Inhibition of Matrix Metalloproteinase Activity Inhibits Smooth Muscle Cell Migration but Not Neointimal Thickening After Arterial Injury
Abstract 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 lesion growth was retarded by continuous treatment with GM 6001 for up to 10 days after injury. At 10 days, intimal area in GM 6001–treated rats was 0.035±0.008 mm2 compared with 0.095±0.01 mm2 in the control group. Neither intimal nor medial 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 in 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.
The intima of a normal rat carotid artery does not contain SMCs, but within days after balloon catheter injury a neointima develops, consisting predominantly of SMCs that have migrated across the internal elastic lamina from the media and then replicated.1 SMC migration is therefore a key step in the development of intimal lesions. The process of migration presumably requires degradation of basement membrane and extracellular matrix surrounding the cell, and there is increasing evidence that SMCs produce extracellular matrix–degrading proteinases after arterial injury. These cells express mRNA for urokinase and tissue-type plasminogen activators after balloon catheter injury,2 3 4 5 and tissue-type plasminogen activator activity is increased at 4 days after arterial injury, coincident with SMC migration to the intima. Further, the addition of tranexamic acid, a plasminogen activator inhibitor, inhibited SMC migration.3 Plasminogen activators catalyze the conversion of plasminogen to plasmin, which in turn can degrade several matrix molecules and can activate MMPs, a family of molecules involved in degradation and remodeling of connective tissues.6 Indeed, we have shown induction of an 88-kD gelatinolytic enzyme (MMP-9) and increased activity of a 62-kD gelatinase (MMP-2), coincident with SMC migration after balloon catheter injury of the rat carotid artery.7 After vascular injury, SMCs should be capable of digesting most if not all matrix components of the arterial wall, and we believe this proteolytic activity is necessary to permit their migration into the intima.
One question not yet answered is whether the MMPs play a role in the development of the intimal thickening of atherogenesis and restenosis after angioplasty. Some support for a role of MMPs in lesions has come from recent studies that have demonstrated MMP-1, -2, -3, and -9 by immunocytochemistry and by in situ hybridization in human atherosclerotic plaque specimens and in diffuse and thickened intimas.8 9 10 In the present study we administered an MMP inhibitor, GM 6001, to investigate the role of MMPs in intimal lesion development after balloon catheter injury of the rat carotid artery. Inhibition of MMP activity resulted in a nearly complete inhibition of SMC migration into the intima, and at early times after injury, a significant reduction in neointimal thickening was observed. This study suggested that MMPs are important mediators of tissue remodeling after arterial injury, permitting the migration of SMCs into the newly formed intima. The intimal lesion eventually increased to control levels because of increased SMC replication rate in the GM 6001–treated rats.
Materials and Methods
Male Sprague-Dawley rats (3 to 4 months old) from Bantin and Kingman Laboratories (Edmonds, Wash) were used in all experiments. Rats were anesthetized by intraperitoneal injection of xylazine (Anased; Lloyd Laboratories), 4.6 mg/kg body weight, and ketamine (Ketaset; Aveco Co Inc), 70 mg/kg body weight. A midline incision in the neck was made to expose the left external carotid artery. A 2F balloon catheter (Baxter Healthcare Co) was introduced through the left external carotid artery and passed into the common carotid artery. The balloon was distended with saline until a slight resistance was felt, then was rotated while pulling back through the common carotid to denude the vessel of endothelium. This procedure was repeated two more times; then the catheter was removed, the external carotid ligated, and the wound closed.
A peptide hydroxamic acid (GM 6001), supplied by Glycomed Inc, was used to inhibit MMP activity in injured rat carotid arteries.11 The ability of this inhibitor to effectively inhibit rat arterial MMPs was tested by incubating a zymogram gel containing rat arterial extracts with incubation buffer containing 0.5 mmol/L GM 6001. GM 6001 completely inhibited all gelatinolytic activity on the zymogram (results not shown). Immediately before balloon catheter injury, rats were injected IP with 100 mg/kg GM 6001 dissolved in 4% CMC. Control rats were injected with 4% CMC vehicle. Plasma GM 6001 concentration was measured by high-performance liquid chromatography in four rats at 5, 14, and 24 hours after initial IP injection of GM 6001 and injury. The rats were injected daily with either GM 6001 or CMC until they were killed at 2, 4, 7, 10, or 14 days after carotid artery injury. For all time points, to label all cells entering S-phase during the last 24 hours before sacrifice, three injections of BrdU (Boehringer Mannheim Corp) were given subcutaneously (25 mg/kg body weight) at 17, 9, and 1 hour(s) before death. Rats were killed by IV injection of sodium pentobarbitol (Anthony Products Co). Lactated Ringer’s injection USP (Baxter) was infused at a pressure of 120 mm Hg retrogradely via a catheter placed in the abdominal aorta. The vessels were then perfusion fixed with 0.1 mol/L phosphate-buffered 4% paraformaldehyde at 110 mm Hg. Vessels were excised and immersed in 4% paraformaldehyde for 1 hour, and then transferred to Ringer’s solution.
In the rats killed 4 days after balloon injury, a 1-cm length of vessel was excised from the middle of the fixed common carotid and used to determine SMC migration as previously described.7 Briefly, migration was measured by staining the intimal cell nuclei with an antibody against histone H1 (MAB 1276, Chemicon International Inc) and counting the number of cells in the intima at 4 days after injury. In rats killed at 2, 4, 7, 10, and 14 days after injury, 5-mm lengths of the carotid arteries were cut and embedded in paraffin, and histological cross sections were prepared. We measured medial (2- and 4-day injury) and intimal (7-, 10-, and 14-day injury) SMC replication rates by immunostaining for BrdU and determining the percentage of BrdU-labeled cells present, as previously described.12 Intimal lesion area was measured by photographing the vessel cross sections, scanning the photographs with a Hewlett Packard Scan Jet IIp scanner, and measuring intimal and medial areas by using public domain NIH Image (written by Wayne Rasband at the US National Institutes of Health and available from the Internet by anonymous FTP from zippy.nimh.nih.gov or on floppy disk from NTIS, 5285 Port Royal Rd, Springfield, VA 22161, part number PB93-504868) version 1.55 software run on a Macintosh IIsi computer. Lumen area was determined by tracing around the inside edge of the vessel and quantitating the area inside the circle. Intimal area was measured as the area encompassed by the internal elastic lamina minus the lumen area.
GM 6001 Delay Experiments
In an attempt to define a critical period for SMC migration from the media to the intima, we altered the time of administration of the MMP inhibitor. In one group (First 7D GM 6001), GM 6001 was administered daily at a dose of 100 mg/kg per day for the first 7 days after balloon catheter injury, then stopped before the rats were finally killed at 14 days postinjury. In a second set of experiments (Second 7D GM 6001), we balloon injured the carotid artery and delayed GM 6001 administration for 7 days. GM 6001 was administered daily at a dose of 100 mg/kg per day between 7 and 14 days postinjury, before the rats were killed at 14 days. The controls for these two groups included a group administered 4% CMC for the first 7 days after injury (First 7D Control) and a group in which 4% CMC was delayed until 7 to 14 days after injury (Second 7D Control). BrdU was administered, and the rats were killed and the carotids perfusion fixed as described above. We measured intimal area on cross sections and determined intimal SMC replication rates as described above.
Differences in SMC migration at 4 days and differences in medial SMC replication rates between control and GM 6001–treated rats at 2 and 4 days after injury were analyzed by unpaired Student’s t test. Differences between control and GM 6001–treated rats in carotid intimal area, intimal SMC replication rate, and the total number of intimal cells at 7, 10, and 14 days after injury were assessed by unpaired Student’s t test. Differences in carotid intimal area and SMC replication rate in the 14-day-delay experiments were analyzed by ANOVA followed by Fisher’s protected least significant difference for pairwise comparisons.
Previous studies have shown that MMP-2 and MMP-9 activities were increased coincident with SMC migration into the intima after balloon injury of the rat carotid.7 These data led us to hypothesize that the MMPs are functionally important in mediating this cell migration. In the present studies, an MMP inhibitor, GM 6001,11 almost completely inhibited the migration of SMCs to the intima after administration for 4 days after balloon catheter injury (Fig 1⇓). The number of intimal SMCs was decreased from 99.7±30.8 cells/mm2 in vehicle-infused control rats to 2.84±0.79 cells/mm2 in GM 6001–treated rats (P=.007). No significant difference in medial SMC replication rate was observed between the control and GM 6001–treated groups at 2 or 4 days after injury (Fig 2⇓).
Growth of the intimal lesion occurs through migration of SMCs from the media, as well as intimal SMC replication and matrix synthesis. Since migration of SMCs by necessity precedes intimal SMC replication, we asked whether inhibition of SMC migration would retard the growth of intimal lesions. Intimal thickening was greatly reduced after treatment with GM 6001 at early times after injury (Fig 3⇓). At 7 days intimal area in the GM 6001–treated rats was 0.023±0.004 mm2, significantly less than the intimal area in the control rats, 0.046±0.009 mm2 (P=.0281). At 10 days after injury intimal area in the GM 6001 rats was still significantly lower than in control rats (P=.005). By 14 days, however, intimal areas were not different in control and GM 6001–treated rats.
Although we know that GM 6001 inhibits early migration of SMCs from media to intima, inhibition of lesion growth could also be affected by a change in intimal SMC replication. To test whether GM 6001 had such an effect, intimal SMC replication rates were measured at 7, 10, and 14 days after injury, and the results are shown in Fig 4⇓. At no time was SMC replication rate inhibited in the GM 6001–treated groups. In fact, at 10 days after injury SMC replication rate was 3.3 times greater in the GM 6001 group than the control group (P=.0045). By 14 days after injury, SMC replication rate in the GM 6001–treated rats declined to values not significantly different from the control rats. We were concerned that the high replication rate in GM 6001–treated rats might result in increased intimal SMC number; however, total intimal SMC number was not significantly different in GM 6001–treated and control rats at 14 days after injury (Fig 5⇓).
Since inhibiting MMP activity blocked cell migration but did not decrease SMC replication, we used GM 6001 to try to define the critical period over which SMC migration occurred. GM 6001 was administered daily for the first 7 days after balloon catheter injury, then stopped, and the rats were finally killed at 14 days postinjury (First 7D GM 6001). In a second set of experiments, the carotid artery was injured, and GM 6001 was administered for 7 to 14 days postinjury (Second 7D GM 6001). The controls for these last two groups included a group administered 4% CMC for the first 7 days after injury (First 7D Control) and a group in which 4% CMC was delayed until 7 to 14 days after injury (Second 7D Control). No significant difference was noted between these two control groups for any parameter measured, so the values were combined to simplify representation in figures.
If GM 6001 treatment was administered for the first 7 days after injury and the rats killed at day 14 (First 7D GM 6001), no significant difference in lesion area compared with control rats was observed (Fig 6⇓). Intimal lesion area was 0.180±0.031 mm2 in control rats and 0.195±0.018 mm2 in First 7D GM 6001 rats. When GM 6001 treatment was given for days 7 through 14 (Second 7D GM 6001), the final lesion size at 14 days, 0.173±0.022 mm2, was not different from controls or the First 7D GM 6001 rats. In all groups, intimal SMC replication rates at 14 days declined to low levels and were not significantly different from control rates: control, 8.10±1.96%; First 7D GM 6001, 10.42±1.47%; and Second 7D GM 6001, 7.46±1.52% (Fig 7⇓).
Rats do not have SMCs normally resident in the arterial intima, yet the thickened intimal lesions that develop after injury consist predominantly of SMCs and matrix. Therefore, the migration of SMCs from media to intima is a necessary step for the development of lesions in rat arteries. In previous work, we have found that migration of SMCs in the injured carotid artery was stimulated by platelet-derived growth factor B, and involvement of the plasmin-plasminogen activator system was critical.3 In the present study we show that MMP activity also plays a critical role in lesion development but is not able to ultimately affect the size of these lesions.
Our aim in this study was to block the activity of those MMPs that we believe are important in SMC migration. The metalloproteinase inhibitor used, GM 6001, is not specific for individual members of the MMP family, and the steady state plasma concentrations achieved in the present experiments, ≈50 nmol/L, were well in excess of the Ki values for MMP-9 and MMP-2 (0.2 and 0.5 nmol/L, respectively; R.E. Galardy, unpublished data, 1995). This lack of specificity of the inhibitor was not a major concern to us, since we have found that expression of active MMP-9 is induced after balloon catheter injury to rat arteries and also that there is increased activation of constitutive MMP-2 coincident with SMC migration into the intima.7 Further, we have found no evidence of MMP-3 or MMP-1 synthesis by rat vascular smooth muscle in vivo7 ; consequently, we believe that MMP-9 and MMP-2 are the principle mediators of migration for arterial SMCs. Our findings are supported by the work of Jenkins et al,13 who observed a similar pattern of MMP-9 and MMP-2 expression after balloon catheter injury of the rat carotid artery.
There is considerable evidence that MMPs mediate cell migration during tumor cell invasion and metastasis, blastocyst implantation, and placentation.14 15 16 17 Our hypothesis that MMPs are important in SMC proliferation and migration is supported by the recent observations of Southgate et al18 that SMCs migrating from rabbit aortic explants in vitro expressed both MMP-2 and MMP-9 and synthetic MMP inhibitors blocked both SMC proliferation and migration from the explants. Similarly, others have shown that invasion of SMCs through basement membranes in chemotaxis chamber assays was dependent on production of MMP-2 and could be inhibited by treatment with peptide MMP inhibitors.19 Taken together with our current results, these prior studies provide evidence for MMP mediation of SMC migration both in vivo and in vitro.
Our data showed that a significant inhibition of migration at day 4 and significant decreases in lesion size at 7 and 10 days after injury were obtained with the MMP inhibitor. Thus, MMP-mediated migration significantly contributes to lesion growth, at least for the first 10 days after injury. The continued presence of the inhibitor, however, did not ultimately reduce the intimal lesion size, since 14 days after injury the intima in the GM 6001–treated rats was equal to that of control rats. From our data it would appear that a prolonged peak of intimal SMC replication rate in GM 6001–treated rats was responsible for this “catch-up” in lesion size. We do not understand the factors mediating the prolonged increase in intimal SMC replication, although it is unlikely that the MMP inhibitor is directly involved, since administration of the drug made no difference to the replication rates of medial cells 2 and 4 days after injury or to intimal cells 7 and 14 days after injury. One possibility is that since matrix degradation is inhibited by GM 6001, there is a change in the cell–extracellular matrix interactions. In vitro, cell attachment to extracellular matrix components is a prerequisite for proliferation in response to growth stimuli and also prevents apoptosis.20 21 In our study, blocking MMP activity may have prevented cells from detaching themselves from the matrix, rendering them susceptible to continued growth stimulation, and hence an enhanced replication was observed. Other possibilities are that changes in extracellular matrix composition directly influenced cell replication or altered the balance of active growth factors sequestered in the matrix. Additional growth regulatory mechanisms must have been present, because SMC replication rate did ultimately decrease by 14 days despite continued inhibition of MMP activity.
It is difficult to measure SMC migration in vivo, since both cell migration and replication contribute to growth of the intima.22 Our data showed that GM 6001 acted as a specific inhibitor of migration at 4 days after injury, and this finding led us to attempt to determine the duration of migration in the injured rat carotid artery. Delaying GM 6001 treatment for the first week after injury and then giving the drug between 7 and 14 days had no effect on lesion size measured at 14 days. This result implies that migration of SMCs into the intima did not contribute significantly to lesion growth during the second week after injury. Somewhat puzzling are the data on GM 6001 administration for the first 7 days after injury and then withdrawal for the next 7 days. We have shown that after 7 days a smaller lesion would be expected (Fig 3⇑), and yet when these lesions were examined at 14 days, no significant difference was observed. This result might imply that SMC migration still occurs during this time, but as pointed out above, the SMC replication of these treated intimas is increased within this time frame; thus, it is not possible to draw any firm conclusions as to whether migration occurs at this time. Considering the data from the 7-day continuous administration and the delay experiments together, we suggest that SMC migration occurs within the first 7 days in the injured artery, since blocking migration over this time led to a significant decrease in lesion size. However, any migration occurring between day 7 and day 14 is relatively insignificant compared with intimal cell replication, and so inhibition of cell migration for the second week after injury has no effect on overall lesion size.
Finally, these data show that any early change in the growth of arterial lesions can be compensated for by intimal cell replication. Over the past 10 years we have characterized lesion growth in the rat carotid artery injury model and divided the response to injury into three discrete phases.23 24 25 Medial SMC replication begins early after injury1 and is followed at 4 days by SMC migration into the intima.3 Our previous studies showed that inhibition of the first phase, medial cell replication, with basic fibroblast growth factor antibodies had no effect on the ultimate size of the lesion.26 In the present study, we now find that inhibition of phase two, SMC migration into the intima, also does not lead to a reduction in lesion size over the long term. Therefore, even though these stages in lesion growth are important for the development of intimal lesions, it would appear that once SMCs arrive in the intima their replication rate is the ultimate determinant of lesion size. A final issue is that the lesions, under all conditions, after either continuous or delayed administration of GM 6001, achieve the same cell number and size as the control lesions. Thus, there is a suggestion of a predetermined set-point for intimal growth after arterial injury. What controls this set-point is at present unknown, but it is apparent that unless intimal cell replication can be blocked it is unlikely that lesion size will be reduced.
In summary, we have shown that a significant reduction in SMC migration resulted in a significant decrease in lesion size at early times after injury. However, prolonged SMC replication in the GM 6001–treated rats resulted in lesion size catching up to controls. These experiments suggest that although MMPs are important mediators of cell migration, inhibiting both migration and intimal cell replication will be necessary to inhibit lesion growth.
Selected Abbreviations and Acronyms
|SMC||=||smooth muscle cell|
This study was supported by grant HL-03174 from the National Institutes of Health and by Bayer AG. Dr Bendeck was supported by a fellowship from the Medical Research Council of Canada. We would like to acknowledge the expert technical assistance of Lisa Stiffler and Jeff Kozlowski. We are grateful to Glycomed Inc for supplying GM 6001, the peptide MMP inhibitor used in these studies.
Reprint requests to Michelle P. Bendeck, PhD, St Michael’s Hospital Division of Cardiology, 30 Bond St, Room 811F, Toronto, Ontario, Canada M5B 1W8.
- Received April 13, 1995.
- Accepted October 3, 1995.
- © 1996 American Heart Association, Inc.
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