smooth muscle cells. FGF2 activates ERKs 1 and 2, and Western blot analysis showed that cyclin D, cyclin E, and cyclin-dependent kinase (CDKs) 2 and 4 were expressed in intimal smooth muscle cells after FGF2 infusion. FGF2 stimulation, however, did not lead to phosphorylation of the retinoblastoma protein (Rb), CDK 2 activation, or expression of cyclin A. Western blot analysis showed that intimal smooth muscle cells express elevated levels of the cell cycle inhibitors p15(INK4b) and p27(Kip1), compared with medial smooth muscle cells, and that FGF2 stimulation does not reduce the level of these inhibitors. These studies suggest that despite activation of ERKs 1 and 2 and expression of the cell cycle activators, cyclin D and cyclin E, high levels of cell cycle inhibitors may inhibit cell cycle transit in FGF2-stimulated intimal smooth muscle cells.

Dissociation of mesangial cell migration and proliferation in experimental glomerulonephritis.

BACKGROUND: Recently, we documented that following in vivo mesangial cell (MC) ablation in the Thy1 model, reconstitution of the mesangium occurs by a coordinated proliferation and migration of Thy1 (OX-7)-positive cells originating from the hilus and extraglomerular mesangium. We investigated the role of basic fibroblast growth factor (bFGF) in the mediation of these events. METHODS: Rats were injected with antithymocyte serum and 48 hours later were pulsed with 3H-thymidine to label proliferating cells. Ninety minutes later, a baseline renal biopsy was obtained, and rats were injected with neutralizing anti-bFGF antibodies or control IgG. Sacrificial biopsies were obtained at 96 hours of disease. Using computer image analysis, biopsies from both time points were quantitated for the number of radiolabeled MC (proliferation) and their mean distance from the hilus (migration). The effect of bFGF on the migration of MCs in culture was examined using a chemotactic assay. RESULTS: At sacrifice, autoradiographs of rats receiving anti-bFGF had significantly fewer radiolabeled MCs as compared with rats receiving control IgG (8.7+/-1.9 vs. 14.7+/-3.5, P = 0.0001), yielding an overall 40% reduction in proliferation. There was no difference, however, in the final distance of radiolabeled MCs from the glomerular hilus in the two groups, indicating that the administration of anti-bFGF did not effect MC migration in this model. In an in vitro chemotactic assay, MCs migrated in response to platelet-derived growth factor (PDGF) BB (20 ng/ml), but did not migrate in response to bFGF at a wide range of concentrations (0.5 to 50 ng/ml). CONCLUSIONS: These studies demonstrate that bFGF is an important mediator of MC proliferation but that it does not significantly influence MC migration. This is the first demonstration showing that the mediators effecting proliferation can be dissociated from those mediating migration in renal injury.

Expression of extracellular matrix proteins accompanies lesion growth in a model of intimal reinjury.

Reinjury of rat arterial lesions induces an increase in lesion size that is not associated with an increase in cell number. In this study, matrix volume was examined after reinjury to preexisting lesions, and the kinetics of matrix gene expression and activity of proteolytic enzymes in the lesion were evaluated. Volume densitometry in intima showed a significant increase in matrix volume 28 days after the reinjury, although no change was observed at 14 days. Three common vascular matrix molecules, alpha1(I)procollagen, tropoelastin, and fibronectin, were expressed highly at 7 days after the reinjury. Expression of tropoelastin remained upregulated for the entire 28 days after the reinjury, whereas alpha1(I)procollagen and fibronectin returned to the control level by 28 days. Protease activity was also increased after reinjury. Within days, a marked increase in urokinase plasminogen activator activity was observed in intima, and this activity decreased to control level by 14 days. The activity of tissue plasminogen activator did not change. The 95-kDa gelatinolytic activity was increased 1 to 2 days after the reinjury, but no change in other gelatinolytic activities was observed. These findings demonstrate that the accumulation of extracellular matrix is important in the increase in lesion size after reinjury and that a balance of matrix synthesis and degradation may explain why no change in matrix volume was detected until 28 days after the reinjury.

Cell replication in the arterial wall: activation of signaling pathway following in vivo injury.

This study examined intracellular signal events of arterial cells following balloon catheter injury to rat carotid artery. Within 30 minutes, a marked increase in extracellular signal-regulated kinase-1/2 (ERK1/2) activity was observed. This activity remained elevated for 12 hours but had decreased to control levels by day 1. No increase in ERK1/2 was detected at any later times. Injection of anti-fibroblast growth factor 2 antibody (60 mg i.v.) significantly inhibited the activation of ERK1/2 at 30 minutes after the injury. PD98059 (80 micromol/L), a selective inhibitor of mitogen-activated protein kinase/ERK kinase-1 (MEK1), decreased ERK1/2 activity in injured arteries and also reduced the medial cell replication. In contrast, PD98059 did not block the intimal cell replication at day 8. Mitogen-activated protein kinase phosphatase-1 (MKP-1) was expressed within hours after injury but only weakly at later times; MKP-1 was again expressed after 7 and 14 days. The expression of MKP-1 was associated with an activation of c-Jun amino-terminal kinase. Injury to the arterial wall also stimulated the activity of p70 S6 kinase from 30 minutes to 12 hours, suggesting an alternative pathway in mitogenic signaling of early cell replication. These findings demonstrate that fibroblast growth factor 2-induced ERK1/2 activation promotes medial cell replication after balloon injury; however, signaling of intimal cell replication may not be linked to the MEK1-dependent ERK pathway.

Mitogenic effect of angiotensin II on rat carotid arteries and type II or III mesenteric microvessels but not type I mesenteric microvessels is mediated by endogenous basic fibroblast growth factor.

In this study, anti-basic fibroblast growth factor (anti-bFGF) antibody was used to determine whether the mitogenic effect of angiotensin II in vivo could be blocked by neutralizing bFGF in the vessel wall. Animals, divided into six experimental groups, were given (1) angiotensin II, (2) angiotensin II + anti-bFGF antibody, (3) angiotensin II + normal goat IgG (ngIgG), (4) anti-bFGF antibody, (5) ngIgG, and (6) Ringer's solution. Angiotensin II at 435 ng x kg(-1) x min(-1) was infused into rats continuously for 1 week to induce smooth muscle cell replication, and anti-bFGF antibody or ngIgG was injected intravenously 4 times over the 1-week period at a dose of 60 mg/injection. Bromodeoxyuridine (30 mg/mL) was also continuously infused during the 1-week period. The left carotid artery of all animals was balloon-injured on day 4 of the treatment, and all groups were killed for study on day 7. The results showed that angiotensin II significantly stimulated smooth muscle replication in the balloon-injured carotid artery, intact carotid artery, and three branch levels of the mesenteric vascular tree. Anti-bFGF was able to block the mitogenic effect of angiotensin II in larger vessels but not the smallest (type I) microvessels of the mesenteric arterial tree. This differential response may be attributable to the nature of the lesions in type I vessels versus larger vessels: the type I vascular lesion has a large component of proliferating macrophages, whereas the larger vessels show less injury, few macrophages, and varying levels of smooth muscle replication. Our data suggest that the vessel wall remodeling in the angiotensin II-treated larger vessels involves DNA replication that is dependent on the presence of bFGF.

Effect of phosphorothioated oligonucleotides on neointima formation in the rat carotid artery. Dissecting the mechanism of action.

Several studies have shown that single-dose administration of agents that inhibit medial cell replication, such as antisense oligonucleotides to cell replication genes, can inhibit neointima formation after arterial injury. However, the precise mechanism of action of these agents is unknown. We analyzed the effect of phosphorothioated oligonucleotides delivered periadventitially on the response to injury in the balloon-injured rat carotid artery. Antisense oligonucleotides to c-myc suppressed medial replication 2 days after injury, but this effect was not present at 4 or 14 days. Endothelial cell proliferation was not affected by antisense oligonucleotides. There was, however, a significant suppression of intimal area and intima/media ratio at 14 days and an increase in lumen area in the antisense-treated group. Indeed, an increase in the number of medial cells at 14 days in the antisense group indicated that most of the effect of the agent was due to the suppression that most of the effect of the agent was due to the suppression of cell migration. No effect was noted on expression of two genes, osteopontin and tropoelastin, used as markers of modulation of smooth muscle cells to a "neonatal" phenotype at 4 days after injury. Because no effect on cell proliferation could be demonstrated after 2 days, our data indicate that an early effect of the antisense agent mediates its longer-term effects. We suggest that this effect may be due to the suppression of migration of medial smooth muscle cells rather than the suppression of medial or intimal cell proliferation.

Reinjury of arterial lesions induces intimal smooth muscle cell replication that is not controlled by fibroblast growth factor 2.

In this study we have examined the response of rat carotid arteries with intimal lesions to an angioplasty injury. Rat carotid arteries were subjected to injury with a 2F Fogarty catheter (first injury), and 28 days later the same arteries were subjected to reinjury with a 1.5-mm-diameter coronary dilation catheter (second injury) or a sham operation. After the second injury, the injured arterial surfaces were covered by a platelet monolayer, with occasional small thrombi. The size of the intimal area was significantly increased 28 days after the second injury, although the luminal area was not changed at this time. Intimal and medial cell replication, measured by 5-bromo-2'-deoxyuridine labeling, was significantly increased at 2 days after the second injury but was markedly reduced by 7 days. Addition of fibroblast growth factor-2 (FGF2, 60 micrograms i.v.) did not increase smooth muscle cell (SMC) replication in arteries subjected to the second injury, and replication was not inhibited with an antibody against FGF2 (120 mg i.v.). Both these reagents, however, did significantly affect SMC replication in normal carotid arteries subjected to Fogarty catheter injury. In a similar manner, heparin (888 UPS units/kg body wt i.v.) did not inhibit cell replication after second injury, although it did suppress SMC replication after a single injury. One conclusion is that rat intimal cells in vivo are different from medial SMCs and that other, as-yet-unknown, factors are important for their proliferation.

Factors important in arterial narrowing.

BACKGROUND: Vascular narrowing in hypertension presents conflicting hypotheses about structural narrowing, vasconstriction, volume expansion, contractile function and other issues more obvious to the physiologist than the cell biologist. Even the cell biology in the injured large arteries used to study atherosclerosis is surprisingly complex. The key issue is that changes in mass, here mainly intimal mass or atherosclerotic plaque, correlate poorly with loss of lumen caliber. An analogy to remodeling of microvessels begs the issue of mechanism. RECENT STUDIES: To explore this, we have focused on two issues: cell death and intramural coagulation. It is likely that cell death, along with tissue factor, promotes coagulation and, in previously injured vessels, fibrin forms in the wall after a repeat injury. In vitro, fibrin promotes smooth muscle gel contraction mediated by an as yet unidentified beta 1 integrin. In vivo, inhibition of coagulation not only prevents vascular narrowing after a reinjury, but may even result in dilation. HYPOTHESIS: We suggest that injury responses, that is, classical wound contracture mechanisms, can be explained as potential pathways for pathologic vascular remodeling.

The role of plasminogen, plasminogen activators, and matrix metalloproteinases in primate arterial smooth muscle cell migration.

The migration of arterial smooth muscle cells (SMCs) plays an important role in normal vessel development as well as the pathobiology of blood vessels. Because it is difficult to study cell migration in primates, we used ex vivo explants. The response of baboon aortic medial explants incubated in vitro in a serum-free medium with insulin and transferrin was compared with the response of whole artery injured in vivo by a balloon catheter to establish the validity of the explant model. Both the time course of entry of SMCs into the S phase and the changes in matrix metalloproteinase 9 were similar in the artery and the explants. SMCs began migrating from explants after a lag of 3 days. By day 11, > 90% of the explants exhibited SMC migration from the tissue (percent of explants with > or = 1 migrating cell). Basal migration was inhibited by antibodies to urokinase and tissue-type plasminogen activator, whereas addition of plasminogen to the explants increased migration. An inhibitor of matrix metalloproteinases. BB-94 (Batimistat), decreased migration, as did alpha 2-macroglobulin. These data demonstrate that proteinases of the matrix metalloproteinase and plasminogen/plasminogen activator families play an important role in the migration of primate arterial SMCs through the extracellular matrix.

Expression of VEGF receptors in arteries after endothelial injury and lack of increased endothelial regrowth in response to VEGF.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific factor with angiogenic effects in vivo and mitogenic effects in vitro. Administration of VEGF has been reported to stimulate endothelial growth in denuded arteries and new blood vessel formation in models of induced tissue ischemia. In the present study, expression of VEGF and its receptors flk-l and flt-l was determined in injured aortas and carotid arteries of rats and mice. Neither VEGF nor flk-l mRNA was detectable in vascular cells. mRNA levels for flt-l were dramatically upregulated at the leading edge of a growing endothelial monolayer in vivo; however, these cells did not demonstrate increased replication after VEGF infusion. Furthermore, all doses and treatment protocols of VEGF failed to promote reendothelialization in denuded arteries. At sites of flt-l expression, VEGF increased permeability. These areas revealed a loss of endothelial contacts at the ultrastructural level. These findings suggest that VEGF is not a direct mitogen for large-vessel endothelium in vivo and that VEGF may play a role in abolishing contact inhibition, which may be a prerequisite for endothelial proliferation.

Differential expression of alpha 1 type VIII collagen in injured platelet-derived growth factor-BB--stimulated rat carotid arteries.

Migration of smooth muscle cells from media to intima is critical for the development of neointimal thickening after balloon catheter injury of the rat carotid artery. The present experiments were designed to identify molecules expressed by smooth muscle cells migrating in vivo in the injured artery. Cell migration was maximized by infusing recombinant platelet-derived growth factor-BB (PDGF-BB) after a minimal filament denudation of the rat carotid artery, whereas cell proliferation was minimized by injecting an antibody against basic fibroblast growth factor (bFGF). This treatment caused an eightfold increase in smooth muscle cell migration into the intima but only a twofold increase in intimal smooth muscle cell replication rates. Differential display screening was used to isolate cDNAs that were overexpressed in the injured PDGF-BB-treated versus unmanipulated rat carotids. One of the clones isolated hybridized to a 4.2-kb mRNA species and shared 90% sequence homology to mouse alpha 1 type VIII collagen. Northern and Western blots confirmed overexpression of type VIII collagen in the injured PDGF-BB-treated vessels. In a separate series of experiments, we performed filament denudation injury and administered antibodies to inhibit the actions of endogenous bFGF and PDGF-BB, thereby decreasing smooth muscle cell migration, and found that type VIII collagen mRNA expression varied with migration. Using a different arterial injury model (balloon catheter injury), we showed that expression of type VIII collagen was maximal 2 to 4 days after injury, in coincidence with cell migration from the media to the intima. This molecule constitutes an important component of smooth muscle cell response to vessel injury and may play an important functional role in mediating migration.

Smooth muscle cells isolated from the neointima after vascular injury exhibit altered responses to platelet-derived growth factor and other stimuli.

A variety of evidence suggests that vascular smooth muscle cells (SMC) exhibit a more immature phenotype when stimulated by injury to replicate in the adult. One growth characteristic common to immature (embryonic, fetal, and neonatal) SMC is a markedly reduced responsiveness to platelet-derived growth factor (PDGF) and other mitogenic stimuli. Here we demonstrate that SMC isolated from the 14-day neointima of experimentally injured carotid arteries exhibit a similar growth phenotype. The proliferative responses of neointimal cells to the BB homodimer of PDGF, which interacts with both forms of the PDGF receptor, were up to twenty-fold less (as assessed by BrdU immunocytochemistry) than that of adult control tunica media cells over a wide range of PDGF concentrations. Paradoxically, these cells expressed abundant mRNA for the alpha- and beta-subunits of the PDGF receptor (by RT-PCR) and expressed abundant PDGF receptor protein (by Western blotting). Addition of PDGF-BB to neointimal SMC induced significant autophosphorylation of the PDGF receptor, suggesting that the PDGF receptors were fully functional. The chemotactic responses of neointimal SMC to PDGF, in in vitro migration assays, were identical to that of control medial cells. The data further establish the existence of vascular SMC phenotypes characterized by a refractoriness to growth stimulation by specific mitogens, and provide further evidence for the reiteration of developmentally regulated programs following vascular injury in vivo.

Tropoelastin gene expression in individual vascular smooth muscle cells. Relationship to DNA synthesis during vascular development and after arterial injury.

After vascular injury, quiescent adult smooth muscle cells (SMCs) revert to a more immature synthetic-state phenotype concomitant with the onset of cell replication. The relationship between SMC proliferation and the reexpression of genes characteristic of immature SMCs (eg,tropoelastin [TE]), on an individual cell basis, has not been determined. Using a combined bromodeoxyuridine (BrdU) immunocytochemistry-TE in situ hybridization technique, we determined the relationship between DNA synthesis and TE gene expression in the rat vascular wall during development of the aortic media (embryonic days 13 to 18), low but detectable levels of TE expression occurred equally in both replicating and nonreplicating SMCs. TE message levels dramatically increased in the late fetal and early postnatal periods (fetal day 19 to 1 month postpartum), after a precipitous drop in SMC replication, and then decreased to undetectable levels by postpartum day 60. After a balloon catheter injury in the adult, a developmental sequence of SMC replication followed by TE gene expression was reiterated in both the media and in the developing neointima. On an individual cell basis, adult SMCs replicating after injury expressed little or no TE message; detectable TE gene expression occurred only in nonreplicating SMCs. The most important implications of these data are that (1) adult SMCs replicating after injury appear to revert to a pre-elastogenic embryonic phenotype; (2) maximal TE expression occurs in SMCs only after the cessation of cell replication; and (3) in both the media and the neointima, adult SMCs responding to injury undergo temporarily sequential changes in phenotype reflective of SMC development.

Migration of arterial wall cells. Expression of plasminogen activators and inhibitors in injured rat arteries.

The expression of plasminogen activators and inhibitors was examined in denuded arteries. Within 5 days, smooth muscle cells (SMCs) on the luminal surface expressed the mRNA for tissue-type plasminogen activator (TPA), urokinase type plasminogen (UPA), the receptor for UPA (UPAR), and plasminogen activator inhibitor type-1 (PAI-1). Similar results were seen after 8 days. Six weeks later, only TPA mRNA was still expressed by SMCs on the luminal surface. En face casein zymograms revealed a net fibrinolytic activity in areas covered with luminal SMCs. Reverse zymography showed no antifibrinolytic activity in these zones. Quiescent endothelial cells did not express TPA, UPA, UPAR, or PAI-1 mRNA. Regenerating endothelium at the wound edge strongly expressed TPA. UPA, and UPAR, as well as PAI-1. UPA and UPAR expression was highly restricted to cells at the wound edge and was not present elsewhere. En face zymography showed no plasmin activity in endothelialized areas, and reverse zymography showed a net fibrinolytic activity in endothelialized zones. These results suggest that plasminogen activator and inhibitor expression correlates with the migration of both SMCs and endothelial cells into an arterial wound.

Inhibition of matrix metalloproteinase activity inhibits smooth muscle cell migration but not neointimal thickening after arterial injury.

Smooth muscle cell (SMC) migration and replication are important for neointimal formation after arterial injury. Migration of SMCs requires degradation of basement membrane and extracellular matrix surrounding the cell, and our previous work has shown a correlation between expression of two matrix-degrading metalloproteinases (MMPs), MMP-2 and MMP-9, and smooth muscle migration into the intima in the balloon catheter-injured rat carotid artery. In the present study, an MMP inhibitor, GM 6001, was administered to rats for various times after balloon injury of the carotid artery. Inhibition of MMP activity resulted in a 97% decrease in the number of SMCs that migrated into the intima by 4 days after injury, and lesions growth was retarded by continuous treatment with GM 6001-treated rats was 0.035 +/- 0.008 mm2 compared with 0.095 +/- mm2 in the control group. Neither intimal nor medical SMC replication rates were decreased by GM 6001 treatment, supporting our hypothesis that the decrease in lesion size was due to inhibition of MMP-mediated migration and not inhibition of replication. By 14 days after injury, however, intimal area and SMC number were the same control and inhibitor-treated rats. An increased rate of SMC replication in the GM 6001 rats (replication rates at 10 days were 56.7 +/- 10.0% in the GM 6001 group and 16.97 +/- 1.73% in the control group) contributed to "catch-up" growth of the neointima. Thus, it appears that inhibiting SMC migration with MMP inhibitors is not sufficient to inhibit lesion growth, and lesion size eventually catches up to control via increased SMC replication.