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(Circulation. 1997;96:3555-3560.)
© 1997 American Heart Association, Inc.

Articles

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

R. D. Kenagy, PhD; C. E. Hart, PhD; W. G. Stetler-Stevenson, MD, PhD; ; A. W. Clowes, MD

From the Department of Surgery, University of Washington (R.D.K., A.W.C.), and Zymogenetics, Inc, Seattle, Wash (C.E.H.), and Laboratory of Pathology, Division of Clinical Sciences, National Cancer Institute, Bethesda, Md (W.G.S.-S.).

Correspondence to Richard Kenagy, PhD, University of Washington School of Medicine, Department of Surgery, Box 356410, 1959 NE Pacific St, Seattle, Wash 98195-6410. E-mail rkenagy{at}u.washington.edu

	Abstract

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Background Migration of arterial smooth muscle cells (SMCs) is regulated by basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and matrix metalloproteinases (MMPs) in the injured rat carotid artery. We have recently shown that migration of SMCs from baboon aortic explants depends on the activity of MMPs, but the identity of the stimulatory MMPs and the role of bFGF and PDGF in this primate system are not known.

Methods and Results These experiments were designed to determine whether MMP2, MMP9, bFGF, or PDGF plays a role in SMC migration from medial explants of baboon aorta. Explants were cultured in serum-free medium with insulin, transferrin, and ovalbumin. Neutralizing antibodies to MMP2 and antibodies that inhibit activation of proMMP9 decreased SMC migration from the aortic explants. Antibodies to bFGF and to the - and ß-subunits of the PDGF receptor also inhibited migration from the explants. Addition of bFGF and PDGF-BB but not PDGF-AA increased migration. The antibodies to bFGF but not the antibodies to the PDGF receptor subunits decreased the levels of MMP9, whereas all the antibodies decreased activated MMP2.

Conclusions These data demonstrate that SMC migration from primate aortic explants is dependent on endogenous MMP2, MMP9, PDGF, and bFGF. The data also suggest that PDGF-induced (PDGF-BB or possibly PDGF-AB) migration is dependent on MMP2, whereas bFGF-induced migration depends on both MMP2 and MMP9.

Key Words: atherosclerosis • metalloproteinases • muscle, smooth • platelet-derived factors • tissue

	Introduction

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Smooth muscle cell migration, an important factor in the development and pathophysiology of blood vessels,^1,2 is regulated in vitro by various proteinases, cytokines, and growth factors.^3–6 In the balloon-injured rat carotid artery, the correlation of changes in plasminogen activators^7–9 and MMPs^10,11 with SMC migration has suggested a role for plasminogen activators and MMPs. The inhibition of SMC migration in vivo in the rat by treatment with the plasminogen inhibitor tranexamic acid⁸ and MMP inhibitors^11,12 supports these conclusions. In addition, endogenous bFGF¹³ and PDGF⁸ have been clearly shown to stimulate SMC migration from the media to the developing neointima.

We recently developed a primate model of migration, because the observations in the rat might not be relevant for humans.^14–19 We have demonstrated that urokinase plasminogen activator, tissue plasminogen activator, and an unidentified MMP are needed for primate SMC migration in baboon aortic explants.²⁰ In this report, we demonstrate the importance of MMP2, MMP9, bFGF, and PDGF in the migration of SMCs out of baboon aortic explants.

	Methods

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Reagents and supplies were purchased from Sigma Chemical Co unless indicated otherwise. Electrophoresis supplies were from BioRad and National Diagnostics. bFGF and rabbit anti-human bFGF were obtained from R&D Systems. The anti-bFGF at 2 µg/mL neutralizes 50% of the bioactivity of 0.5 to 1.5 ng bFGF/mL and has no activity against acidic fibroblast growth factor. Recombinant PDGF-BB was purified as described previously.²¹ Monoclonal antibodies to the PDGF- (169.3.1.1.1) and PDGF-ß (163.3.1.1.1) receptors were produced as previously described.^22,23 Antibodies to MMP9 (6–6B and 7–11C) were generously provided by Deborah French, Naomi Ramos-DeSimone, and James Quigley.²⁴ These antibodies do not cross-react with other MMPs. IgG was purified from rabbit antiserum to MMP2 (Ab IVase²⁵) by protein A affinity chromatography (Pierce). This antibody has been used previously to specifically inhibit the activity of MMP2 in SMCs and in HT-1080 cells.^26,27 Normal rabbit IgG (R&D Systems, Inc) and a monoclonal antibody against bovine liver carboxylase (an IgG1; No. 170.3.1; Zymogenetics Inc) were used as control antibodies.

Explants were prepared from baboon thoracic aortas as previously described.²⁰ After the endothelial layer was removed, the inner media was dissected from the adventitia and chopped into 1-mm² pieces. Explants were then distributed to 25-cm² tissue culture flasks (15 per flask) in DMEM with 5 µg transferrin/mL, 6 µg insulin/mL, 1 mg ovalbumin/mL, and any test factors. Explants were examined daily and counted as positive for migration if one or more cells were observed on the plastic culture surface. This method of quantification precludes any involvement of proliferation of cells outside of the explants. In some experiments, the number of migrating cells per explant was determined at day 7.

Gelatin zymography for MMPs was performed as described previously²⁸ on medium harvested on day 7. Because the DNA content per flask²⁰ was not altered by any treatment (data not presented), equal volumes of medium were loaded per lane. Explants from each flask 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) with a Teflon pestle in a 1.5-mL microfuge tube. The extract was dialyzed overnight against 500 to 1000 volumes of 50 mmol/L Tris/0.2% Triton X-100 (pH 7.4) twice. Equal amounts of protein were loaded per lane for extracts of explants. Bands were quantified by scanning of gels with an HP3C Deskscan (Hewlett Packard) and analyzed with NIH Imagequant software. MMP2 and MMP9, purified as complexes of tissue inhibitor of metalloproteinases 2 and 1, respectively, were used as standards (a gift from H.G. Welgus, Washington University, St Louis, Mo)

Analysis of results was performed with the Wilcoxon signed-rank test (SPSS/PC+). The Bonferroni correction was used for multiple comparisons. In the experiments designed to test whether PDGF-AA altered the stimulatory effect of PDGF-BB, results were analyzed by repeated-measures ANOVA (SPSS/PC+). Explant experiments were done with single or multiple flasks for each condition with explants from a single animal. All values are the mean±SEM of the indicated number of animals.

	Results

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Role of MMP2 and MMP9 in SMC Migration
Because MMP activity is needed for migration of SMCs from baboon aortic explants and MMP2 and MMP9 are the major MMPs we have detected,²⁰ we tested the effect of antibodies to MMP2 and MMP9 on migration. We have previously observed that SMCs migrate out of the explants at a steady rate after a lag of 3 days. Approximately 50% of explants exhibit migrating SMCs at day 7, and >90% are positive by day 10.²⁰ The antibody to MMP2 blocked migration dose-dependently (Fig 1A). The antibodies to MMP9, which inhibit the activation of proMMP9,²⁴ also decreased the rate of SMC migration but to a lesser extent (Fig 1B).

View larger version (19K): [in this window] [in a new window]   Figure 1. Role of MMP2 and MMP9 in SMC migration. Results are expressed as % of control migration at day 7. A, Effect of anti-MMP2 (solid bars) or rabbit IgG (open bars). n=9 or 10 for 25 to 150 µg/mL and 4 for 200 µg/mL; *P<.05. On average, 9 or 10 of 15 explants were positive for migration for control groups of 25, 150, and 200 µg/mL, and for 50 µg/mL, 7 of 15 control explants were positive. B, Antibodies to MMP9 (6–6B and 7–11C) and an irrelevant IgG1 were used at 40 µg/mL. On average, 7 of 15 control explants were positive. n=5 to 9; *P<.05.

Role of bFGF and PDGF in SMC Migration
To determine whether endogenous bFGF and PDGF stimulate migration of SMCs in primate arterial tissue as is observed in the rat, we studied the effects of blocking antibodies to bFGF and the PDGF receptor subunits. Addition of antibodies to bFGF decreased migration by 50% at day 7 (Fig 2). Antibodies to the - and ß-chains of the PDGF receptor each decreased migration by 30%. A mixture of anti–- and anti–ß-antibodies at 25 µg/mL each did not inhibit SMC migration from explants to any greater extent than either antibody alone at 50 µg/mL (Fig 2). These concentrations were chosen to be greater than the maximally inhibitory concentrations on the basis of previous studies in which the anti–-chain antibody (2.5 µg/mL) inhibited PDGF-AA (100%) and PDGF-BB (80%) –mediated mitogenesis in baboon SMCs.²² The anti–ß-chain antibody does not block PDGF-AA–mediated mitogenesis because PDGF-AA activates only the PDGF -receptor. The antibody does block 50% of PDGF-BB–mediated baboon SMC mitogenesis at 5 µg/mL and up to 60% at higher concentrations.²²

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of antibodies to bFGF at 100 µg/mL and to - and ß-chains of PDGF receptor at 50 µg/mL individually or combined at 25 µg/mL each on migration as % of control migration (*P<.05; n=9 to 13; on average, 8 of 15 control explants had migrating cells on day 7).

The addition of either PDGF-BB or bFGF stimulated migration of SMCs from the explants at day 7 (Fig 3A). The relative effect of bFGF and PDGF-BB was greater at earlier times when control migration was low (eg, at day 5, migration with bFGF and PDGF-BB, each at 50 ng/mL, was 269±51% and 343±64% of control, respectively, when 4 of 15 control explants had migrating cells; n=19). Qualitatively similar results were observed when the number of cells migrating from explants was counted (Fig 3B), although this form of quantification includes migration and proliferation of cells on the plastic. In contrast to PDGF-BB, PDGF-AA (50 ng/mL) did not significantly alter migration, nor did it alter the effect of PDGF-BB when added with PDGF-BB (Fig 4).

View larger version (19K): [in this window] [in a new window]   Figure 3. Dose-response effects of bFGF (open bars) and PDGF-BB (solid bars) on migration at day 7. A, Migration measured as % of explants showing migrating cells and expressed as % of control values (*P<.05 vs control; n=7 to 19). On average, 9 of 15 control explants showed migrating cells. B, Effects on number of migrating cells per explant expressed as % control values (*P<.05 vs control; n=7 to 13). Number of cells per explant in control explants was 8.5±2.8 (mean±SEM; n=13).

View larger version (27K): [in this window] [in a new window]   Figure 4. Time course of effects of PDGF-BB, PDGF-AA, or both together (each at 50 ng/mL) on SMC migration (as % of migration-positive explants; n=6). By repeated-measures ANOVA, PDGF-AA neither had an effect of its own nor did it affect stimulatory action of PDGF-BB.

Effects of bFGF and PDGF on MMP2 and MMP9
Because MMP2, MMP9, bFGF, and PDGF activity are required for SMC migration in this model, we performed gelatin zymography to determine whether bFGF or PDGF might act by increasing either of the MMPs. Addition of 50 ng/mL bFGF increased levels of MMP9 and 60-kD MMP2 (activated MMP2) by >60%, whereas 50 ng/mL PDGF-BB had no significant effect on either (Figs 5A and 6). The antibody to bFGF decreased levels of MMP9 by 45% and the activated 60-kD form of MMP2 by 36% (Figs 5B and 6). Antibodies to the PDGF receptor subunits had no effect on MMP9 but decreased levels of the 60-kD MMP2 by 52% (Figs 5B and 6). Levels of proMMP2 (70-kD) were not changed by any of the treatments (data not presented).

View larger version (51K): [in this window] [in a new window]   Figure 5. Gelatin zymograms of 7-day conditioned medium after treatment with (A) 50 ng/mL bFGF or PDGF-BB or with (B) anti-PDGF receptor- plus anti-PDGF receptor-ß (25 µg/mL each or a control IgG1at 50 µg/mL) or anti-bFGF (or a control rabbit IgG [RbIgG] at 100 µg/mL).

View larger version (14K): [in this window] [in a new window]   Figure 6. Levels of MMP9 (A) and 60-kD MMP2 (B) by densitometric scanning of gelatin zymograms of explant conditioned medium. Explants were treated with anti-bFGF (100 µg/mL; n=5 to 6) antibodies to - and ß-chains of PDGF receptor (a combination of 25 µg/mL each; n=5; *P<.05 vs IgG control) or 50 ng/mL of either bFGF or PDGF-BB (n=10 to 20; *P<.05 vs control). Results (mean±SEM) are presented as % of control or % of appropriate IgG control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The migration of vascular SMCs plays an important role in the production of the thickened intima after vascular injury²⁹ and also in the neovascularization that occurs during wound healing, ischemia, and the formation of advanced atherosclerotic plaques.^30–32 Less is known about the regulation of SMC migration in vascular tissue of primate origin compared with the vascular tissue of other experimental animals. For example, SMC migration from human restenotic arterial tissue is faster than from primary atherosclerotic tissue or undiseased tissue.^33–35 We and others have previously shown that a general MMP inhibitor blocks migration in the injured rat carotid artery^12,36 and also in baboon aortic explants.²⁰ We have now identified MMP2 and MMP9 as MMPs that mediate primate SMC migration through native matrix. MMP2 and MMP9 have been shown to be involved in the invasion through matrix of various other cell types,³⁷ including cytotrophoblasts,³⁸ fibrosarcoma cells,²⁷ glioblastoma cells,³⁹ U937 cells,⁴⁰ and T cells.⁴¹ Rat SMCs have been shown to require MMP2 to invade Matrigel in vitro.²⁶ MMP activity is required for migration in vitro only when SMCs migrate through matrix 1,^12,26 indicating that it is the ability of the MMP2 and MMP9 to degrade one or more of their described substrates (including collagens I, III, IV, V, and XI, gelatins I and V, and elastin^42–45) that increases migration.

MMP2 and MMP9 are secreted as inactive proenzymes that must be activated by cleavage of the N-terminal prosegment. How the MMPs are being activated in the explants is not known. MMP2 can be activated by the membrane-type MMPs,^46,47 whereas binding to _Vß₃ integrin⁴⁸ may promote autoactivation. Whether membrane-type MMPs and _Vß₃ are expressed in the normal baboon artery or after injury is not known. MMP9 can be activated by MMP3, cathepsin G, tissue kallikrein, MMP2, and high concentrations of plasmin.^42,49–52 The antibodies 6–6B and 7–11C, which inhibited SMC migration from explants, inhibit activation by MMP2, MMP3, and tissue kallikrein (N. Ramos-deSimone, PhD, and J. Quigley, PhD, personal communication, 1997).

Our observation that migration of SMCs through explants is stimulated by endogenous PDGF and bFGF confirms in a primate model the results obtained in the balloon-injured rat carotid artery.^8,13,53 The inhibition of migration with antibodies to the PDGF receptor subunits was only partial, because in some experiments, antibodies were completely depleted by 7 days. However, there was a significant negative correlation between migration and the concentration of antibodies (R.D.K., C.E.H., A.W.C., unpublished data). PDGF might also be involved in the formation of a "neointima" in cultured human saphenous veins.⁵⁴ These observations support the conclusion reached in the rat experiments that a major effect of PDGF in arterial tissue is to stimulate cell migration. In addition, our results suggest that part of the stimulatory effect on migration caused by bFGF or by PDGF is mediated by MMP2 and MMP9. The induction of MMP9 that occurs after arterial injury in the baboon²⁰ and rat^10,11 may be at least partly caused by bFGF released from injured SMCs.^55,56 It is of interest that cultured rat, baboon (R.D. Kenagy, N. Zempo, and A.W. Clowes, unpublished data), and human SMCs⁵⁷ do not make MMP9 constitutively or in response to either bFGF or PDGF. These observations are consistent with reports that SMCs on plastic respond differently than when in matrix⁵⁸ and that passaged SMCs are different from primary SMCs.⁵⁹ In addition, induction of MMP9 might depend on activation by several growth factors simultaneously.^60,61

Because PDGF-BB but not PDGF-AA stimulates migration of SMCs from explants, it is likely that the BB (or possibly AB) isoform of PDGF is active in the explants. This is supported by the inhibitory effects on SMC migration of antibodies against the - and ß-PDGF receptor subunits, because only PDGF-B chain can bind to both subunits.⁶² PDGF-BB but not PDGF-AA also stimulates migration of cultured baboon SMCs.²² We have previously reported that PDGF-AA can inhibit PDGF-BB–mediated chemotaxis but not chemokinesis of SMCs in vitro.²² The lack of an effect of PDGF-AA on PDGF-BB–mediated migration from explants may be because a chemotactic gradient might not be present in this system. Our results are also consistent with the observation that injury induces PDGF-B–chain expression in rat,⁶⁴ rabbit,⁶⁵ and human⁶⁶ arteries. This leaves the role of PDGF-A chain, which is also induced after arterial injury,^66–68 less clear. The expression of PDGF-A does not correlate with proliferation in vivo,⁶⁹ even though PDGF-A chain mediates proliferation in vitro.^70–73

The use of primate models is attractive compared with the commonly used rat model because of differences between rats and humans and the similarities between nonhuman primates and humans with regard to vascular responses. We have demonstrated similar responses in the injured baboon artery and arterial explants for SMC entry into the S phase and production of urokinase plasminogen activator and MMP9.²⁰ Arterial explants may prove to be a promising model of arterial injury, which is difficult to study in primates, particularly in humans.

	Selected Abbreviations and Acronyms

 

bFGF = basic fibroblast growth factor MMP = matrix metalloproteinase PDGF = platelet-derived growth factor SMC = smooth muscle cell

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health (HL-30946, HL-18645, and RR-00166). Our thanks to Randolph Geary, MD, Thomas R. Kirkman, Larry Kraiss, MD, Sandro Lepidi, MD, Erney Mattsen, MD, and Selina Vergel for procuring aortic specimens; to Debra Gilbertson for anti-PDGF receptor antibody generation; and to Kitty Ratcliff and Holly Lea for technical assistance. We also thank Deborah French, Naomi Ramos-DeSimone, and James Quigley for their advice and for providing the antibodies to MMP9.

	Footnotes

 
Presented in part in abstract form at Experimental Biology 93, March 28–April 1, 1993, New Orleans, La (FASEB J. 1993;7:A637) and the Meeting of ASBMB, AAIP, and AAI, June 2–6, 1996, New Orleans, La (FASEB J. 1996;10:A1297).

Received May 6, 1997; revision received August 6, 1997; accepted August 13, 1997.

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