© 1996 American Heart Association, Inc.
Articles |
Overexpression of Tissue Inhibitor of Matrix Metalloproteinase-1 Inhibits Vascular Smooth Muscle Cell Functions In Vitro and In Vivo
the Departments of Surgery (R.F., N.K., H.L., M.C., S.T.N., A.W.C.) and Pathology (D.H.), University of Washington, School of Medicine, Seattle; Eisai Co, Tsukuba, Japan (N.K.); and the Department of Medical Biochemistry (S.T.N.), University of Tampere, Finland.
Correspondence to Dr Reza Forough, Department of Medical Physiology, Texas A&M University, Health Science Center, College Station, TX 77843.
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Abstract |
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Arterial smooth muscle cells (SMCs) are in a quiescent growth state under normal physiological conditions, but they can be stimulated to proliferate and migrate from one tissue compartment to another if the vessel is injured. This response might require a selective and focal increase in tissue degradation, which might be mediated through the increased production of matrix metalloproteinases (MMPs). Blockade of MMP activity might therefore inhibit the SMC response to injury. To test this hypothesis, we developed clones of rat SMCs that overexpress baboon tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), using retrovirally mediated gene transfer, and characterized the functional capacity of these cells in vitro and in vivo. SMCs transduced with the TIMP-1 vector (LTSN) grew more slowly and also migrated through a gel matrix in a Boyden chamber assay more slowly than the vector alone (LXSN) cells. The conditioned medium from LTSN cells completely inhibited the platelet-derived growth factor-BB–induced migration of normal SMCs across a matrix-coated filter, while the LXSN cell conditioned medium had no effect. The inhibitory activity in the LTSN conditioned medium could be neutralized with an antibody to TIMP-1. In vivo, local overexpression of TIMP-1 using LTSN cells implanted onto balloon-injured rat carotid artery inhibited intimal hyperplasia. Neutralizing antibodies against TIMP-1 suppressed the effect of LTSN cell seeding on intimal thickening. These data support the conclusion that the process of SMC activation leading to a thickened intima is dependent on MMP activity and that TIMP-1 could be utilized to inhibit this process.
Key Words: balloon angioplasty • gene therapy • matrix metalloproteinase • restenosis • tissue inhibitor of matrix metalloproteinase
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Introduction |
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Smooth muscle cells in the media of normal adult arteries proliferate at a very low rate (<0.1%/d) but can switch very rapidly from quiescence to a proliferative state in response to appropriate stimuli.1 For example, balloon injury to the rat carotid artery causes DNA replication in 10% to 30% of medial SMCs within 24 hours.2 Migration of SMCs from the media to the intima starts 3 days after injury, and subsequent proliferation of these SMCs results in intimal thickening and an increase in vessel mass. A similar transition of arterial SMCs to an active state has also been implicated in the failure of human vascular reconstructions after angioplasty3 and arterial bypass grafting.4 5
To proliferate and migrate, SMCs must be able to sever their attachments by focally degrading extracellular matrix without producing a general dissolution of the vascular structure. It is likely that SMCs accomplish this task in the same way as other cells by expressing simultaneously proteinases and their inhibitors.6 The relative proteolytic balance may be of great importance.7 8
MMPs are a class of matrix-degrading enzymes that might play a role in SMC activation. Increased expression of these proteinases has been detected in cultured SMCs stimulated with a variety of cytokines and also in SMCs in injured arteries and atherosclerotic plaques.9 For example, the expression of the 92-kD gelatinase (MMP-9) is induced in the rat carotid artery after balloon-catheter injury.10 Stromelysin-1 (MMP-3) and interstitial collagenase (MMP-1) are expressed in atherosclerotic plaques by SMCs and macrophages.11 12 The importance of these proteinases is evident, since their pharmacological inhibition results in decreased cell movement and reduced intimal thickening after experimental arterial injury.10 13 14 15 16 However, many of the effective drugs are relatively toxic.
Inhibition of SMC migration and proliferation might be achieved more physiologically by increasing the expression of natural inhibitors of MMPs in the SMCs themselves. This could shift the proteolytic balance toward an antiproteolytic state in the pericellular space. TIMPs, the natural inhibitors of MMPs, are secreted by many cells17 18 and are also present in serum.19 Three forms of TIMP have so far been described (TIMP-1, TIMP-2, and TIMP-3)20 21 22 23 24 ; TIMP-1 and TIMP-2 have been found in SMCs. TIMPs inhibit MMP function by forming a 1:1 molar ratio complex.25
TIMP-1 is produced by virtually all mesenchymal cells.18 26 27 28 The cDNA encoding for human TIMP-1 has been cloned and sequenced; it is identical to the gene encoding erythroid-potentiation activity.22 TIMP-1 is a secreted protein with a molecular weight of about 28 kD containing two sites of N-linked oligosaccharide linkage and six disulfide bridges.21 TIMP-1 inhibits all MMPs but has a particular affinity for interstitial collagenase (MMP-1), type IV and V collagenases (MMP-2 and MMP-9), and stromelysin (MMP-3).29 TIMP-1 expression can be induced by cytokines and growth factors such as IL-1,30 IL-6,30 and TGF-ß31 in different tissues.
Relative overexpression of the MMPs might lead to proliferation and migration, while overexpression of TIMP-1 might keep cells in the quiescent state. For example, MMPs are necessary for tumor cell invasion and migration through extracellular matrixes associated with increased MMP expression32 and can be inhibited by TIMP-1.33 Also, murine B16 metastatic cell lines transfected to overexpress recombinant TIMP-1 do not form as large tumors as the control untransfected cells when implanted subcutaneously in nude mice.34 Likewise, inhibition of TIMP-1 expression with antisense oligonucleotides35 or through targeted gene disruption by homologous recombination36 enhances the invasive properties of tumor cells in vitro and their metastatic potential in vivo and promotes invasive behavior of embryonic stem cells.
To define further the importance of MMPs and the balance of proteolytic and antiproteolytic activity for SMC activation, we have generated strains of rat aortic SMCs that overexpress TIMP-1. Our studies demonstrate that the overexpressed TIMP-1 protein inhibits SMC function in an autocrine and paracrine fashion. We have extended this observation to our previously described model of cell seeding onto rat carotid artery after balloon injury37 to demonstrate that the local overexpression of TIMP-1 inhibits intimal hyperplasia in the short term.
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Materials and Methods |
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Materials
The retroviral vector LXSN and the cell lines PE501, PA317, and NIH 3T3 TK- were all generously provided by Dr A.D. Miller, Fred Hutchinson Cancer Research Center, Seattle, Wash. The polyclonal antibody against human TIMP-1 was a generous gift of Dr H. Welgus, Washington University, St Louis, Mo. The neutralizing monoclonal antibody against bovine TIMP-1, capable of recognizing human TIMP-1, was a generous gift of Prof T. Hayakawa, Aichi-Gakuin University, Japan, and Fuji Chemical Industries, Ltd, Toyama, Japan. Recombinant human TIMP-1 made in CHO cells was kindly provided by Dr Chris Fisher, The Upjohn Company, Kalamazoo, Mich. Fischer rats were purchased from Simonsen Inc, Gilroy, Calif.
Construction of Recombinant TIMP-1 (LTSN) Retrovirus
A PCR-generated baboon TIMP-1 coding sequence38 was introduced into the unique EcoRI site of the retroviral vector LXSN to construct the recombinant TIMP-1 retroviral vector. DNA sequencing was performed to confirm the integrity of the LTSN. Viral packaging was performed according to Miller and Rosman.39 Virus titers were 7x105 cfu/mL for PA317/LTSN and 6x105 cfu/mL for PA317/LXSN.
Rat Smooth Muscle Cell Transduction
Rat smooth muscle cells were isolated from a male Fischer 344 rat and propagated in culture. Sixteen-hour virus harvests from the PA317/LXSN and PA317/LTSN cells were independently used to infect the rat SMCs. Colonies of G418-sulfate (Geneticin, GIBCO/BRL)–resistant cells were isolated as described elsewhere.40
RNA Isolation and Northern Analysis
Total cellular RNA was extracted by using the protocol of Chomczynski and Sacchi.41 RNA was then processed for Northern analysis using the method of Church and Gilbert.42
Media Collection and Western Analysis
LTSN and LXSN cells were starved for 48 hours in 100-mm dishes containing 10 mL of the starvation medium per plate. The starvation medium consisted of DMEM/F-12, supplemented with 5 µg/mL transferrin, and 6 µg/mL insulin.43 Conditioned media from each plate containing 3x106 transduced cells were collected (10 mL) and concentrated to 100 µL by centrifugation at 4°C using Centricon-10 (Amicon) filters. The concentrated samples were divided into aliquots of 25 µL and stored at -20°C for future assay.
For Western analysis, a 25-µL aliquot of the concentrated CM per lane was used, according to the manufacturer's recommendations for the ProtoBlot Western Blot AP system (Promega). The primary antibody was used at a 1:2000 dilution. A 1:7500 dilution of anti-rabbit IgG alkaline phosphatase conjugate (Promega) was used as the secondary antibody.
Reverse Gelatin Zymography and Gelatin Zymography
For reverse gelatin zymography, sample buffer without the reducing agent was added to each aliquot of the concentrated CM. Each sample was loaded without heating onto a 10% SDS–polyacrylamide gel containing 1 mg/mL bovine skin type II gelatin (Sigma Chemical Company) as substrate and 4 mL of serum-free medium conditioned by exposure to 24-hour postpartum rat uterus (see below) as the protease source in the resolving gel. The protease source is at a concentration of 26% (vol/vol) in the gel. After gel electrophoresis, the gel was rinsed in 2.5% Triton X-100 at room temperature with gentle shaking for 30 minutes to remove the SDS. Subsequently, the gel was developed for 17 hours at 37°C in a solution containing 50 mmol/L Tris-HCl (pH 7.8) and 10 mmol/L CaCl2. Next, the solution was discarded and the gel was stained with Coomassie blue.
Protein extracts from stripped-off seeded intimas were prepared and processed for reverse zymography as described elsewhere.44 A reverse zymography kit (University Technologies INTL Inc) was used when performing the assay on tissue extracts. Gelatin zymography was used to determine MMP profiles.45 46
Preparation of a Protease Source From the Rat Uterus
Uteri isolated from 24-hour-postpartum rats were minced into 
3-mm pieces and placed in a 150-mm dish containing 20 mL of the DMEM/F-12 medium for 3 days. The CM was collected and clarified by centrifugation at 1500 rpm for 7 minutes before being divided into aliquots and stored at -20°C.
Cell Growth Assay
LTSN and LXSN cells were seeded in triplicate at 1.5x104 cells per well in a 24-well plate in DMEM supplemented with 10% FBS. The media were changed after 24 hours and then left unchanged for the remaining period of the experiment. At the designated times, cells were trypsinized and quantitated by using a hemocytometer.
Invasion Assay for SMCs
The ability of SMCs to invade matrix was assayed by a modification of the Boyden chamber method, using a 48-well microchemotaxis chamber (Neuro Probe Inc) and polycarbonate filters (Nucleopore Corp) with pores of 10 µm diameter.47 The filter was precoated with 2.7 µg per well of basement membrane gel matrix (Matrigel, Collaborative Research Inc) and dried. Thirty minutes before use, a gel matrix layer was reconstituted with 10 µL of distilled water at 37°C. Cultured SMCs were trypsinized and suspended at a concentration of 5x105 cells per milliliter in serum-free DMEM. A volume of 40 µL of SMC suspension was placed in the upper compartment (cell number applied was 20 000 per well), and 25 µL DMEM with or without 10 ng/mL PDGF-BB was placed in the lower compartment. In some experiments, CM from LTSN or anti–TIMP-1 antibody was applied to the upper compartment. The chamber was incubated at 37°C under 5% CO2 in air for 6 hours. The SMCs that had invaded through the gel matrix layer 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 quantitation of SMC migration. Invasion activity was expressed as the mean number of cells that had migrated per high-power field.
Animal Seeding
Male Fischer 344 rats (300 g) were anesthetized exactly as described by Zempo et al.48 The left common carotid artery was surgically exposed. Balloon catheterization and subsequent cell seeding were performed as previously described.37 40 Approximately 105 vascular SMCs transduced with the baboon TIMP-1 vector (LTSN) were seeded into the denuded area. Controls were seeded with LXSN cells. At various times, the animals were killed, and carotid arteries were removed for further analysis. The animals 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).
Bilateral Seeding
Rats were anesthetized and their left common carotid arteries ballooned and seeded either with the LXSN or LTSN cells as described above. Serum prepared from tail vein bleeding at various times was tested by dot blot analysis for the generation of antibody to baboon TIMP-1.49 We have previously shown that antibody to baboon TIMP-1 is detectable by 3 weeks.49 Eight weeks after the initial seeding, rats were reanesthetized and their left common carotids were ballooned and seeded with LXSN or LTSN cells. After a 10-minute cell-attachment period, the circulation was restored. Two weeks later, the rats were killed and the left carotid arteries of each group removed and processed for morphometric analysis.
Morphometry
LTSN and LXSN cell–seeded carotid arteries were fixed by perfusion at 120 mm Hg pressure with 1% paraformaldehyde in PBS (pH 7.4) at 2 and 14 days. The tissue was cross-sectioned at two sites and analyzed by making tracings of individual sections by means of a camera lucida and a Hewlett-Packard digitizing pad.
DNA Synthesis
For measuring DNA synthesis in animals, groups of LTSN and LXSN cell–seeded rats received BrdU (50 mg per rat SC) 24 hours before they were killed. The carotid arteries were fixed by perfusion and removed. Sections were cut from paraffin-embedded tissues and stained with a specific monoclonal antibody to BrdU (Boehringer Mannheim). BrdU-positive and -negative nuclei were counted under a microscope, and the BrdU labeling index (percent of total nuclei) was determined separately for the intima and media.50
Electron Microscopy
For electron microscopy, LTSN and LXSN cell–seeded arteries were prepared by dehydrating cross-sectional samples through graded alcohols and embedding in Poly/Bed (Polysciences, Inc). Thin sections were cut and electron micrographs taken at x10 000 magnification from four quadrants of the entire intima as described previously51 on a JOEL 100B electron microscope (Japan Optics Electron Laboratory) at 60 kV.
The fraction of intima occupied by SMCs was determined as previously described.51 The actual smooth muscle cross-sectional volume per centimeter of carotid was obtained by multiplying the SMC fraction by the intimal area obtained using a Hewlett-Packard 9830A digitizer.
Statistical Analysis
Mann-Whitney U (SPSS 6.0, IBM) test was used to determine statistical differences between LXSN- and LTSN-seeded carotids. A value of P<.05 was considered significant.
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Results |
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Construction and Expression of Baboon TIMP-1 in the Retroviral Vector
We have previously cloned and sequenced baboon TIMP-1 coding sequence from an SMC cDNA library by using the PCR technique.38 The PCR-generated TIMP-1 coding sequence was cloned into a unique EcoRI site of the retroviral vector pLXSN (Fig 1
). The chimera pLTSN was processed in the packaging lines PE501 and PA317 to generate stable virus-producing cell lines. Virus harvested from PA317 cell line media was then used to transduce rat SMC.
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Several independent clones of G418-resistant rat SMCs transduced with the baboon TIMP-1 (LTSN) or the vector alone (LXSN) were obtained. Total RNA obtained from these cells was analyzed by Northern blotting for TIMP-1 (Fig 2A). A TIMP-1 transcript of 4.0 kb was easily identified in the LTSN cells. This transcript was of the expected size and was larger (4.0 kb) than normal TIMP-1 mRNA (0.9 kb). The additional 3.1 kb consists of the retroviral vector coding sequence including neomycin phosphotransferase.22 TIMP-1 mRNA was not detected in the LXSN cells. Western analysis of the media demonstrated the presence of a 28-kD polypeptide only in the concentrated CM collected from LTSN cells (Fig 2B), suggesting that a properly glycosylated recombinant TIMP-1 was synthesized, processed, and secreted. The concentrated CM from these cells was also analyzed for protease-inhibitory activity with a modified reverse zymography technique. This approach permitted us to measure the effectiveness of baboon TIMP-1 against rat MMPs. Two bands of inhibitory activity were detected: a band at 28 kD corresponding to the TIMP-1 seen on the Western blot and a band at 20 kD (Fig 2C). The levels of secreted recombinant TIMP-1 determined by reverse zymography and by immunoblotting were in agreement and were 100 ng/mL TIMP-1 for 3x106 cells in 10 mL over 24 hours.
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In the LXSN CM, bands were detected at Mr=28 kD and 20 kD on reverse zymograms. The 28-kD band was extremely weak. Although TIMP-1 mRNA and protein could not be detected on Northern or Western blots (Fig 2A and 2B
), the 28-kD band of inhibitory activity in the LXSN cell CM was probably rat TIMP-1 since it had the same molecular weight as baboon TIMP-1 and was abolished by alkylation-reduction, a chemical treatment known to inhibit TIMP but not MMP activities (data not shown).52
The medium conditioned by LTSN and LXSN cells was assayed for gelatinolytic activity by zymography. A 72-kD band was detected and expressed at the same levels in both cell types (Fig 3).
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Characterization of Growth Properties of Transduced TIMP-1 Cells
LTSN clones grew at a slower rate than LXSN control cells over a period of a 7-day growth assay (60% inhibition, Fig 4). In addition to exhibiting slower growth, the LTSN cells saturated at a lower cell density. A similar growth reduction at confluence has been observed in human melanoma cell line stably transfected with TIMP-2.53 The response of nonmanipulated Fischer rat SMCs in culture was not different when they received serum-free CM from LTSN or LXSN cells in the growth assay (data not shown). Furthermore, the incorporation of a neutralizing antibody to TIMP-1 in this paracrine model of growth assay did not result in any significant changes for growth of nonmanipulated normal SMCs (data not shown).
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Characterization of Migration Properties of Transduced TIMP-1 Cells
We studied the effects of the concentrated starved CM from LTSN and LXSN cells on the migration of normal rat SMCs across a matrix-coated filter in a modified Boyden chamber assay. The CM from LTSN but not LXSN cells completely inhibited the migration of normal Fischer SMCs across the matrix-coated membrane induced by PDGF-BB (Fig 5). The effect on the cell migration was observed in a 6-hour incubation period at 37°C. The preincubation of LTSN cell CM with a neutralizing monoclonal antibody against TIMP-1 reversed the inhibitory effect (Fig 5). As expected, this antibody did not alter the activity of LXSN cell CM (Fig 5). We also measured the migration of LTSN and LXSN cells in this assay. LTSN cells migrated less well in response to PDGF-BB across the gel matrix in the Boyden chamber assay than the LXSN cells. However, the addition of TIMP-1 neutralizing antibody did not reverse the migration profile of LTSN cells, although it did block the migration inhibitory activity in the LTSN CM (Fig 5). The failure of the antibody to promote the migration of the LTSN cells might be due to its poor penetration into the pericellular matrix.
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The retardation in migration was affected by the thickness of the gel matrix used for the invasion assay. LTSN cells but not LXSN cells exhibited a significant reduction in invasion through a thick gel matrix layer (2.7 µg per well, Fig 6A). The inhibition of migration was not significant when a thin gel layer (0.5 µg/mL) was employed in the Boyden chamber assay (Fig 6B).
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Recombinant TIMP-1 in the Vessel
The presence and activity of the baboon TIMP-1 were confirmed by Northern blotting and gelatin reverse zymography of the extracts prepared from LTSN cell–seeded rat carotid arteries. TIMP-1 was not detected in extracts of arteries seeded with LXSN cells (Fig 7). Furthermore, MMP-2 activity was detected in all arteries, while MMP-9 activity was detectable in LXSN cell–seeded but not in uninjured or LTSN cell–seeded arteries (Fig 8).
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Reduction of Intimal Hyperplasia in LTSN Cell–Seeded Vessels
Histological examination and morphometric quantification of the formalin-fixed cross-sections of seeded vessels revealed a 40% reduction in the intimal area of LTSN animals at 2 weeks (LTSN cell–seeded, 0.123±0.049 mm2; LXSN cell–seeded, 0.201±0.043 mm2; P<.05; Fig 9). Morphometric examination of medial areas showed no significant differences between the two tested groups (data not shown).
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DNA Synthesis and Total Intimal SMC Volume
To determine the mechanisms contributing to the intimal reduction in LTSN cell–seeded vessels, we used BrdU labeling to measure DNA synthesis in the rats. We did not find a significant difference in BrdU labeling between LXSN- and LTSN-seeded groups postseeding (Table 1). Intimal BrdU labeling was maximal when measured 2 days after the seeding and declined at 2 weeks later (Table 1). The total volume of intimal SMCs was decreased in LTSN cell–seeded arteries (Table 1), even though the fraction of the intimal area occupied by SMCs as determined morphometrically by transmission electron microscopy was the same (LXSN, 0.51±0.03; LTSN, 0.47±0.03). Thus, the relative proportion of cells to matrix was the same in both groups of animals.
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TIMP-1 Antibodies Block the Effect of LTSN Cell Seeding on Intimal Hyperplasia
The decrease in intimal hyperplasia in LTSN cell–seeded arteries was probably attributable to baboon TIMP-1 overexpression. To test this hypothesis, we attempted to block the LTSN cell effect by immunizing the animals in advance against baboon TIMP-1. We have shown that blocking antibodies can be detected at 4 weeks and later in the sera of rats seeded with LTSN cells.49 Therefore, the right carotid arteries were seeded and at 8 weeks, when a high titer of anti–TIMP-1 antibodies was present, the left carotid arteries were seeded with LXSN or LTSN cells (Table 2). Animals seeded first with LXSN cells were used as controls. Two weeks after the second seeding, the intimal thickening in the left carotid artery was analyzed.
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Immunization with LTSN cells (LTSN/LTSN group) but not with LXSN cells (LXSN/LXSN group) abolished the inhibiting effect of LTSN-seeded cells on intimal thickening in the left carotid arteries (Table 2).
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Discussion |
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Abnormal proteolytic balance might contribute to the development of pathological vascular conditions, including atherosclerotic plaque growth and disruption, aneurysmal dilatation, and intimal hyperplasia, and luminal narrowing after vascular reconstruction.54 55 Since vascular SMCs are the principal cell type in normal and diseased vessels, altered SMC protease/antiprotease expression might be part of the pathology. A shift of the proteolytic balance toward protease inhibition might inhibit growth and migration and restore the cells to a more normal quiescent state. We have attempted to test this hypothesis by constructing and characterizing SMCs that stably overexpress TIMP-1.
A retroviral approach to introducing the TIMP-1 sequence was chosen because of its high efficiency and the persistence of gene expression. The transduced SMCs continued to express TIMP-1 even when the cells were propagated in the absence of G418.
These modified SMCs were then characterized in terms of growth and migration. The significant findings from our in vitro studies were: (1) The SMCs overexpressing TIMP-1 grew more slowly than vector alone–transduced cells; (2) the CM from overexpressing TIMP-1 SMCs but not the vector alone–bearing cells completely inhibited the migration of SMCs induced by PDGF-BB across a matrix (Matrigel)-coated filter in a modified Boyden chamber assay. This effect could be overcome by treatment of the CM with an antibody to TIMP-1; and (3) TIMP-1–overexpressing cells migrated more slowly through a thick layer of matrix than controls, although both cells migrated to the same extent through a thin matrix layer.
Our in vivo studies demonstrate that intimal thickening in arteries seeded with LTSN cells is decreased and that this effect can be blocked by immunizing in advance with baboon TIMP-1. Since SMC proliferation in the media and intima were not different in LTSN and LXSN cell–seeded vessels, we conclude that the principal effect of TIMP-1 overexpression is to block the migration of medial SMCs into the intima.
Effects of TIMP-1 Overexpression on Cell Migration and Proliferation In Vitro
Much of what we know about proteolytic balance comes from the study of malignant cells. Overexpression of TIMPs in neoplastic human56 and murine57 cells is associated with decreased MMP activity and a reduction in the malignant behavior of these cells. Our data show that secreted TIMP-1 inhibits the migration of normal SMCs across a matrix-coated filter in a Boyden chamber assay. This inhibition is reversible when a neutralizing antibody is added; this result suggests to us that TIMP-1 protein by one cell might inhibit the migration of its neighbor (paracrine effect) as well as itself (autocrine effect).
We do not fully understand what factors contribute to the reduced growth of TIMP-1–overexpressing cells. However, we propose that TIMP-1 overexpression interferes with the intracellular signaling cascades required for growth of these cells. Reports from other laboratories support the possibility that the effects of TIMP-1 on SMC growth may be independent of its MMP inhibitory activity. For example, TIMP-2 inhibits basic fibroblast growth factor–induced endothelial cell proliferation, but the effect cannot be mimicked with BB-94, a synthetic inhibitor of MMPs.58 A cell surface receptor might be involved in mediating the TIMP-1 effect on different cell types.59 Transduced TIMP-1 SMCs might block growth-stimulatory signals in the cytosol and suppress cell growth in this fashion. This concept is supported by our data that growth inhibition of LTSN cells could not be attenuated by a neutralizing monoclonal antibody to TIMP-1.
Role of MMPs in Intimal Hyperplasia
Migration of SMCs from the media to the intima is an important part of intimal thickening in injured arteries of animals and is probably important in humans as well.60 Pericellular proteolysis might be required for this event to take place. MMP-2 and MMP-9 are increased during SMC outgrowth from rabbit and baboon aortic explants in vitro13 61 and in balloon-injured carotid arteries of rat.10 16 Rat SMC migration in vitro can be blocked with antibody against MMP-2.15 Migration of SMCs and intimal thickening in vivo can be inhibited with synthetic MMP inhibitors, albeit at somewhat toxic doses.62 63
To demonstrate further the importance of MMPs for intimal thickening in injured arteries, we have used a local pharmacological approach for the delivery of MMP inhibitors. We have described in previous publications a cell-based method for gene delivery to injured arteries. Retrovirally transduced SMCs can be seeded into deendothelialized Fischer rat carotid arteries and express the transduced gene over the long term (up to 12 months).37 40 49 64 The transduced SMCs seeded into the rat carotid arteries maintain a normal nontransformed phenotype.37 The intima formed after seeding contains transduced SMCs and SMCs that have migrated from the media.40
In the in vivo experiments, LTSN cells seeded onto balloon-injured rat carotid arteries reduced intimal hyperplasia by 40% compared with LXSN controls. This effect is due to the overexpressed TIMP-1. Since neither SMC proliferation nor the relative proportion of cells and matrix in the intima were altered by TIMP-1 overexpression, we surmise that the decrease in intimal thickening might have been because of decreased medial SMC migration.
In the balloon-injured rat carotid artery, intimal thickening is the result of cell proliferation, cell migration, and matrix deposition.
TIMP-1 overexpression did not block SMC proliferation in the media and intima at 2 and 14 days. These results are in agreement with some but not all studies with synthetic inhibitors of MMP.62 63 65 We also attempted to determine whether TIMP-1 overexpression would increase overall matrix accumulation by inhibiting MMP-mediated matrix degradation. We did not detect any differences in the volume densities of SMC and extracellular matrix compartments in the two groups by transmission electron microscopy. However, we cannot rule out the possibility that TIMP-1 overexpression may result in changes in specific extracellular matrix components.
Since there were no differences in relative matrix volume or DNA synthesis, we assume that the migration of medial cells might have been affected by the TIMP-1 overexpression. The observation of in vitro migration supports this possibility.
We seeded right and left carotid arteries of individual rats with LXSN, LTSN, or both sets of cells to confirm that the effect of the LTSN cells on intimal thickening could be attributed to the baboon TIMP-1. The results of the experiments support this conclusion and agree with similar in vitro data.
Our observations support the concept that proteolytic balance can have a profound effect on normal as well as malignant cell growth and migration and that overexpression of an antiprotease markedly retards these activities. How TIMP-1 and related inhibitors achieve this result is not yet evident. The simplest explanation is that the proteases and their inhibitors modify the way cells interact with surrounding matrix, but it is also possible that the signals generated by cell contact with matrix modify the proteolytic balance in the pericellular space. These cell-matrix-protease interactions might also determine whether a cell is primed to respond to a growth factor. In diseased arteries, these signaling pathways could regulate the extent of the SMC response to the growth and migratory signals generated by injury, and a deliberate intervention such as antiprotease overexpression might be a novel way to suppress this response.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by NIH grant HL18645. Dr Forough is supported by NIH training grant HL07312-15 and a grant from F. Hoffmann-La Roche Ltd, Basel, Switzerland. We thank Dr A.D. Miller, Fred Hutchinson Cancer Research Center, Seattle, Wash, for providing the retroviral vector LXSN and the cell lines PE501, PA317, and NIH 3T3 TK-; Dr H. Welgus, Washington University, St Louis, Mo for providing the polyclonal antibody against human TIMP-1; Prof T. Hayakawa, Aichi-Gakuin University, Japan, and Fuji Chemical Industries, Ltd, Toyama, Japan, for providing the neutralizing monoclonal antibody against bovine TIMP-1, capable of recognizing human TIMP-1; and Dr Chris Fisher, The Upjohn Company, Kalamazoo, Mich, for providing the recombinant human TIMP-1 made in CHO cells.
Received June 17, 1996; accepted July 24, 1996.
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References |
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L. Cheng, G. Mantile, R. Pauly, C. Nater, A. Felici, R. Monticone, C. Bilato, Y. A. Gluzband, M. T. Crow, W. Stetler-Stevenson, et al. Adenovirus-Mediated Gene Transfer of the Human Tissue Inhibitor of Metalloproteinase-2 Blocks Vascular Smooth Muscle Cell Invasiveness In Vitro and Modulates Neointimal Development In Vivo Circulation, November 17, 1998; 98(20): 2195 - 2201. [Abstract] [Full Text] [PDF]
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R. P. Fabunmi, G. K. Sukhova, S. Sugiyama, and P. Libby Expression of Tissue Inhibitor of Metalloproteinases-3 in Human Atheroma and Regulation in Lesion-Associated Cells : A Potential Protective Mechanism in Plaque Stability Circ. Res., August 10, 1998; 83(3): 270 - 278. [Abstract] [Full Text] [PDF]
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R. Forough, H. Lea, B. Starcher, E. Allaire, M. Clowes, D. Hasenstab, and A. W. Clowes Metalloproteinase Blockade by Local Overexpression of TIMP-1 Increases Elastin Accumulation in Rat Carotid Artery Intima Arterioscler Thromb Vasc Biol, May 1, 1998; 18(5): 803 - 807. [Abstract] [Full Text] [PDF]
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S. Baek and K. L. March Gene Therapy for Restenosis : Getting Nearer the Heart of the Matter Circ. Res., February 23, 1998; 82(3): 295 - 305. [Abstract] [Full Text] [PDF]
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A. H Baker, D. Mehta, S. J George, and G. D Angelini Prevention of vein graft failure: potential applications for gene therapy Cardiovasc Res, September 1, 1997; 35(3): 442 - 450. [Abstract] [Full Text] [PDF]
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