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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:28-33

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:28-33.)
© 1996 American Heart Association, Inc.

Articles

Regulation of Vascular Smooth Muscle Cell Migration and Proliferation In Vitro and in Injured Rat Arteries by a Synthetic Matrix Metalloproteinase Inhibitor

Nobuya Zempo; Noriyuki Koyama; Richard D. Kenagy; Holly J. Lea; Alexander W. Clowes

From the Department of Surgery, University of Washington, Seattle.

Correspondence to Alexander W. Clowes, MD, Department of Surgery, RF-25, University of Washington, Seattle, WA 98195.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Abstract Smooth muscle cell (SMC) migration and proliferation and extracellular matrix remodeling are essential aspects of the arterial response to injury, vessel development, and atherogenesis. Matrix metalloproteinase (MMP) expression is associated with SMC proliferation and migration after arterial injury. To assess the role of MMPs in SMC proliferation and migration and intimal thickening, we measured the effect of the synthetic MMP inhibitor BB94 (Batimastat) on DNA synthesis and migration of SMCs in vitro as well as the formation of a neointima after balloon injury to the rat carotid artery. BB94 dose-dependently inhibited SMC migration induced by platelet-derived growth factor (PDGF)–BB through a filter coated with a thick basement membrane matrix (Matrigel) layer but did not show any inhibitory effect on SMC migration through a lightly coated filter. At concentrations up to 1 µmol/L, BB94 did not alter DNA synthesis induced by PDGF-AA or PDGF-BB. Treatment with 30 mg BB94·kg-1·d-1 IP for 7 or 14 days after balloon injury to the rat carotid artery decreased the total number of intimal SMC nuclei and suppressed intimal thickening. SMC proliferation (5-bromo-2'-deoxyuridine labeling) was decreased in the media at 2 days, whereas it was increased in the intima at 7 but not 14 days. These results suggest that BB94 inhibits intimal thickening after arterial injury by decreasing SMC migration and proliferation and support the conclusion that MMPs play a significant role in regulating intimal thickening in injured arteries.


Key Words: metalloproteinases • smooth muscle cells • rats • arterial injury • migration


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
SMC proliferation and migration and ECM remodeling are important for intimal hyperplasia after arterial injury1 2 3 and vascular grafting4 5 and in atherosclerosis.6 These events are regulated by various cytokines and growth factors secreted by platelets and vascular cells6 and might depend on matrix degradation by enzymes such as MMPs, plasminogen activators, and plasminogen. This hypothesis is based on observations in other physiological settings requiring tissue growth (tumor metastasis, wound healing, and organ remodeling).7 8 9 10 11 MMPs expressed in human atherosclerotic lesions include interstitial collagenase (MMP-1), 72-kD type IV collagenase (MMP-2), stromelysin (MMP-3), and 92-kD type IV collagenase (MMP-9).12 13 14 We reported15 16 that the expression and activation of 72-kD type IV collagenase and 92-kD type IV collagenase by SMCs are associated with increased SMC proliferation and migration after arterial injury. SMC expression of tissue-type plasminogen activator is associated with migration and that of urokinase-type plasminogen activator with mitogenesis in the balloon-injured rat carotid artery.17 Plasminogen increases SMC migration from baboon arterial explants in a urokinase-type and tissue-type plasminogen activator–dependent manner.18 19 This is of particular interest because plasminogen, when converted to plasmin by plasminogen activators, degrades a broad range of matrix molecules and can activate interstitial collagenase, stromelysin, and 92-kD type IV collagenase.20 21 These reports suggest that MMPs and other proteases may play a role in vascular remodeling.

To test the hypothesis that MMPs are required for SMC proliferation and migration, we determined whether a synthetic metalloproteinase inhibitor, BB94, inhibits these processes in vitro and in vivo.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Reagents
A synthetic MMP inhibitor, BB94 (Batimastat) {[4-(N-hydroxyamino)-2R-isobutyl-3S-(thienyl-thiomethyl)-succinyl]-L-phenylalanine-N-methylamide, MW 478}, was provided by British Biotechnology Limited. BB94 contains a peptide backbone that binds to the MMP and a hydroxamic acid group that binds to the catalytically active zinc atom. The concentrations of BB94 producing 50% inhibition against the MMPs are interstitial collagenase, 5 nmol/L; 72-kD type IV collagenase, 4 nmol/L; stromelysin, 20 nmol/L; and 92-kD type IV collagenase, 1 to 10 nmol/L.22 The activity of 72-kD and 92-kD type IV collagenases in gelatin zymograms of rat SMC–conditioned medium was inhibited by 1 to 10 nmol/L BB94 (unpublished data). Recombinant human PDGF-BB was kindly supplied by Dr Charles Hart (Zymogenetics, Inc); it was produced in a yeast expression system as described previously23 and purified to >95% homogeneity. 3H-Thymidine was purchased from Amersham.

Cell Culture
Rat aortic medial SMCs grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 200 µg streptomycin/mL, and 200 U penicillin/mL (GIBCO Laboratories) were isolated by the explant method. Cells were used between passages 3 and 15. In immunohistochemical preparations, SMCs stained positive for smooth muscle {alpha}-actin (mouse anti-human smooth muscle {alpha}-actin, Boehringer Mannheim Corp).

SMC DNA Synthesis
Confluent SMCs were trypsinized, suspended in DMEM plus 10% fetal bovine serum with streptomycin and penicillin, and seeded at 2.5x104 cells per well into 24-well plates (Corning Co). After 6 hours, the cells were washed with PBS, and the medium was changed to DMEM containing insulin (10 µg/mL), transferrin (5 µg/mL), and ovalbumin (1 mg/mL) to allow the cells to become quiescent. After 2 days, PDGF-BB at 10 ng/mL was added directly to the serum-free medium, and the cells were incubated an additional 20 hours at 37°C. 3H-Thymidine (1 µCi/well) was then added for an additional 6 hours. Incorporation of 3H-thymidine into the trichloroacetic acid–precipitable fraction was determined as previously described.24 BB94 was solubilized in ethanol at a concentration of 10 mmol/L. BB94 was added to the cells at final concentrations between 1 nmol/L and 1 µmol/L 1 hour before the addition of PDGF-BB.

SMC Migration Assay
Migration of SMCs was assayed with polycarbonate filters (Nucleopore Corp) having 5.0-µm pores in 48-well chemotaxis chambers (Neuro Probe Inc).25 The filters were coated in one of two ways. Either 2.7 µg/well of Matrigel (Collaborative Research), a basement membrane preparation, was dried at room temperature and reconstituted with distilled water before the assay,26 or filters were incubated in 100 µg/mL of Vitrogen (mainly type I collagen; Celtrix Pharmaceuticals, Inc) overnight at room temperature and dried. Cultured SMCs were trypsinized and suspended at a concentration of 5.0x105 cells/mL in serum-free DMEM with streptomycin and penicillin. A volume of 50 µL of SMC suspension was placed in the upper chamber, and 25 µL of DMEM containing 10 ng/mL of PDGF-BB was placed in the lower chamber. In some experiments, the SMC suspension was mixed with increasing concentrations of BB94 before it was placed in the upper chamber. The chamber was incubated at 37°C for 24 hours. For the migration experiments with filters coated with a thin layer of Vitrogen, the chamber was incubated for 4 hours. After incubation, the filters were removed and the SMCs on the upper side of the filter were scraped off. The SMCs that had migrated to the lower side of the filter were fixed in methanol, stained with Diff-Quick staining solution (Baxter), and counted under a microscope (x100) for quantification of SMC migration. Migration activity was expressed as the mean number of cells that had migrated per x100 field.

Animal Model
Three-month-old male Sprague-Dawley rats (370 to 400 g) were obtained from Bantin & Kingman, Inc. Rats were anesthetized with 1.0 mL/kg IM of a solution containing acepromazine 1 mg/mL (Fermenta Animal Health Corp), ketamine 50 mg/mL (Aveco, Inc), and xylazine 5 mg/mL (Mobay Corp) in saline and were cared for according to the `Principles of Laboratory Animal Care' (formulated by the National Society for Medical Research) and the Guide for the Care and Use of Laboratory Animals (NIH publication 86-23, revised 1985). Endothelial denudation was performed in the left common carotid artery by the passage of a 2F balloon embolectomy catheter (V. Mueller) as described previously.1 A 2.5-mg/mL suspension of BB94 was made by sonication in PBS, pH 7.2, with Tween-80 (0.01%).22 Rats received a daily injection of the BB94 suspension at 1 to 30 mg·kg-1·d-1 IP from 2 to 14 days. Control animals were given vehicle alone.

Morphometry
Animals were killed with an overdose of pentobarbital, and the carotid arteries were fixed by perfusion with 10% formalin or 4% paraformaldehyde in PBS, pH 7.4, at 100 mm Hg. The arteries were embedded in paraffin (two segments per animal), cross-sectioned, and stained with hematoxylin-eosin. Intimal and medial cross-sectional areas were measured as described previously,27 and the total number of nuclei in each cross section was also counted.

Measurement of SMC Proliferation in Tissues
The thymidine analogue BrdU (Boehringer Mannheim Corp) was injected (30 mg/kg IP) at 1, 9, and 17 hours before death. In some experiments, a tablet containing 50 mg of BrdU was placed subcutaneously 20 hours before death. The BrdU labeling index (the fraction of labeled nuclei times 100) was determined as described previously28 by use of a monoclonal antibody against BrdU (Boehringer Mannheim Corp) on histological cross sections.

Statistical Analysis
In vitro experiments were analyzed by one-tailed t tests. In vivo experiments were analyzed by the Mann-Whitney U test for comparisons of two groups or, for comparing multiple groups, the Kruskal-Wallis test (an extension of the Mann-Whitney test) with Dunn's correction for multiple comparisons. To determine whether BB94 increased the 7-day intimal BrdU labeling index, post hoc analysis was performed with a two-tailed t test. This analysis was confirmed (P<.04, BB94 versus control) by permutation analysis,29 with the test statistic being the difference between the means.


*    Results
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*Results
down arrowDiscussion
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Effects of BB94 on Migration and Proliferation of Cultured SMCs
SMC migration through Matrigel was stimulated by PDGF-BB at the maximally effective dose of 10 ng/mL.30 BB94 dose-dependently suppressed the migration through a thick layer of Matrigel (Fig 1Down). In contrast, BB94 at concentrations up to 100 nmol/L did not suppress SMC migration through filters lightly coated with Vitrogen. In either type of coating, BB94 did not alter cell attachment to the upper side of the membrane (unpublished data).



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  		Figure 1. Bar graph showing effect of BB94 on the migration of SMCs in vitro. SMCs were stimulated by 10 ng/mL of PDGF-BB, and the effect of BB94 on SMC migration was determined in a Matrigel invasion assay (filled columns) and with uncoated filters (open columns) as described in `Methods.' Migration in the absence of BB94 was 77.3 cells per x100 field with filters coated with 2.7 µg of Matrigel and 247.3 cells per x100 field with those coated with a thin layer of Vitrogen. Results are expressed as a percentage of PDGF-stimulated migration (mean±SD). *P<.01; **P<.05.

Proliferation (3H-thymidine incorporation) was stimulated by 10 ng/mL of either PDGF-AA or PDGF-BB. PDGF-AA increased 3H-thymidine incorporation by 2.1-fold over control, and PDGF-BB increased it by 8.2-fold. BB94 at concentrations from 1 nmol/L to 1 µmol/L had no effect on 3H-thymidine incorporation in either unstimulated SMCs or SMCs stimulated by PDGF-AA or PDGF-BB (Fig 2Down).



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  		Figure 2. Bar graph showing effect of BB94 on 3H-thymidine incorporation by unstimulated SMCs (open columns) or SMCs stimulated by PDGF-AA (solid columns) or PDGF-BB (hatched columns) at 10 ng/mL. Results are expressed as mean±SD in counts per minute (cpm) of triplicate determinations. There was no significant effect of BB94.

Suppression of Intimal Thickening in Balloon-Injured Rat Carotid Artery by BB94
Seven days after balloon injury to the left carotid artery, intimal area was increased to 0.037±0.013 mm2. Uninjured controls did not exhibit intimal thickening.1 Intraperitoneal administration of BB94 at 30 mg·kg-1·d-1 decreased intimal area by 50% (Fig 3ADown versus 3B; Fig 4ADown). BB94 also decreased the total number of SMC nuclei in the intimal layer by 40% at 7 days (Fig 4BDown). Medial area was not changed (0.135±0.009 and 0.124±0.013 mm2 for control and BB94, respectively; n=10). At 14 days, the effect of BB94 on intimal area was less, with a decrease of only 26% (Figs 3CDown, 3DDown, and 5ADown), whereas the total number of intimal SMC nuclei was decreased by 30% (Fig 5BDown). Medial area was not altered by treatment with BB94 (0.145±0.057 and 0.126±0.019 mm2 for control and BB94, respectively; n=10).



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  		Figure 3. Histological cross sections demonstrating the effect of BB94 on intimal thickening of the balloon-injured rat carotid artery at 7 days (A and B) and 14 days (C and D). Arrows pointing out the internal elastic lamina and asterisks marking the lumen demarcate intimal thickness. Rats received a daily injection of either BB94 (30 mg·kg-1·d-1 IP) (B and D) or vehicle alone (A and C) starting on the day of injury.



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  		Figure 4. Bar graphs showing (A) dose-response effect of BB94 on carotid intimal area 7 days after balloon injury. Values are mean±SD; n=5 for 1 and 10 mg·kg-1·d-1 and n=10 for control and 30 mg·kg-1·d-1. *P<.05 vs vehicle. B, Effect of BB94 (30 mg·kg-1·d-1) on the number of SMC nuclei in the intima 7 days after injury. Values are mean±SD. *P<.05 vs control; n=9 for control and 30 mg·kg-1·d-1.



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  		Figure 5. Bar graphs showing effect of BB94 (30 mg·kg-1·d-1) on intimal area (A) and the number of SMC nuclei (B) in the intima 14 days after injury. Values are mean±SD. *P<.05 vs vehicle; n=13.

To determine whether BB94 inhibited SMC proliferation, we measured the fraction of cells labeled with BrdU at 2, 7, and 14 days after carotid artery injury. BB94 decreased the BrdU labeling index in the media at 2 days after injury (Fig 6Down) but not at 7 or 14 days. The intimal BrdU-labeling index was increased by BB94 at 7 days but not altered at 14 days (Fig 6Down).



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  		Figure 6. Line graph showing effect of BB94 (30 mg·kg-1·d-1) on medial SMC proliferation at 2, 7, and 14 days (control, solid squares; BB94, open squares) after balloon injury to the rat carotid artery. Intimal SMC proliferation at 7 and 14 days (control: closed triangles, dotted lines; BB94: open triangles, dotted lines) is also presented. The BrdU labeling index is the percent labeled nuclei (mean±SEM; n=5 for days 2 and 7, n=8 for day 14). *P<.05 BB94 vs control.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
In this study, we demonstrate that the potent MMP inhibitor BB94 suppresses intimal thickening after arterial injury. BB94 also inhibited the initial wave of proliferation in the media from day 1 to day 2 after injury, although it had no significant effect on medial proliferation at later times. The synthetic MMP inhibitors Ro31-4724 and Ro31-7467 were reported to decrease entry into the S phase by SMCs in rabbit aortic explants.31 In contrast, previous results using different MMP inhibitors showed no effect on medial SMC proliferation in vivo after labeling between days 3 and 4.16 BB94 had no effect on SMC proliferation in vitro but inhibited SMC migration through Matrigel. Whether the lack of an effect of BB94 on proliferation is the result of phenotypic changes in SMCs caused by culture in vitro is not known, but this result confirms previous observations on other cell lines, such as Chinese hamster ovary cells, human foreskin fibroblasts, and various cancer cells.22 We have also observed that BB94 inhibits the migration of SMCs from baboon arterial explants in vitro.32 Together, these observations suggest that MMP inhibitors may inhibit migration and only the first wave of SMC proliferation in vivo. The maintenance of inhibitory levels of BB94 for as long as 6 months of treatment in rats (Alan Galloway, PhD, British Biotech, Ltd, personal communication, 1995) indicates that the lack of an effect of BB94 on medial proliferation after 2 days does not result from increasing metabolism of BB94.

In contrast to the effect of BB94 on medial SMC proliferation, proliferation in the intima was increased by BB94 at 7 days but not 14 days. The reason for this difference is not clear but may be the result of differences in the cells or the matrix surrounding the cells or may be a compensatory response to hemodynamic forces that results from the reduced migration of SMCs into the intima. Regarding the former possibility, intimal and medial SMCs demonstrate many differences. For example, intimal SMCs express less {alpha}1-integrin33 and can produce more PDGF34 and migrate faster35 in culture than medial SMCs. Concerning the matrix, it is clear that matrix components can signal cells (for example, via integrins) and influence SMC differentiation and growth.36 BB94 might alter turnover of matrix binding sites. BB94 can inhibit the turnover of proteoglycans in cartilage,37 and if this is true for SMCs, the balance of growth-inhibitory and -stimulatory factors in the matrix (eg, heparan sulfate proteoglycans and fibroblast growth factor) may be altered to favor proliferation in the intima and inhibition in the media. Finally, vascular growth is regulated by hemodynamic forces such as flow and shear stress38 39 40 that may determine final intimal size. These factors may cause increased proliferation in the intima as a compensatory response to decreased SMC migration from the media.

BB94 had less effect on intimal area and SMC number at 14 days than 7 days. In a preliminary report, Prescott and colleagues41 found that an MMP inhibitor also significantly decreased intimal thickening in the injured rat carotid artery after 7 days but had no effect at 21 days. One explanation for this is the increased intimal proliferation at 7 days after treatment with BB94. Another possibility is that BB94 increased ECM by decreasing matrix degradation. However, BB94 did not alter the number of nuclei per square millimeter in the intima at 14 days (ie, cell density) and by inference did not affect the amount of matrix per square millimeter. So even though BB94 decreased the absolute number of cells in the intima, there was a proportional decrease in matrix. In addition, there was no effect of treatment with BB94 on histochemical staining for collagen, elastin, and proteoglycans (unpublished data, 1995). These results indicate to us that BB94 did not increase the accumulation of matrix.

These effects of BB94 are probably mediated through the inhibition of MMP activity. BB94 inhibited SMC migration in vitro when a thick layer of Matrigel was present on the filter but failed to do so when the membranes were lightly coated. A matrix-dependent inhibitory effect on SMC migration in vitro was also demonstrated for antibodies to 72-kD type IV collagenase and peptides of the amino terminus of pro–72-kD type IV collagenase.42 Wang and colleagues43 reported that the blood levels of BB94 administered at 30 mg·kg-1·d-1 IP in mice were 10-fold higher than the IC50 for various MMPs; rats given 25 mg·kg-1·d-1 BB94 also attain levels >50 nmol/L in whole blood (Alan Galloway, PhD, British Biotech, Ltd, personal communication, 1995), well in excess of the IC50s of 72- and 92-kD type IV collagenase, interstitial collagenase, and stromelysin.22

It is not clear which MMPs play a role in the formation of the neointima. However, we recently showed that activation of 72-kD type IV collagenase is associated with SMC migration into the intima 4 to 5 days after arterial injury.15 16 This finding is consistent with the report of Pauly and coworkers,42 who found that antibodies to 72-kD type IV collagenase inhibit rat SMC migration through Matrigel in vitro. In addition, the expression of 92-kD type IV collagenase is increased within 24 hours after arterial injury as SMCs are entering the S phase. The activity of 92-kD type IV collagenase decreases thereafter.15 16 Our present results showing inhibition of medial SMC mitogenesis at 2 days suggest that 92-kD type IV collagenase may be involved in entry into the cell cycle by medial SMCs. Although these data do not rule out a role for other MMPs in SMC proliferation and migration, we have been unable to detect interstitial collagenase (MMP-1), stromelysin (MMP-3), or matrilysin (MMP-7) in the injured rat carotid artery.15

Inhibition of MMP activity by BB94 may alter cell function in more dynamic ways than just blocking the degradation of a simple physical barrier to migration. It is possible that some ECM proteins provide inhibitory signals to migration. Elastin peptides and fibronectin decrease SMC migration.44 In addition, we recently reported that heparan sulfate proteoglycans potentiate a migration-inhibitory signal induced by PDGF-AA in cultured SMCs.45 Matrix factors like fibronectin, elastin, and proteoglycans are substrates for MMPs and would be protected by treatment with BB94. A second way BB94 might act could be by changing the matrix that SMCs interact with, which in turn might alter the ability of SMCs to produce MMPs.46 Attachment of human alveolar macrophages to collagen types I and III but not type IV, laminin, fibronectin, or elastin augments the production of interstitial collagenase.47 Similar results have been reported for keratinocytes.48 The interactive effects of matrix proteins are illustrated by the observation that expression of several MMPs is increased in human synovial fibroblasts when cells are plated on a mixture of fibronectin and tenascin but not on fibronectin alone.49 These effects of the ECM on MMP expression are mediated in part by integrins.46 50 51 52

The effects of MMPs and other proteinases on cell migration, tissue development, and tissue remodeling depend on the balance of these proteinases with their endogenous inhibitors, such as TIMP-1 and -2 and PAI-1.53 54 55 56 For example, increased activity of interstitial collagenase, 72-kD and 92-kD type IV collagenases, and stromelysin is associated with accelerated tumor metastasis as well as angiogenesis.53 54 57 58 59 60 Members of the TIMP family of proteinase inhibitors and synthetic metalloproteinase inhibitors block tumor invasion and metastasis in vitro and in vivo,22 43 61 62 angiogenesis,63 64 65 collagen degradation by endothelial cells,66 and mammary gland involution.11 SMCs that overexpress TIMP-1 have reduced capacity to migrate through ECM (unpublished data, 1995). TIMP-1, TIMP-2, PAI-1, plasminogen activators, and several MMPs are expressed in human atherosclerotic plaques12 13 14 67 68 ; TIMP-2, PAI-1, plasminogen activators, and several MMPs are increased with different kinetics after injury in rat carotid arteries.15 17 69 These observations suggest that there is a complex interplay among proteinases and their inhibitors in the vascular wall.

In conclusion, the synthetic MMP inhibitor BB94 inhibits SMC migration through ECM but has no effect on DNA synthesis in vitro. BB94 also suppresses intimal thickening after injury in rat arteries and has complex effects on SMC proliferation. Thus, MMPs may play an important role in SMC migration and proliferation during the arterial response to injury.


*    Selected Abbreviations and Acronyms
 
BrdU = 5-bromo-2'-deoxyuridine
ECM = extracellular matrix
MMP(s) = matrix metalloproteinase(s)
PAI = plasminogen activator inhibitor
PDGF = platelet-derived growth factor
SMC = smooth muscle cell
TIMP = tissue inhibitor of metalloproteinase


*    Acknowledgments
 
This study was supported by grant HL-18645 from the National Institutes of Health. We thank Reza Forough and David Hassenstab for valuable discussions and Monika M. Clowes for technical advice regarding animal experiments. We also thank Paul Sampson, Chris Volinsky, and Juanjuan Fan, Department of Statistics, University of Washington, for performing the permutation analysis.

Received June 23, 1995; accepted October 20, 1995.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

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