Richard D. Kenagy

Matrix metalloproteinases of vascular wall cells are increased in balloon-injured rat carotid artery * ** *

PURPOSE Although matrix metalloproteinase (MMP) expression has been correlated with proliferation and migration of various tumor cells, the relation between MMP expression and smooth muscle cell (SMC) proliferation and migration has not been established. METHODS We measured MMP expression (gelatin, casein, and elastin zymography) by vascular wall cells in balloon-injured carotid artery during the period of medial SMC proliferation, migration of SMC from the media to the intima, and subsequent intimal SMC proliferation. RESULTS The 72 and 64-kd gelatinases (presumably 72 kd type IV collagenase or MMP 2) were constitutively expressed in normal carotid arteries, and the activated (59 and 54 kd) forms of this enzyme were increased at 5 days when SMCs start to migrate. A 92 kd gelatinase (presumably 92 kd type IV collagenase or MMP 9) was increased at 24 hours, when SMCs entered the growth cycle, and decreased thereafter. A low-molecular-weight metalloproteinase with elastolytic activity was present in the adventitia, and the activity was increased at 5 days after surgery. CONCLUSIONS These results suggest that the 72 kd and 92 kd gelatinases may be involved in basement membrane and matrix degradation in the media in relation to SMC proliferation and migration, whereas the low-molecular-weight metalloproteinase may have a role in elastin turnover in the adventitia.

Matrix Metalloproteinase-9 Overexpression Enhances Vascular Smooth Muscle Cell Migration and Alters Remodeling in the Injured Rat Carotid Artery

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of atherosclerosis as well as intimal hyperplasia after vascular injury. We used Fischer rat smooth muscle cells (SMCs) overexpressing MMP-9 to determine the role of MMP-9 in migration and proliferation as well as in vessel remodeling after balloon denudation. Fischer rat SMCs were stably transfected with a cDNA for rat MMP-9 under the control of a tetracycline-regulatable promoter. In this system, MMP-9 was overexpressed in the absence, but not in the presence, of tetracycline. In vitro SMC migration was determined using a collagen invasion assay as well as a Boyden chamber assay. In vivo migration was determined by measuring the invasion into the medial and intimal layers of transduced SMCs seeded on the outside of the artery. Transduced SMCs were also seeded on the luminal surface, and the effect of local MMP-9 overexpression on vascular structure was measured morphometrically at intervals up to 28 days. MMP-9 overexpression enhanced SMC migration in both the collagen invasion assay and Boyden chamber in vitro, increased SMC migration into an arterial matrix in vivo, and altered vessel remodeling by increasing the vessel circumference, thinning the vessel wall and decreasing intimal matrix content. These results demonstrate that MMP-9 enhances vascular SMC migration in vitro and in vivo and alters postinjury vascular remodeling.

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

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 alpha- and beta-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.

Heparin inhibits the induction of three matrix metalloproteinases (stromelysin, 92-kD gelatinase, and collagenase) in primate arterial smooth muscle cells.

Heparin inhibits the migration and proliferation of arterial smooth muscle cells and modifies the extracellular matrix. These effects may be the result of heparins effects on proteinases that degrade the matrix. We have previously reported that heparin inhibits the induction of tissue-type plasminogen activator and interstitial collagenase mRNA. We have investigated the possibility that heparin affects other members of the matrix metalloproteinase family. Phorbol ester increased the levels of mRNA of collagenase, 92-kD gelatinase and stromelysin as well as the synthesis of these proteins. These effects were inhibited by heparin, but not by other glycosaminoglycans, in a dose-dependent manner. The induction of these matrix metalloproteinases was also inhibited by staurosporine and pretreatment with phorbol ester indicating the involvement of the protein kinase C pathway. In contrast, the 72-kD gelatinase was expressed constitutively and was not affected by phorbol ester or heparin. Tissue inhibitor of metalloproteinase-1 was expressed constitutively and was slightly increased by phorbol ester. It was not affected by heparin. Thus, heparin inhibits the production of four proteinases (tissue plasminogen activator, collagenase, stromelysin and 92-kD gelatinase) that form an interdependent system capable of degrading all the major components of the extracellular matrix.

The Role of Plasminogen, Plasminogen Activators, and Matrix Metalloproteinases in Primate Arterial Smooth Muscle Cell Migration

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

PDGFβ Receptor Blockade Inhibits Intimal Hyperplasia in the Baboon

BACKGROUND We have evaluated the use of a mouse/human chimeric anti-platelet-derived growth factor-beta receptor antibody in combination with heparin to inhibit intimal hyperplasia in the saphenous artery of the baboon after balloon angioplasty. METHODS AND RESULTS The study evaluated lesion development in sequential injuries made 28 days apart. Each animal received control treatment after the first injury and antibody/heparin therapy after the second injury to the contralateral artery. The antibody was administered by bolus intravenous injections (10 mg/kg) on study days 1, 4, 8, 15, and 22 and heparin coadministered by continuous intravenous infusion at a dose of 0.13 mg/kg per hour. Morphometric analysis of tissue sections showed a 53% decrease in intimal area after antibody/heparin treatment (P=0.005), corresponding to a 40% decrease in the intima-to-media ratio (P=0.005). Smooth muscle cell proliferation in the injured wall, measured at both 4 and 29 days after balloon injury, were similar in the control and antibody/heparin-treated animals. CONCLUSIONS These data suggest that platelet-derived growth factor plays a key role in the development of intimal lesions at sites of acute vascular injury in the nonhuman primate.

Platelet-Derived Growth Factor-BB–Induced Human Smooth Muscle Cell Proliferation Depends on Basic FGF Release and FGFR-1 Activation

We have shown that the G protein–coupled receptor (GPCR) agonists, thrombin and Factor Xa, stimulate smooth muscle cell (SMC) proliferation through transactivation of the EGF receptor (EGFR) or the FGF receptor (FGFR), both of which are tyrosine kinase receptors. In the present study, we investigated whether platelet-derived growth factor (PDGF), a tyrosine kinase receptor agonist, might transactivate another tyrosine kinase receptor to induce SMC proliferation. Because heparin inhibits PDGF-mediated proliferation in human SMCs, we investigated whether the heparin-binding growth factor basic fibroblast growth factor (bFGF) and one of its receptors, FGFR-1, play a role in the response of human arterial SMCs to PDGF-BB. PDGF-BB induced the release of bFGF and sustained phosphorylation of FGFR-1 (30 minutes to 6 hours). A bFGF-neutralizing antibody inhibited PDGF-BB–mediated phosphorylation of FGFR-1, DNA synthesis, and cell proliferation. In the presence of bFGF antibody, PDGF-BB–induced early activation of ERK (0 to 60 minutes) was not affected, whereas late ERK activation (2 to 4 hours) was reduced. When FGFR-1 expression was suppressed using small interfering RNA (siRNA), ERK activation was reduced at late, but not early, time points after PDGF-BB stimulation. Addition of bFGF antibody to cells treated with siRNA to FGFR-1 had no further effect on ERK activation. Our results provide support for a novel mechanism by which PDGF-BB induces the release of bFGF and activation of FGFR-1 followed by the sustained activation of ERK and proliferation of human SMCs.

Thrombin- and Factor Xa-Induced DNA Synthesis Is Mediated by Transactivation of Fibroblast Growth Factor Receptor-1 in Human Vascular Smooth Muscle Cells

Abstract— Thrombin and factor Xa (FXa) are agonists for G protein–coupled receptors (GPRCs) and may contribute to vascular lesion formation by stimulating proliferation of vascular smooth muscle cells (SMCs). Mitogenic signaling of GPCRs requires transactivation of receptor tyrosine kinases (RTKs). In rat SMCs, thrombin transactivates the epidermal growth factor receptor (EGFR) via a pathway that involves heparin-binding EGF-like growth factor (HB-EGF) as ligand for EGFR. The purpose of this study was to investigate in human SMCs the role of receptor transactivation in the mitogenic response to thrombin and FXa. Thrombin (10 nmol/L) and FXa (100 nmol/L) cause a 3.3- and 2.6-fold increase in DNA synthesis, respectively. In human SMCs, neither thrombin nor FXa causes EGFR phosphorylation, and blockade of EGFR kinase does not inhibit DNA synthesis. However, DNA synthesis and phosphorylation of fibroblast growth factor receptor-1 (FGFR-1) induced by thrombin or FXa are inhibited by antibodies neutralizing basic fibroblast growth factor (bFGF) or by heparin. Hirudin inhibits thrombin-, but not FXa-induced mitogenesis, indicating that FXa acts independently of thrombin. We further demonstrate by ELISA that upon thrombin and FXa stimulation, bFGF is released and binds to the extracellular matrix. Our data suggest that in human vascular SMCs, both thrombin and FXa rapidly release bFGF into the pericellular matrix. This is followed by transactivation of the FGFR-1 and increased proliferation. Heparin may inhibit the mitogenic effects of thrombin and FXa in human SMCs by preventing bFGF binding to FGFR-1.

Heparin inhibits the expression of tissue-type plasminogen activator by smooth muscle cells in injured rat carotid artery.

Smooth muscle cells (SMCs) in balloon-injured rat carotid artery express tissue-type plasminogen activator (t-PA) at a time when they are migrating from the media to the intima. Since heparin inhibits SMC migration and intimal thickening, we have examined the possibility that heparin might also inhibit t-PA expression. Heparin (nonanticoagulant fraction; molecular weight, approximately 6,000) was administered by continuous intravenous infusion (1.0 mg/kg per hour) to Sprague-Dawley rats subjected to balloon injury of the left common carotid artery. At various times up to 14 days after injury, plasminogen activator expression was analyzed by zymography, plasmin generation, enzyme-linked immunosorbent assay, Northern blotting, and in situ hybridization. This dose of heparin inhibited SMC accumulation at 14 days by 60%. Both urokinase plasminogen activator (u-PA) and t-PA activity increased in injured arteries and reached a maximum at 7 days. Heparin treatment decreased t-PA, but not u-PA, activity. Total t-PA protein was decreased by treatment with heparin but not chondroitin sulfate, and the decrease in t-PA protein was associated with decreased t-PA mRNA in the media. These results in the injured rat carotid artery agree with our earlier observations that heparin inhibits t-PA gene expression in cultured baboon aortic SMCs. They also provide support for the hypothesis that heparin interferes with the expression of certain proteases required for SMC migration and proliferation.

Mechanisms of inhibition by heparin of vascular smooth muscle cell proliferation and migration

Heparin, an inhibitor of vascular smooth muscle cell proliferation and migration, affects a number of other cell functions. These effects include inhibition of growth factor binding, deposition of matrix proteins and gene expression. Various mechanisms have been proposed and, yet, how heparin works as an inhibitor remains unclear. We have postulated that heparin inhibits smooth muscle cell growth and migration by suppressing the expression of matrix-degrading enzymes such as plasminogen activators and interstitial collagenase. The molecular mechanism of heparins inhibitory action on these proteases is currently under investigation.