© 1996 American Heart Association, Inc.
Articles |
The Role of Plasminogen, Plasminogen Activators, and Matrix Metalloproteinases in Primate Arterial Smooth Muscle Cell Migration
the Departments of Surgery and Pathology (M.B., M.A.R.), University of Washington, Seattle.
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Abstract |
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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
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 
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.
Key Words: matrix metalloproteinases • plasminogen • urokinase • tissue-type plasminogen activator • smooth muscle cell migration
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Migration of arterial SMCs is considered to be an important aspect of the normal development of blood vessels, the pathogenesis of atherosclerosis, and the arterial response to injury.1 2 In other biological systems, proteinases of the MMP and plasminogen/PA families are required for the migration of tumor cells,3 4 monocytes,5 cytotrophoblasts,6 and ECs7 through the ECM. These enzymes also play a critical role in the involution of mammary and uterine tissue and in ovulation.8 9 Specific inhibitors of the MMPs, TIMP-1 and TIMP-2, and of PAs, PAI-1, inhibit invasion and metastasis.10 11 12
We investigated whether matrix-degrading proteinases also play a role in vascular SMC migration. In SMCs of the rat carotid artery, UPA, TPA, MMP9 (92-kD type IV collagenase), and an unidentified gelatinase are induced after injury.13 14 Levels of TPA and an activated form of MMP2 (72-kD type IV collagenase) increase at about the same time that SMCs appear in the neointima, whereas UPA and MMP9 levels increase at about the same time that proliferation of medial SMC begins,13 14 although it is not clear which enzymes are involved in which process. Inhibition of SMC migration into the intima after treatment with tranexamic acid15 or MMP inhibitors16 17 supports a role for plasminogen/PAs and MMPs in SMC migration. Preliminary results of studies on gene-knockout mice show that deletion of UPA decreases neointima formation after vascular injury, deletion of TPA has no effect, and deletion of PAI-1 increases neointima formation.18 However, the relative contributions of migration, proliferation, and matrix changes to these responses have not been reported. In addition, the response of mouse, rat, human, and other primate vessels to injury may be quite different. Certainly, the difference in response to angiotensin-converting enzyme inhibitors19 20 21 22 and heparin23 24 25 after arterial injury between rat and primate vessels is remarkable. For this reason, it is important to investigate the role of proteinases in the SMC response to injury in primate vessels. Little is known about the PAs and MMPs in primate vascular tissue, other than that MMP1, MMP2, MMP3, MMP9, PAI-1, and TIMP-1 are expressed in human atherosclerotic arterial tissue.26 27 28 29 30 31 In this article, we report the results of our investigations on the role of proteinases in SMC migration through native matrix of baboon aortic explants.
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Materials
Reagents and supplies were purchased from Sigma Chemical Co unless indicated otherwise. Electrophoresis supplies were purchased from Bio-Rad and National Diagnostics. Rabbit anti-human MMP2 and rabbit anti-human MMP9 were kind gifts from William Stetler-Stevenson (National Cancer Institute, National Institutes of Health, Bethesda, Md) and Howard Welgus (Jewish Hospital, Washington University, St Louis, Mo), respectively. A monoclonal anti-propeptide of MMP2 was purchased from Molecular Oncology, Inc.
2-Macroglobulin and 1-proteinase inhibitor (Prolastin) were obtained from Athens Research and Technology, Inc, and Miles, respectively. Recombinant human and bovine lung aprotinin were obtained from NovoNordisk and Sigma, respectively. Human Glu-plasminogen and antibodies to human urokinase (No. 398 and No. 394) and TPA (No. 387 and 374B) came from American Diagnostica. An antibody against bovine carboxylase (No. 170; Zymogenetics, Inc), an irrelevant monoclonal IgG1, was used as a control. BB-94 (Batimistat), a kind gift from British Biotech Pharmaceuticals Ltd, is a hydroxamic acid analogue inhibitor of MMPs and does not affect other metalloproteinases32 or PAs (R.D.K. and A.W.C., unpublished data, 1995). The half-maximal inhibiting concentrations of BB-94 against the MMPs are as follows: 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.32 BB-374 has the same pseudopeptide backbone (including stereochemistry) as BB-94 but has a carboximide residue rather than the hydroxamic acid residue. Also, BB-374 is 1000-fold less potent against MMPs than is BB-94 (personal communication, Alan Drummond, British Biotech Limited, 1995).
Animal Model
Male baboons (Papio cynocephalus), 2 to 3 years old and weighing 9 to 10 kg, were subjected to balloon catheter injury. Anesthesia was induced with ketamine hydrochloride (10 mg/kg body wt) and maintained with inhaled halothane. A side branch artery proximal to the saphenous-popliteal bifurcation was cannulated to introduce a 2F Fogarty embolectomy catheter (V. Mueller). The catheter was passed into the saphenous artery, and the 10-cm segment distal to the bifurcation was injured by three passages of the inflated balloon. The side branch was then ligated and circulation restored. Prior to injury all animals received 100 U heparin/kg IV.
The baboons received IM 30 mg BrdU (Boehringer Mannheim) per kilogram of body weight at 1, 9, and 17 hours before they were killed by barbiturate overdose. Animals were perfused with lactated Ringer's solution at physiological pressure. Segments (5 mm) were taken at the proximal, middle, and distal portions of the injured area and placed in neutral formalin overnight. After the tissue was embedded in paraffin, cross sections were deparaffinized and rehydrated. Sections were stained with anti-BrdU antibody (Boehringer Mannheim) with a horseradish peroxidase–labeled secondary antibody.25 The labeling index for the media and intima was determined by dividing the number of labeled cells by the total number of cells in cross sections and multiplying by 100.
All animal care and experimental procedures were performed at the University of Washington Regional Primate Research Center in accordance with state and federal laws. Animal protocols were approved by the University of Washington Animal Care Committee and conformed with guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and by the National Institutes of Health in publication No. 86-23.
Preparation of Explants and Migration Assay
Thoracic aortas of male baboons (Papio cynocephalus) 
3 years old were obtained, the periadventitial fat was removed, and the endothelial layers were scraped off with a polytetrafluoroethylene (Teflon) scraper. The inner media was dissected from the adventitia without further treatment in DMEM with 25 mmol/L HEPES, pH 7.4. Using a McIlwain tissue chopper (Brinkman) and the method described by McMurray and colleagues,33 we chopped the media into 1-mm2 pieces, 15 of which were then placed into each 25-cm2 tissue culture flask. Excess medium was aspirated from around each explant. The flasks were left open for a few minutes until no moisture was visible around explants but before the explants became desiccated. Then 1 mL DMEM with 5 µg transferrin, 6 µg insulin, and 1 mg ovalbumin per milliliter and any test factors was added, so that each explant was wet. All experiments were performed with continuous exposure to test factors starting at the time of explantation. BB-94 and BB-374 were dissolved in DMSO at 2 mg/mL and diluted in culture medium as required. Controls for DMSO were made with corresponding concentrations of DMSO. Explants were examined daily for evidence of any cell migration onto the plastic culture surface and were counted as positive if 1 cell was observed. Because migration of only 1 cell was required for an explant to be counted as positive, this method precludes any involvement of proliferating cells outside the explants. The medium was changed on day 7.
Immunocytochemistry
To characterize the cells migrating from explants, both cells and explants were fixed in cold methanol after 2 weeks. Fixed cells in flasks were stained for SM -actin or the macrophage marker CD68 with monoclonal antibodies from Boehringer Mannheim or Dako Corp, respectively, and an avidin-biotin-peroxidase system (Vector).
DNA Mass and [3H]Thymidine Incorporation
The time of cell entry into the S phase was determined by exposing the explants to 1.0 µCi [3H]thymidine per milliliter for 24-hour pulses between 0 and 14 days. In other experiments, the effect of various agents on cell entry into the S phase was determined by continuously exposing the explants to [3H]thymidine (0.1 µCi/mL) starting the day after the explants were made and stopping on day 7. To assay DNA mass and thymidine incorporation the medium was first removed. Then the explants were removed from the flasks and placed in 12x75-mm glass tubes. NaOH (1 mL, 1N) was added to the explants for 48 hours or until all tissue was completely digested. The NaOH was then neutralized with 1N HCl. The digest was brought to 6% TCA and left at 4°C overnight. The DNA content of the TCA-precipitable material was determined as described previously for rat carotid arteries,34 and the radioactivity of the TCA-precipitable material was quantitated by liquid scintillation spectroscopy. In some experiments, autoradiography was performed on sections fixed in methanol–Carnoy's solution as described.34
ELISA Analysis
Double-antibody sandwich ELISAs for UPA and TPA were obtained from American Diagnostica, Inc. The UPA assay detects HMW single- and two-chain UPA, while the TPA ELISA detects single- and two-chain TPA. Both assays also detect the PA complexed to inhibitors35 (R.D.K. and A.W.C., unpublished data, 1995).
Immunocapture Assay
An immunocapture assay for UPA was performed as described.36 This assay detects all activatable UPA, including HMW single- and two-chain UPA as well as LMW UPA. In brief, microtiter plate wells were coated with 0.5 µg anti-UPA overnight at 4°C (No. 398, American Diagnostica), washed (in PBS, pH 7.4, with 0.05% Tween 20), and blocked with 0.5% BSA. The samples were bound to the plate for 2 hours at room temperature. After another wash, a buffer containing 0.1 mol/L Tris, pH 8.1, 0.1% Triton X-100, 50 µg/mL Glu-plasminogen, and 0.4 mmol/L S-2251 (Kabi-Vitrum) was added to the wells. The plate was incubated at 37°C and the optical density at 405 nm monitored every 30 minutes for 180 minutes. Human HMW UPA was used as the standard (Calbiochem).
Zymography
At the times indicated, the medium was harvested and explants from flasks were extracted in 200 µL of 2 mol/L guanidine HCl, 0.2% Triton X-100, 10 mmol/L CaCl2, and 50 mmol/L Tris, pH 7.5,37 with a polytetrafluoroethylene (Teflon) pestle in a 1.5-mL microfuge tube. The explant extract was dialyzed overnight twice against 500 volumes of 50 mmol/L Tris and 0.2% Triton X-100, pH 7.4. Samples of injured or contralateral uninjured saphenous arteries were extracted directly into sample buffer for SDS-PAGE (0.1 mol/L Tris, pH 6.8; 2% SDS; 20% glycerol; 0.1% bromphenol blue; and 0.1% pyronin red). Zymography for PAs and MMPs was performed as described previously.13 38 Equal amounts of protein were loaded per lane for extracts of explants. Since the mass of DNA per flask was not altered by any treatment, equal volumes of medium were loaded per lane.
Identification of Proteinases
Identification of PAs was achieved by incorporating specific blocking antibodies (American Diagnostica No. 398 for UPA and No. 387 for TPA at 200 µg/mL) or 1 mmol/L amiloride (which blocks UPA activity) into the casein/plasminogen zymography underlay. To identify MMP9, 0.4 mL of conditioned medium was immunoprecipitated with antiserum to MMP9 or normal rabbit serum as previously described,38 and the precipitates were subjected to gelatin zymography. To identify MMP2, IgG purified by protein A affinity from either normal rabbit serum or antiserum to MMP2 was added to the conditioned medium at a concentration of 50 µg/mL. After a 30-minute incubation at room temperature, the medium was subjected to gelatin zymography. Western blotting was also performed, and the blots were developed with a monoclonal antibody against a proregion peptide of proMMP2 as described previously.38
Statistics
Analysis of results by the paired t test or Wilcoxon signed rank test was performed with SPSS/PC+ (SPSS, Inc) with corrections for multiple comparisons where appropriate. Explant experiments were done with single or multiple flasks for each condition with explants from a single animal. All values from in vivo and in vitro experiments are the mean±SEM from the indicated number of animals.
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Results |
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Effects of Injury on Arterial SMC Proliferation and Proteinase Expression In Vivo
The saphenous artery contained preexisting intimal SMCs (mean±SEM, 152±8 per cross section; n=5), which were only partially removed by balloon catheterization (67±5 per cross section; n=2). The number of intimal SMCs on days 1 and 4 (28±8 and 82±32, respectively; n=3 to 4) was about the same as that immediately after injury. This observation suggests that little or no migration or cell division had occurred, although both medial and intimal SMCs had entered the S phase by day 4 (Fig 1
). The number of intimal SMCs increased by days 7 and 14 (169±76 and 301±64 per cross section, respectively; n=3). Both intimal and medial SMC proliferation decreased by day 14, with the decline in medial proliferation being more precipitous. In this model of arterial injury, a continuous endothelium was restored by day 14 (S.V. et al, unpublished data, 1995).
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Gelatin zymography of arterial extracts showed that MMP9 content increased within 1 day in the injured artery compared with the contralateral control artery, while MMP2 content was not significantly altered (Fig 2). Casein zymography also indicated that UPA activity increased by days 1 and 4 but decreased thereafter to baseline levels. TPA was not detected by zymography (R.D.K. et al, unpublished data, 1995).
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Characterization of Baboon Aortic Medial Explants
We made medial explants from the thoracic aorta because it provides an essentially pure population of SMCs. A typical starting preparation of arterial media (stripped of endothelium and adventitia) ready for cutting into explants is shown in Fig 3A. The media stained uniformly positive for SM -actin and negative for the EC marker von Willebrand factor (S.V. et al, unpublished data, 1995).
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To determine whether explants in vitro behaved similarly to the injured artery in vivo, we measured SMC proliferation and proteinase production in the explants. [3H]thymidine incorporation into DNA increased 10-fold between days 2 and 3 (Fig 4A), which is consistent with in vivo results (Fig 1
). Autoradiographic analysis of sections from the explants showed that labeled SMCs were randomly distributed throughout the explants (H. Lea and R.D. Kenagy, unpublished data, 1994). The thymidine labeling index for 24-hour pulses on days 2 through 7 ranged from 0% to 12% (H. Lea and R.D. Kenagy, unpublished data, 1994). The total DNA content of the explants decreased by 30% by day 7 (Fig 4B; P<.05), but returned to time-0 values by day 14.
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Proteinase Production by Aortic Explants
Bands of gelatinase activity at 60, 65, 70, and 100 kD were observed in gelatin zymograms of the medium 7 days after explantation. An antibody against a peptide of the amino-terminal prodomain of human MMP2 stained a 70-kD band on Western blots of 7-day conditioned medium (Fig 5A), thereby identifying this band as the proenzyme of MMP2. Gelatinase activity at 70, 65 (faint band), and 60 kD was removed (Fig 5B) by preincubation of the medium with an antibody that reacts with all forms of the enzyme.39 This expression of proMMP2 and activated MMP2 has been observed in cultured baboon SMCs38 and other cell types.39 40 41 Additionally, minor bands at >100 kD were removed by the antibody to MMP2, suggesting that multimers or complexes containing MMP2 were also present. Gelatinase activity at 100 kD was identified as MMP9 by immunoprecipitation followed by zymography (Fig 5C). All of the gelatinases were inhibited by 20 mmol/L EDTA. Treatment of the medium with 1 mmol/L p-aminophenyl mercuric acetate, which activates MMPs, increased the activity of the 65-kD form of MMP2 and led to the appearance of an 89-kD band (data not presented).
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MMP2 activity increased in the explants after 1 day and also accumulated in the medium (Fig 6A), in which the 70-kD band predominated and could be detected as early as 1 day after explantation. In the explant extracts, the relative intensity of the three MMP2 bands varied from animal to animal, with the 65- and 60-kD forms predominating (R.D.K. and A.W.C., unpublished data, 1995). MMP9 was consistently visible in the zymograms of 3-day unconcentrated medium, but the signal was too faint to be visible in photographs. After the medium was concentrated, MMP9 was clearly visible as early as 2 days (Fig 6B) and accumulated thereafter.
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In extract explants, UPA (54 kD) and UPA/inhibitor complex (90 to 110 kD) were identified in casein zymograms. The identity of UPA was confirmed by amiloride and antibody inhibition of UPA (data not shown). The level of UPA in the explants increased dramatically after 1 day, with a steady accumulation for at least 7 days in both explants and medium (Fig 6C). The double band of UPA activity frequently observed in explant extracts may represent HMW and LMW forms of UPA complexed to an inhibitor like PAI-1. TPA, as identified by antibody inhibition with anti-TPA, was detected at 7 days in only small amounts in the medium as an HMW complex >100 kD (data not shown). However, similar amounts of TPA and UPA were detected by ELISA in the medium after 7 days (0.36±0.06 and 0.23±0.02 ng/mL, respectively; n=20), presumably because most of the TPA, unlike UPA, is bound to inhibitors and is inactive though still detectable by ELISA.
SMC Migration From Explants
SMCs migrated from the explants after a 3-day lag (Fig 7). SMCs migrated from 60% of the explants at 7 days (8.5±2.8 migrated cells per explant; n=13) and from >90% after 11 days. These migrating cells stained positive for SM -actin but not for the macrophage marker CD68 (Fig 8) or von Willebrand factor (R.D.K. and A.W.C., unpublished results, 1994). Hydroxyurea (5 mmol/L), which inhibits cell entry into the S phase by inhibiting ribonucleotide reductase, had a slight effect on migration by day 7 (79±7% of control; P<.01, n=12), although entry into the S phase clearly decreased (37±7% of control cumulative thymidine incorporation between days 1 and 7; P<.01).
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Role for Proteinases in SMC Migration
To assess the role of proteinases in SMC migration, we tested the effect of several inhibitors. Antibodies to UPA and TPA both inhibited migration at concentrations of 50 µg/mL (Fig 9A). Recombinant aprotinin at clinically therapeutic concentrations (300 µg/mL) and bovine lung aprotinin (1 TIU/mL) had no effect on migration (100±6% and 86±13% of day-7 control migration, respectively; n=3 and 10, respectively). 2-Macroglobulin decreased migration to 44±10% of day-7 control values (n=11, P<.01; on average, 8 of 15 control explants had migrating cells) at a concentration of 200 µg/mL. 1-Proteinase inhibitor also decreased migration (48±14% of day-7 control values; n=4, P=.06) at 6 mg/mL but not at 1 mg/mL.
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Since plasminogen has been reported to facilitate the migration of some cells,42 we tested the ability of exogenously added plasminogen to increase SMC migration from the explants. Increased migration was observed at plasminogen concentrations between 10 and 50 µg/mL (Fig 9B). The effect appears to be greater on day 5 because control values for the number of explants with migrating cells are lower at this time. Stimulation of cell migration by plasminogen (50 µg/mL) was inhibited by the UPA but not the TPA antibody (Fig 9C). Finally, plasminogen at a concentration of 50 µg/mL also had a small (120±11% of control; n=11) but significant (P<.05) stimulatory effect on cumulative thymidine incorporation from days 1 to 7.
We also tested the effect of MMP inhibitors. BB-94 inhibited migration >40% at doses of 0.4 and 4.0 µmol/L compared with the DMSO control. The carboximide BB-374, 1000-fold less potent against MMPs, had no effect (Fig 10) compared with the DMSO control. Interestingly, DMSO alone increased migration. Neither BB-94 nor BB-374 had a significant effect on cumulative [3H]thymidine incorporation or DNA content (all doses averaged between 100 and 117% of control; n=5 to 8).
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Relationship Between Changes in Migration and Proteinase Secretion
To determine whether there was a correlation between cell migration and proteinase secretion into the medium, we performed zymography on media from explants treated with plasminogen (which stimulated migration) and BB-94 and 2-macroglobulin (which inhibited migration). Plasminogen increased levels of the 60-kD form of MMP2 (Fig 11A, lane 5), with no apparent increase in 70-kD MMP2. This effect was decreased by anti-UPA (Fig 11A, lane 6). Basal levels of 60-kD MMP2 were also decreased by the UPA antibody (Fig 11A, lanes 2 and 3). Plasminogen had no effect on TPA levels in the medium as detected by ELISA (109±21% of control; n=6) but did decrease the amount of UPA (21±1% of control; n=4). However, plasminogen converted HMW UPA (54 kD) to LMW UPA (34 kD) (R.D.K. and A.W.C., unpublished data, 1994). LMW UPA is active but not detectable by ELISA. Plasminogen did not alter UPA activity, as detected by immunocapture assay (85±13% of control; n=4), which can measure the activity of both LMW and HMW UPA. BB-94 decreased MMP9 and the 60-kD form of MMP2 compared with the DMSO control, whereas BB-374 had no effect on the MMPs. DMSO alone increased MMP2 and MMP9 (Fig 11B). In addition, BB-94 had no effect on TPA (0.4 µmol/L BB-94, 85±17% of control; n=4) but decreased UPA levels in the medium, as measured by ELISA (0.4 µmol/L BB-94, 67±7% of control; n=6). 2-Macroglobulin had no consistent effect on the MMPs (data not presented) but decreased UPA and TPA levels (26±7% and 53±11% of control, respectively; n=4 and 5, respectively). The effects of BB-94 and 2-macroglobulin on UPA were confirmed by immunocapture assay and zymography (R.D.K. and A.W.C., unpublished data, 1994). In summary, migration inhibitors decreased UPA levels in the medium (ie, 2-macroglobulin, BB-94, and anti-UPA), and the level of the 60-kD MMP2 was positively correlated with migration (ie, increased by plasminogen and decreased by BB-94 and anti-UPA).
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Discussion |
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Arterial Explants Provide a Model of In Vivo Events
Information about the vascular response to injury in vivo and the regulation of SMC proliferation and migration has come primarily from small-animal models. Experiments in vivo in nonhuman primates are difficult. Therefore, to establish a "bridge" between small-animal work and nonhuman primates, we chose to use arterial explants. We measured SMC migration by determining the fraction of explants that exhibited
1 cell on the plastic. This assay is based on the premise that the first cell on the plastic is representative of the migrating cell population. This premise is supported by the observation that our conclusions were the same when the number of cells per explant at 7 days was used as the method of quantifying migration (R.D.K. and A.W.C., unpublished data, 1995). However, to measure migration only (instead of migration plus proliferation), we present "single cell" data. Our results demonstrate that SMCs in explants behave similarly to SMCs in the injured artery. For example, the lag time before SMCs appeared outside the explants was the same as that observed before SMCs migrated into the intima of the balloon-injured rat carotid artery,34 and the steady migration from explants (17% of explants became positive for migration each day from days 5 through 8) fits the steady migration of nondividing SMCs into the intima of the injured rat carotid artery after 3 days.43 There is also a similar delay before SMC entry into the S phase in baboon aortic explants, in the rat,34 and in baboon arteries (Fig 1) after balloon injury. Direct comparison between the baboon artery in vivo and the explants regarding SMC migration is not possible because of the presence of intimal SMCs before and immediately after injury. We do not know how to distinguish between intimal cells that have proliferated from proliferating medial SMCs that migrated and quiescent intimal SMCs from quiescent medial SMCs that migrated. However, the increase in intimal SMCs between days 4 and 7 after injury is consistent with the migration kinetics of explants. Finally, SMCs undergo other phenotypic changes after arterial injury and explantation. In rats and baboons, expression of UPA13 and MMP914 16 is increased in vivo and in explants. Inhibition of mitogenesis in explants by hydroxyurea only partially inhibited migration, a finding consistent with other in vivo and in vitro data43 44 that indicate that migration can occur independently of SMC entry into the S phase. Explants provide a system for studying factors required for cell migration and proliferation. On the basis of the number of intimal and medial SMCs 4 and 7 days after balloon injury in vivo, the fraction of medial SMCs that migrated into the intima is at most 10% (S.V. et al, unpublished data, 1995). This figure is an overestimate because it ignores the contribution of proliferation to the increase in numbers of intimal cells. The fraction of SMCs that migrate from medial explants (<0.1%, if one assumes 6 pg DNA per cell) is much smaller, perhaps because exogenous factors needed for maximal migration are absent. Pituitary-dependent plasma factors are known to play an important role in SMC proliferation after arterial injury.45 Platelet-derived growth factor B dimer, basic fibroblast growth factor,46 and plasminogen stimulate SMC migration from explants. In addition, there is no chemotactic gradient in this system, although it is not clear whether chemotaxis plays a role in vivo.
Other factors such as wall tension, shear force and pressure, neural factors, and cells (eg, ECs and lymphocytes) may play important roles that could be studied in vitro using explants or organ cultures. For example, the prolonged elevation of thymidine incorporation in explants compared with that in the in vivo artery may be explained by the absence of endothelium in the explants. Healing of the endothelium in vivo is complete by 14 days after injury (S.V. et al, unpublished data, 1995). The effects of a preexisting lesion could be explored by using explants made from arteries of atherosclerotic (eg, cholesterol-fed) primates. We believe that explants of human arteries might be useful for modeling the human vascular response to injury. In preliminary experiments we found that explants of human aortic media and intima secreted UPA, MMP2, and MMP9 in the same pattern seen in baboon explants (R.D.K. and A.W.C., unpublished data, 1994).
Role of MMPs and PAs in Migration
Previous observations of the injured rat carotid artery model, which showed correlations between SMC migration and proliferation and the production of PAs,13 47 support the conclusion that plasminogen and PAs promote SMC migration through the matrix. The inhibition of SMC migration in the rat carotid artery by tranexamic acid15 and the decrease in intimal thickening after arterial injury in the UPA-knockout mouse18 are consistent with this hypothesis. In addition, explants made from arteries of UPA- but not TPA-knockout mice show delayed migration by the "first cell out" definition of migration (Peter Carmeliet, personal communication, 1995). Both UPA48 49 and TPA50 have been reported to stimulate EC migration in vitro. For UPA this action is independent of enzymatic activity.49 Both UPA and TPA have also been shown to stimulate proliferation of fibroblasts, renal cells, or tumor cells51 52 53 ; TPA stimulates SMC proliferation in vitro.54 The studies described herein provide conclusive evidence for the role of plasminogen, TPA, and UPA in primate SMC migration and suggest a role for plasminogen in proliferation. The effect of UPA on migration appears to be greater than that of TPA. Whether UPA or TPA acts via plasminogen activation or has a direct effect is not clear, since the concentration of baboon plasminogen in explants is unknown and UPA does have activity against such proteins as fibronectin55 and proMMP2.56 57 The concentration of plasminogen in the human arterial intima has been reported to be much less than that in plasma58 but is similar to the dose at which we can see an effect on migration (ie, 10 µg/mL). Finally, the lack of an effect of aprotinin on migration may be due to protection from inhibition of active proteinases bound to receptors. That aprotinin was active is clear because it prevented the conversion of HMW UPA to LMW UPA caused by the addition of plasminogen to the explants (R.D.K. and A.W.C., unpublished data, 1994).
The UPA receptor has been shown to play a key role in migration and metastasis of various tumor cells.59 60 61 There are also cellular receptors for plasminogen and TPA.62 63 64 These receptors bring plasminogen and its activators into proximity on the cell surface. In addition, binding of ligands to their receptors affords some but not complete protection from some inhibitors. Single-chain UPA is inactive but can acquire some activity after binding to its receptor.65 Single-chain UPA is converted to the active two-chain form by plasmin. SMCs have been reported to possess UPA receptors66 that move to the leading edge during cell migration.67 However, further work is necessary to determine the role of these receptors in SMC migration in vivo.
Our results extend the findings of Southgate et al,68 who found that synthetic MMP inhibitors inhibit SMC proliferation in aortic explants. Our results demonstrate inhibition of SMC migration by an MMP inhibitor. The general endoproteinase inhibitor 2-macroglobulin also inhibited migration, although it can also inhibit migration by binding growth factors such as fibroblast growth factor and platelet-derived growth factor.69 Differences in species, drugs, or drug dosage may be the reason why we did not find a significant effect of MMP inhibitor on [3H]thymidine incorporation. Like Southgate et al,68 we have also shown that MMP inhibitors decrease MMP9 secretion as well as MMP2 activation. Whether this effect changes the net MMP activity has not been established. It is interesting that DMSO alone increases both migration and MMP9 and MMP2 secretion. This is not surprising, given the many effects of DMSO on gene expression.70
Other recent work defines the role of MMPs in vascular SMC migration. Pauly et al71 showed that cultured rat SMCs required active MMP2 for invasion of a reconstituted basement membrane matrix, and Bendeck et al16 and Zempo et al17 showed that synthetic MMP inhibitors decreased rat SMC migration and proliferation in vivo after arterial injury. The observation that an MMP prosegment peptide,71 BB-94 (unpublished observations), and an anti-MMP2 antibody71 affect SMC migration in a chemotaxis chamber system only when a thick ECM is present suggests that these factors act by decreasing MMP degradation activity.
The plasminogen/PA and MMP systems of enzymes are interconnected. Plasmin72 73 74 can activate MMP1, MMP9, or MMP3. We as well as Wong et al75 showed apparent activation of MMP2 to 60 kD by treatment with plasminogen that was mediated by UPA. Other investigators have reported that plasmin has no effect on the activity of purified MMP2, although plasmin can cause limited cleavage from 72 kD to 70 kD.74 If 60-kD MMP2 does become activated, then these data suggest that another factor present in explant and cell culture systems is needed to activate MMP2. A new group of MMPs, called membrane-type MMPs, may act in this way.76 Further work is needed to clarify this question. Because TPA is secreted in its active form, it is possible that TPA is the initial activator of plasminogen. Plasmin might then convert single-chain UPA to active two-chain UPA. In addition, thrombin57 can convert proMMP2 to apparently activated forms. Some MMPs can degrade major inhibitors of serine proteinases, such as 1-proteinase inhibitor.77 Whether a combination of inhibitors of MMP and PAs would have additive effects remains to be determined.
In summary, our results demonstrate the positive role that MMPs, plasminogen, UPA, and TPA play in the migration of primate SMCs through the arterial matrix. There are striking similarities between the response of nonhuman primate arteries to injury in vivo and to explantation in vitro. This suggests that human arterial explants may provide a useful model of the response of human arteries to injury.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health, Bethesda, Md (HL30946, HL18645 [to A.W.C.] and RR00166 [to the Northwest Regional Primate Center]). Our thanks are extended to Randolph Geary, MD, Thomas R. Kirkman, Larry Kraiss, MD, and Sandro Lepidi, MD, for procuring the aortic specimens and to Kitty Ratcliff and Holly Lea for technical assistance.
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Footnotes |
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Reprint requests to Richard Kenagy, PhD, University of Washington School of Medicine, Department of Surgery, AA404 Health Sciences Bldg, Box 356410, Seattle, WA 98195-6410. E-mail rkenagy@u.washington.edu.
Presented in part as abstracts at Experimental Biology 93, New Orleans, La, March 28-April 1, 1993, and at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form in Circulation. 1995;92(suppl II):II-559.
Received July 21, 1995;
revision received April 2, 1996;
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H. Wang and J. A. Keiser Vascular Endothelial Growth Factor Upregulates the Expression of Matrix Metalloproteinases in Vascular Smooth Muscle Cells : Role of flt-1 Circ. Res., October 19, 1998; 83(8): 832 - 840. [Abstract] [Full Text] [PDF]
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 D. R. Todor, I. Lewis, G. Bruno, and D. Chyatte Identification of a Serum Gelatinase Associated With the Occurrence of Cerebral Aneurysms as Pro-Matrix Metalloproteinase-2 Stroke, August 1, 1998; 29(8): 1580 - 1583. [Abstract] [Full Text] [PDF]
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E. Allaire, D. Hasenstab, R. D. Kenagy, B. Starcher, M. M. Clowes, and A. W. Clowes Prevention of Aneurysm Development and Rupture by Local Overexpression of Plasminogen Activator Inhibitor-1 Circulation, July 21, 1998; 98(3): 249 - 255. [Abstract] [Full Text] [PDF]
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M.-L. Bochaton-Piallat, G. Gabbiani, and M. S. Pepper Plasminogen Activator Expression in Rat Arterial Smooth Muscle Cells Depends on Their Phenotype and Is Modulated by Cytokines Circ. Res., June 1, 1998; 82(10): 1086 - 1093. [Abstract] [Full Text] [PDF]
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S. Banai, Y. Wolf, G. Golomb, A. Pearle, J. Waltenberger, I. Fishbein, A. Schneider, A. Gazit, L. Perez, R. Huber, et al. PDGF-Receptor Tyrosine Kinase Blocker AG1295 Selectively Attenuates Smooth Muscle Cell Growth In Vitro and Reduces Neointimal Formation After Balloon Angioplasty in Swine Circulation, May 19, 1998; 97(19): 1960 - 1969. [Abstract] [Full Text] [PDF]
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 A Delannoy-Courdent, V Mattot, V Fafeur, W Fauquette, I Pollet, T Calmels, C Vercamer, B Boilly, B Vandenbunder, and X Desbiens The expression of an Ets1 transcription factor lacking its activation domain decreases uPA proteolytic activity and cell motility, and impairs normal tubulogenesis and cancerous scattering in mammary epithelial cells J. Cell Sci., January 6, 1998; 111(11): 1521 - 1534. [Abstract] [PDF]
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R. D. Kenagy, C. E. Hart, W. G. Stetler-Stevenson, and A. W. Clowes Primate Smooth Muscle Cell Migration From Aortic Explants Is Mediated by Endogenous Platelet-Derived Growth Factor and Basic Fibroblast Growth Factor Acting Through Matrix Metalloproteinases 2 and 9 Circulation, November 18, 1997; 96(10): 3555 - 3560. [Abstract] [Full Text]
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S. J. George, J. L. Johnson, G. D. Angelini, and J. Y. Jeremy Short-term Exposure to Thapsigargin Inhibits Neointima Formation in Human Saphenous Vein Arterioscler Thromb Vasc Biol, November 1, 1997; 17(11): 2500 - 2506. [Abstract] [Full Text]
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