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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1640-1649

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1640-1649.)
© 1999 American Heart Association, Inc.

Vascular Biology

Increased Secretion of Basement Membrane–Degrading Metalloproteinases in Pig Saphenous Vein Into Carotid Artery Interposition Grafts

Kay M. Southgate; Dheeraj Mehta; Mohammed B. Izzat; Andrew C. Newby; Gianni D. Angelini

From the Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, UK.

Correspondence to Dr Kay Southgate, Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK. E-mail k.m.southgate{at}bris.ac.uk


*    Abstract
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Abstract—Late saphenous vein bypass graft failure in humans involves medial and neointimal thickening as the result of migration and proliferation of vascular smooth muscle cells (SMCs). Recent work on angioplasty indicates that basement membrane–degrading metalloproteinases (MMPs) cooperate with growth factors to mediate SMC migration and proliferation. We sought evidence here for a similar role in experimental vein grafts in pigs. Tissue levels and secretion of MMP-2 and MMP-9 were compared by quantitative zymography in veins and grafts removed 2 to 168 days after implantation. Pro and active forms of MMP-2 were present in veins, but levels were increased in vein grafts after 7 days (4- and 6-fold, respectively) and 28 days (3-fold for both), returning to values in veins after 168 days. MMP-9 was not detected in veins, was increased in grafts after 2 days, further increased after 7 days (6-fold) and 28 days (15-fold), and declined to undetectable levels by 168 days. Immunocytochemistry detected increased expression of MMP-2 and MMP-9 with the same time course. MMP-2 was widely distributed, whereas MMP-9 was concentrated in highly proliferative SMCs at the superficial layers of the thickened neointima. We conclude that increased production of the basement membrane–degrading MMP-2 and MMP-9 spanned the period of neointima formation and SMC proliferation in experimental vein grafts. MMPs therefore constitute new therapeutic targets for reducing late vein graft failure.


Key Words: vascular smooth muscle • cell proliferation • gelatinases • vein grafts


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Despite increased use of the internal mammary artery, autologous saphenous vein continues to be the most commonly used conduit for coronary artery by pass grafting into multiple sites.1 2 The patency rates of saphenous vein grafts after 5 years are {approx}50% compared with 90% for internal mammary artery grafts.2 3 The histological changes associated with vein graft failure are well defined. They include acute thrombosis, early medial and neointimal thickening that may be focally progressive, and atheroma formation, which is the most important cause of failure beyond 5 years after implantation.4 Early thrombosis can be reduced by atraumatic surgical preparation and by the use of antiplatelet agents.5 Few studies, however, have aimed to define the mechanisms of graft wall thickening at the molecular level and hence, identify unique targets for therapeutic intervention. In experimental models as in humans, neointimal thickening continues after endothelial regeneration,6 implicating chemotactic and mitogenic factors produced by cells in the blood vessel wall.7 Platelet-derived growth factor (PDGF) is 1 such factor, the secretion of which is increased in animal8 and organ culture9 models of neointima formation in veins. PDGF is also a key factor after balloon injury,10 11 12 making it an attractive target for intervention. Growth factors are not, however, the only potential therapeutic target, since there is strong evidence that they need to interact with other factors to initiate neointima formation in veins.13 Components of the basement membrane surrounding vascular smooth muscle cells (SMCs) normally suppress migration and proliferation.14 15 16 17 Proteolytic remodeling of the basement membrane may be required, therefore, to allow SMC migration and proliferation responses to growth factors. The basement membrane–degrading metalloproteinases (gelatinases) play the key role in basement membrane turnover.17 18 Evidence implicating gelatinases in neointimal formation has come from several previous studies, which have demonstrated that (1) vascular SMCs derived from different animal species can synthesize gelatinases16 19 20 21 ; (2) inhibitors of gelatinases inhibit the migration and proliferation of rabbit and rat vascular SMCs in vitro16 21 and in vivo21 22 23 ; and (3) upregulation of gelatinases parallels vascular SMC migration and proliferation in rat and pig balloon-injured carotid arteries22 24 25 and in organ cultures of human saphenous vein.26 The relevance of these observations to neointima formation in vein grafts in vivo has not, however. been established.

In the present study, we used a pig model of saphenous vein into carotid artery interposition grafting to investigate whether there were time-dependent increases in tissue levels and secretion of gelatinases after grafting. We also used immunocytochemistry to identify proliferating SMCs and to investigate their relationship to the spatial localization of the cells responsible for gelatinase production.


*    Methods
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Reagents
All reagents were purchased from Sigma Chemical Co except for those listed below. The MMP inhibitors Ro 31-9790 and Ro 31-4724 were gifts from Roche Products Ltd (Hertfordshire, UK); stock solutions in neat dimethyl sulfoxide (DMSO) were diluted 200-fold immediately before use to give final concentrations of 10 µmol/L and 0.5% DMSO. MMP-2 and MMP-9 standard proteins and sheep polyclonal antibodies to human MMP-2 (X670/10) and pig MMP-9 (A560/8) were generous gifts of Dr Gillian Murphy (Strangeways Research Laboratory, Cambridge, UK). Anti–human MMP-2 antisera for immunocytochemistry was produced in rabbits with synthetic peptides synthesized to the C-terminus of the enzyme as described previously.26 Mouse monoclonal {alpha}-smooth muscle actin antibody (HHF35) and goat, rabbit, and horse sera were from Dako Ltd. Monoclonal antibody to proliferating cell nuclear antigen (PCNA, clone PC10) was from Zymed Laboratories Inc. Biotinylated rabbit anti-sheep IgG, biotinylated Dolichos biflorus agglutinin, biotinylated horse anti-mouse IgG, and Vectashield Fluoromount were purchased from Vector Laboratories Ltd. Optimal cutting temperature (OCT) compound and Coomassie blue R250 were from Merck Ltd. Dulbecco's modified Eagle's medium (DMEM), Dulbecco's PBS, FCS, and L-glutamine were obtained from Gibco BRL Ltd. Gentamicin was from Roussel. HT1080 cells were obtained from the European Collection of Animal Cell Cultures, Salisbury, UK.

Surgical Technique
The study was conducted in 28 Land Race pigs 3 to 4 months old weighing 25 to 30 kg, all of which received humane care according to the Home Office Animals (Scientific Procedure) Act of 1986. Premedication, anesthesia, and autologous saphenous vein into carotid artery interposition grafting were performed as detailed previously.27 28 Animals were then assigned to groups designated for graft retrieval on days 2 (n=4), 7 (n=4), 28 (n=14), and 168 (n=6). They were then reanesthetized, and patent grafts were removed for further analysis. Segments of freshly isolated, ungrafted saphenous vein (obtained from the hind leg not used for grafting) and carotid artery were also removed for comparison.

Organ Culture
Vessel segments were transported to the laboratory in warm (25°C to 30°C), sterile, 20 mmol/L HEPES-buffered DMEM supplemented as detailed previously.8 Tissue segments were then transferred into a Petri dish containing a wash medium consisting of HEPES-buffered DMEM and antibiotics as above but with the addition of 2 mmol/L L-glutamine and 8 µg/mL gentamicin. As much as possible of the adventitia was removed with microvascular scissors. Ring sections (3x0.5 cm) were then obtained from the end of each tissue segment and weighed; they were then snap-frozen in LN2 for tissue extraction, frozen in isopentane cooled over LN2, or fixed in 10% formalin and embedded in paraffin wax for immunocytochemistry, as detailed below. The remaining tissue segments were subjected to organ culture by using the method of Francis et al8 with the following slight modifications. To allow secretion of MMPs, 10-mm tissue segments were cultured individually for 2 days at 37°C in DMEM-HCO3 tissue culture medium supplemented as above but also containing 2.5 g/L lactalbumin. Conditioned media were centrifuged at 1500g for 10 minutes to remove any cells and debris, 0.1% NaN3 was added, and they were stored at -20°C. The cultured tissue segments were then washed twice with Dulbecco's PBS and snap-frozen in LN2 for determination of tissue viability by measurement of purine metabolites as described previously.27

Extraction of Tissue
Preweighed frozen segments of vein grafts, ungrafted saphenous veins, and arteries (both before and after grafting) were crushed under LN2 and extracted in medium (200 µL/100 mg of tissue) as detailed previously.25 The tissue extracts were then stored at -20°C.

Zymography
Gelatinase activity in conditioned media collected from 2-day organ cultures, HT1080 cells, and tissue extracts was measured by zymography. To correct for variations in tissue segment size, all conditioned media were diluted to the equivalent of 2 mg/mL with serum-free incubation medium. All tissue extracts were diluted to the equivalent of 65 mg/mL with extraction medium. Conditioned media and tissue extracts were then subjected to electrophoresis on SDS/polyacrylamide gels containing 2 mg/mL gelatin as described previously.25 Zones of gelatinolytic activity appeared as clear bands against the Coomassie blue–stained background. All gels were calibrated with high-molecular-weight standards. One well of each gel also included conditioned medium from the HT1080 fibrosarcoma cell line diluted to a concentration previously determined to degrade {approx}50% of the gelatin substrate. This cell line, which is known to secrete both MMP-2 and MMP-9, thereby acted as an internal positive control and allowed quantification of proteolytic activity across several gels to be statistically compared.26

To study inhibition of the enzymes, the reaction buffer was supplemented with 20 mmol/L EDTA, a nonspecific metalloproteinase inhibitor; 1 mmol/L PMSF, a serine protease inhibitor; 5 mmol/L N-ethylmaleimide, a cysteine protease inhibitor; 5 mmol/L pepstatin A, an aspartic protease inhibitor; and either Ro31-9790 (10 µmol/L) or Ro31-4724 (10 µmol/L), both of which are specific synthetic metalloproteinase inhibitors, as indicated in the text. The gels were then stained with a 0.1% aqueous solution of Coomassie blue. For further identification, immunoprecipitation of gelatinase isoforms was conducted using sheep polyclonal antibodies to human MMP-2 (X670/10) and purified pig MMP-9 (A560/8) or nonimmune sheep sera as detailed previously.25 The resultant supernatants were then removed and subjected to zymography as described above.

MMP-2 and MMP-9 proteins detected by zymography were quantified by densitometric scanning of wet gels by using a GS-690 imaging densitometer (Bio-Rad). The results were expressed as optical density multiplied by mm2, and the means were calculated. To correct for variation between zymograms, the optical density mm2 results were expressed as a percentage of the standard HT1080 sample.

Determination of Cell Proliferation Indexes
The presence of cells proliferating through the cell cycle was detected by indirect immunocytochemistry for PCNA28 in paraffin sections of vein grafts removed 2 to 168 days later, ungrafted saphenous vein, and arteries (both before and after grafting) by using a monoclonal antibody (clone PC10) at a 1:100 dilution as described previously.29 The PCNA index was determined by counting only those cells with strongly positive brown nuclear PCNA staining and expressing it as a percentage of the counterstained nuclei in both intima and media in 5 microscopic fields per section with the use of a 40x objective and an image analysis system (Image-Pro Plus, version 3.0, Media Cybernetics).

Histological Methods
Adjacent paraffin sections of all recovered vessels were examined by Miller's elastic van Gieson's and Harris' hematoxylin and eosin staining. Vascular SMCs were identified by indirect immunocytochemistry with a monoclonal anti–{alpha}-smooth muscle actin antibody (clone HHF35) and 3,3'-diaminobenzidine (DAB) as the substrate (brown color) as described previously.30 The presence of an intact endothelial layer was identified in all recovered vessels by immunocytochemical staining with 5 µg/mL of biotinylated lectin from Dolichos biflorus agglutinin for 30 minutes at room temperature,31 followed by ExtrAvidin–alkaline phosphatase (1:100) for a further 30 minutes at room temperature. The color was developed using fast red TR/naphthol AS-MX phosphate tablet sets as the substrate (rose-pink).

Immunocytochemistry for MMP-2 and MMP-9
Paraffin wax– and frozen OTC–embedded sections (5 µm) were mounted on 3-aminopropyltriethoxysilane–coated glass slides and air-dried. MMP-2 protein was immunolocalized in paraffin sections by using the primary rabbit anti-human C-terminus MMP-2 antiserum (diluted 1:1000) and visualized with DAB substrate as described previously.26 MMP-9 was identified in frozen sections by indirect immunofluorescence. In brief, the sections were fixed with 4% paraformaldehyde in Tris-buffered saline, pH 7.6, for 30 minutes and then permeabilized with 0.1% Triton X-100 in Tris-buffered saline for 5 minutes. All subsequent incubations included 1% BSA and were performed at room temperature in a humidified chamber. After blocking nonspecific sites and autofluorescence with a mixture of 5% rabbit serum and 0.1% pontamine sky blue for 30 minutes, we incubated the sections with 25 µg/mL sheep anti–pig MMP-9 antibody for 30 minutes and then with 1:200 biotinylated rabbit anti-sheep conjugate for a further 1 hour. ExtrAvidin–FITC-labeled conjugate (1:200) was then added for a further 30 minutes. The sections were counterstained with 0.7 µg/mL propidium iodide, mounted in Vectashield, and examined using an Olympus BX40 fluorescence microscope fitted with a wide-blue filter. The specificities of these immunocytological techniques were demonstrated by the inclusion of negative controls, in which the primary antibodies were preadsorbed with 10 µg/mL of the appropriate synthetic peptide or substituted by either nonimmune serum or isotype-specific nonimmune IgG. In addition, a positive control of frozen or paraffin-embedded HT1080 cells was always included.

Statistical Analysis
All values given are mean±SEM, with numbers of observations in parentheses. Paired analysis between 2 groups was performed using the Student's paired t test, and unpaired data for different time points were compared using Student's unpaired t test when the ANOVA indicated significance for the multiple comparisons. Statistical significance was accepted when P<=0.05.


*    Results
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 *Results
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Secretion of MMP-2 and MMP-9 Is Elevated in Vein Grafts Removed After 28 Days
Measurement of ATP concentrations, performed to examine the viability of the predominant SMC type within the cultured tissue segments, demonstrated that there was no difference in the ATP concentrations of any tissue segment before and after culture in accordance with published data.8 The secretion of MMP-2 and MMP-9 into serum-free conditioned media of organ cultures of vein grafts removed 28 days after surgery was investigated first by gelatin zymography and densitometric scanning. The gelatin zymography technique enables the detection of the latent as well as the activated forms of MMPs because of molecular conformational changes that occur in the presence of SDS, allowing digestion of the substrate incorporated into the gels at the respective molecular weights. Using this approach, we found that this conditioned medium contained 3 major bands of metalloproteinase activity with molecular weights of 68, 72, and 95 kDa and a minor band of 205 kDa (Figure 1aDown). Heating vein graft segments to 60°C before culture or culturing the grafts in the presence of 100 µmol/L cycloheximide for 48 hours abolished metalloproteinase activity in the conditioned medium (data not shown), demonstrating that these activities arose through new synthesis and secretion. The enzyme activities were identified as MMPs by inhibition with EDTA or the specific synthetic inhibitors of MMPs, Ro31-9790 and Ro31-4724, but not with N-ethylmaleimide, pepstatin A, or PMSF (Figure 1aDown). Immunoprecipitation with antibodies to human MMP-2 almost completely removed the 72- and 68-kDa activities without affecting the 95-kDa activity. The 205- and 95-kDa activities were completely immunoprecipitated by antibodies to purified pig MMP-9, whereas the 72- and 68-kDa activities were not affected (Figure 1bDown). These data indicate clearly that the 72- and 68-kDa activities result from the pro and active forms, respectively, of MMP-2 (gelatinase A) and that the 95-kDa activity results from the pro form of MMP-9 (gelatinase B). This assignment of activities is identical to the molecular weights of pro and active MMP-2 and of pro-MMP-9, which we reported in our previous studies of balloon-injured pig carotid arteries.25



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  		Figure 1. Characterization of gelatinase activities. Pooled serum-free conditioned media (15 µL) from organ cultures of saphenous vein grafts removed 28 days after surgery were subjected to gelatin zymography. a, In inhibition studies, strips of gel were incubated with 0.5% DMSO vehicle and lane 1, HT1080 standard; lane 2, no addition (vehicle alone); lane 3, 5 mmol/L N-ethylmaleimide; lane 4, 5 mmol/L pepstatin A; lane 5, 0.5 mmol/L PMSF; lane 6, 10 µmol/L Ro31-4724; lane 7, 10 µmol/L Ro31-9790; and lane 8, 20 mmol/L EDTA. b, In immunoprecipitation, to identify the gelatinases positively, the same pooled samples in panel a were immunoprecipitated with anti-gelatinase antibodies or nonimmune globulin, and the supernatants were subjected to zymography: lane 1, MMP-2 standard; lane 2, MMP-9 standard; lane 3, empty; lane 4, vehicle alone; lane 5, 50 µg anti–MMP-2; lane 6, 50 µg anti–MMP-9; and lane 7, 50 µg nonimmune globulin. Each panel shows a representative of 3 similar experiments.

Secretion of gelatinases into conditioned media was compared on the same zymograms from ungrafted veins and vein grafts removed 28 days later from the same animal (Figure 2Down) and quantified for 8 pairs by densitometric scanning (Figure 3Down). Secretion of 95-kDa pro-MMP-9, 72-kDa pro-MMP-2, and 68-kDa active MMP-2 was significantly increased relative to ungrafted vein segments (Figures 2Down and 3Down). As an additional control, ungrafted veins were also removed from animals not subjected to vein grafting. The same levels of MMP-2 and MMP-9 secretion were observed in these veins (data not shown) as in ungrafted veins removed 28 days after surgery (Figures 2Down and 3Down).



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  		Figure 2. Measurement of gelatinase activity in conditioned media by zymography. In pigs, carotid arteries were subjected to bilateral autologous saphenous vein interposition grafting. Segments of ungrafted saphenous vein and vein grafts were removed 28 days (n=8) after surgery. Segments were subjected to organ culture in serum-free medium for 2 days as described in Methods. Conditioned media were subjected to gelatin zymography. Samples (2 mg/mL) of conditioned media from organ cultures were applied as follows: lane 1, high-molecular-weight standard; lane 2, empty; lane 3, HT1080 standard; lane 4, ungrafted saphenous vein; and lane 5, saphenous vein graft. Each panel shows a representative of 8 similar experiments.



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  		Figure 3. Densitometry of zymograms for conditioned media. The zymograms referred to in Figure 2Up were subjected to densitometry. Segments of ungrafted saphenous vein and vein grafts removed 28 days after surgery were always compared on the same zymograms, which always contained an HT1080 internal control standard. Units for transmission were optical densityxmm2; these values were expressed as a percentage of the densitometric readings of MMP-2 and MMP-9 for the internal HT1080 standard. Data from 8 different vessels were then subjected to statistical analysis with Student's paired t test. *P<0.05 vs ungrafted saphenous vein on the same day.

Time Course of Increased Tissue Levels of MMP-2 and MMP-9 in Vein Grafts
In a separate series of pigs, levels of MMP-2 and MMP-9 in tissue extracts of vein grafts removed 2, 7, 28, and 168 days after surgery were compared with carotid arteries and ungrafted veins from the same animals. Samples of these 3 tissues from the same animal were compared on the same gels. Densitometric scanning of these zymograms revealed that tissue levels of pro and active MMP-2 and pro-MMP-9 were similar in carotid arteries and contralateral ungrafted veins removed at any time in the interval of 2 to 168 days after implantation of the other saphenous vein or from animals not subjected to vein grafting (day 0, Figure 4aDown through 4c). Tissue extracts showed similar patterns of changes in both MMP-2 and MMP-9 levels as in conditioned media when we compared 28-day vein grafts and ungrafted veins from the same animals (Figure 4aDown through 4c). Paired analysis again revealed that levels of pro-MMP-9 and active MMP-2 were significantly increased after 28 days. Pro-MMP-9 levels were also significantly increased in vein grafts compared with levels in ungrafted vein and carotid artery tissues removed 2 and 7 days after surgery (Figure 4aDown). Levels of pro and active MMP-2 were not significantly increased 2 days after surgery but became so 7 days after surgery (Figure 4bDown and 4cDown). Our use of wet weight as the normalizing parameter for these quantitative studies has limitations, in particular if the cellularity of the vein changes. We therefore determined the cellularity (total number of cells per mm2) directly in tissue sections (n=4) for the media of ungrafted vein (1655±110), carotid arteries (1673±162), and vein grafts recovered at 2 days (1675±48), 7 days (1650±166), 28 days (1534±67), and 168 days (1050±96; where P<0.02 versus all other time points). The cellularity of the neointima was also determined, when it was sufficiently large to analyze, in vein grafts recovered at 28 days (9447±473) and 168 days (7328±316; P<0.01 versus 28-day vein grafts). The increase in pro-MMP-9 levels at 2 and 7 days was clearly not the result of increased cellularity, although increased numbers of neointimal cells may have contributed to the further rise at 28 days. The decline in pro-MMP-9 at 168 days cannot be explained wholly by decreased cellularity. Similarly, the increase in pro-MMP-2 levels at 7 days cannot be explained by an increase in cellularity, and its decline at 28 and 168 days occurred despite increased cellularity in the neointima.



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  		Figure 4. Densitometry of zymograms for tissue extracts. Segments of carotid artery, ungrafted saphenous veins, and vein grafts removed at 2 (n=4), 7 (n=4), 28 (n=6), and 168 (n=6) days after surgery were always compared by quantitative zymography on the same gels, which also usually contained a day-0 control sample (carotid artery and ungrafted vein removed from animals not subjected to vein grafting), as well as the HT1080 internal control standard. Units for transmission were optical densityxmm2; these values were expressed as a percentage of the densitometric readings of MMP-2 and MMP-9 for the internal HT1080 standard, and then the mean±SEM was calculated. Data from 4 to 6 different vessels were then subjected to statistical analysis with Student's paired t test. Unpaired data for different time points were compared with Student's unpaired t test and by ANOVA, both of which yielded similar conclusions. *P<0.05 vs ungrafted saphenous vein on the same day; {dagger}P<0.05 vs carotid artery on the same day; $P<0.0003 vs vein graft removed after 28 days; §P<0.05 vs vein graft removed after 7 days.

Figure 5Down shows a representative zymogram of the time course of MMP-2 and MMP-9 activities in tissue extracts of vein grafts from separate animals compared with either ungrafted veins or carotid artery segments removed from animals not subjected to vein grafting (day 0). The use of separate animals at different times required the use of less powerful unpaired analysis to compare different time points. Even so, the levels of MMP-9 activity observed in 28-day vein graft tissues were significantly greater than in 2-day vein grafts (P<0.05) and in ungrafted veins removed from animals not subjected to vein grafting (P<0.01, Figure 4aUp). Moreover, levels of MMP-9 activity from tissue extracts of vein grafts removed 168 days after surgery were significantly less than in 28-day grafts (P<0.01) and had returned to the normal levels observed in ungrafted vein and carotid artery tissues (Figure 4aUp). Similar unpaired analysis revealed that the increased levels of both pro and active MMP-2 observed in 7-day vein graft tissues were significant when compared either with levels in 2- and 168-day vein grafts (P<0.05) or with ungrafted veins removed from animals not subjected to vein grafting (P<0.01, Figure 4bUp and 4cUp). In addition, increased levels of active MMP-2 observed in 7-day vein graft tissues were also significant when compared with levels in 28-day vein grafts (P<0.05, Figure 4cUp). Levels of both active and pro-MMP-2 in vein grafts removed 168 days after surgery were the same as those in ungrafted vein and carotid artery tissues (Figure 4bUp and 4cUp).



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  		Figure 5. Time course of MMP-2 and MMP-9 activities in tissue extracts by zymography. A representative zymogram showing the time course of MMP-2 and MMP-9 activities in tissue extracts of vein grafts (n=4 to 6) from separate animals compared with either carotid artery (n=6) or ungrafted vein (n=6) segments removed from animals not subjected to vein grafting (control). Gelatinase activity was extracted by homogenization in a buffer, as described in Methods. Extracts equivalent to 65 mg/mL were subjected to gelatin zymography as follows: lane 1, carotid artery control; lane 2, ungrafted vein control; lane 3, vein graft at 2 days; lane 4, vein graft at 7 days; lane 5, vein graft at 28 days; and lane 6, vein graft at 168 days. Each panel shows a representative of 4 or 6 similar experiments.

Time Course of Cell Proliferation Indexes Within Media and Intima of Vein Grafts
The number of cells progressing through the cell cycle was measured by immunocytochemistry for PCNA. PCNA-positive cells were rarely detected in carotid arteries (data not shown) and ungrafted veins (Figure 6Down). Unpaired analysis across the time points revealed that the percentage of PCNA-labeled cells increased significantly in the media of all vein grafts removed up to 168 days after surgery compared with ungrafted veins (day 0, Figure 6Down). In addition, the percentage of PCNA-labeled cells in the neointima of 7- and 28-day vein grafts was not only as high as that observed in the media of these grafts, respectively (Figure 6Down), but also was localized to both endothelial cells and SMCs at the most luminal aspect of the neointima of 28-day vein grafts, as has been reported previously28 (Figure 7ADown). The PCNA index of the neointima of vein grafts removed 168 days after surgery was significantly less than that observed in the media and neointima of 7- and 28-day vein grafts and had almost returned to normal levels observed in ungrafted veins (Figure 6Down), in agreement with previous data.32



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  		Figure 6. Time course of cell proliferation indexes within media and intima of pig vein grafts. Medial and neointimal cell proliferation was detected by immunocytochemistry for PCNA in sections of ungrafted saphenous vein from animals not subjected to vein grafting (day 0, n=4) and vein grafts removed at 2, 7, 28, and 168 days after surgery (n=4). The PCNA index (percentage of proliferating cells) was then calculated as described in Methods. Unpaired data for different time points were compared with Student's unpaired t test and by ANOVA, both of which yielded similar conclusions. *P<0.006 vs media of ungrafted saphenous vein (day 0); {dagger}P<0.0001 vs neointima of vein graft at 168 days.



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  		Figure 7. Morphological characteristics of pig vein grafts. Immunocytochemical identification of cell types in adjacent sections of a representative pig saphenous vein graft removed 28 days after surgery. Panels illustrate (A) nuclei of proliferating cells stained dark by DAB for PCNA antiserum. Note that large arrowheads indicate PCNA-positive SMCs and small arrows indicate PCNA-positive endothelial cells at the superficial layer of the neointima. B, SMCs stained dark by DAB for anti–{alpha}-smooth muscle actin; note that endothelial cells indicated by the small arrows are negative. C, Endothelial cells stained dark by fast red for Dolichos biflorus lectin. In all panels, the scale bar in panel A represents 25 µm and applies to all panels. Large arrows indicate the position of the internal elastic lamina, which separates the neointima (NI) and medial (M) layers.

Morphological Characteristics of Vein Grafts
Consistent with previous work,6 vein grafts removed over a time course of 2 to 168 days after surgery showed a progressive increase in both medial and intimal thickness compared with ungrafted veins (Figure 8ADown through 8E). Immunocytochemistry with a monoclonal antibody against {alpha}-actin confirmed the presence of vascular SMCs in both the thickened media and intima (Figure 7BUp). Staining with Dolichos biflorus lectin, but not by {alpha}-actin, also confirmed that endothelial cells lined all of the recovered vessels (Figure 7CUp).



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  		Figure 8. Localization of MMP-2 in pig vein grafts. Detection of MMP-2 in vein grafts by immunoperoxidase staining. One of 4 ungrafted saphenous veins (A). One of 4 saphenous vein grafts removed 2 days (B), 7 days (C), 28 days (D), or 168 days (E) after surgery. Adjacent section of vein graft from D was stained for MMP-2 antiserum, which had been preadsorbed with the corresponding synthetic peptide (F). Note cells expressing MMP-2 protein had brown-stained cytoplasm and blue-counterstained nuclei. Scale bar in panel A applies to all panels and represents 25 µm. Small arrows indicate positive endothelial cells; large arrows indicate the position of the internal elastic lamina, which separates the neointima (NI) and medial (M) layers.

Localization of MMP-2 and MMP-9 in Vein Grafts
We sought to identify the cellular source of MMP-2 and MMP-9 in vein grafts. The available antibodies to MMP-2 were found to be suitable for immunoperoxidase staining of paraffin-embedded tissue. Western blot analysis confirmed that the rabbit anti-human C-terminus MMP-2 antibody recognized the pig protein (data not shown). In all vein grafts removed up to 168 days after surgery, MMP-2 expression was high in a population of surface cells identified as luminal endothelial cells by immunocytochemistry (Figure 8BUp through 8E). A lower level of widespread MMP-2 staining was observed throughout the medial layers of ungrafted veins (Figure 8AUp) and vein grafts removed 2 days after surgery, when intimal thickening was minimal (Figure 8BUp). In vein grafts removed after 7 days, when intimal thickening had clearly developed, increased MMP-2 expression by SMCs appeared throughout the thickened intima compared with the media (Figure 8CUp). MMP-2 staining was still prominent in the grossly thickened intima after 28 days (Figure 8DUp) but declined again by 168 days after surgery to similar levels seen in ungrafted veins (Figure 8EUp).

In contrast, the MMP-9 antibodies available to us did not give a clear immunoperoxidase stain in paraffin-embedded tissue, because the antigenic target for this antibody was sensitive to fixation. However, a combination of frozen sectioning and immunofluorescence did yield specific staining. MMP-9 expression was not detectable in sections from 6 ungrafted saphenous veins (Figure 9ADown). By 2, 7, and 28 days after surgery, however, SMCs highly immunopositive for MMP-9 were seen throughout the medial layer of grafted veins (Figures 9BDown, 9CDown, and 10ADown, respectively). MMP-9 expression was greatest at the superficial layer of the thickened intima in 28-day vein grafts (Figure 9DDown). This distribution was similar to that of the highly proliferating SMCs identified by staining with PCNA (Figure 7AUp). Such high expression of MMP-9 was not detectable at the superficial layer of the thickened intima (Figure 9EDown) nor in the media (Figure 10BDown) of vein grafts removed 168 days after surgery.



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  		Figure 9. Localization of MMP-9 in pig vein grafts. Detection of MMP-9 in vein grafts by FITC-immunofluorescence staining. One of 4 ungrafted saphenous veins (A). One of 4 vein grafts removed 2 days (B), 7 days (C), 28 days (D), or 168 days (E) after surgery. Adjacent section of vein graft from D was stained for MMP-9 antiserum, which had been preadsorbed with the corresponding synthetic peptide (F). Note the green immunofluorescence staining for MMP-9 in the cytoplasm and the orange nuclear counterstaining of propidium iodide. Scale bar in panel A applies to all panels and represents 25 µm. Large arrows indicate the position of the internal elastic lamina, which separates the neointima (NI) and medial (M) layers.



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  		Figure 10. Localization of MMP-9 in the media of 28- and 168-day vein grafts. FITC-immunofluorescence detection of MMP-9 in the medial layers of 28-day (A) and 168-day (B) vein grafts referred to in Figure 9DUp and 9EUp, respectively. Note the high expression of MMP-9 in medial layer of 28-day vein grafts compared with the residual amount present in 168-day vein grafts. Scale bar in A applies to both panels and represents 25 µm. Large arrows indicate the position of the internal elastic lamina, which separates the neointima (NI) and medial (M) layers.

MMP-2 and MMP-9 expression was not detectable in sections when the antibodies had been either preadsorbed with the corresponding synthetic peptide (Figures 8FUp and 9FUp, respectively) or substituted for nonimmune IgG (data not shown), thereby demonstrating the specificity of the technique.


*    Discussion
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 *Discussion
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Previous studies have established that MMP-2 and MMP-9 expression is upregulated in models of atherosclerosis formation26 33 and angioplasty restenosis.22 24 25 There have not, however, been any previous studies of gelatinase activity in animal models of vein grafting. The present study demonstrates clearly that grafting saphenous veins into the carotid arteries of pigs increases the tissue levels and secretion of both pro and active forms of MMP-2 and of pro-MMP-9. The time course of increase in MMP-2 and MMP-9 activity spans the period of medial and intimal thickening in this model, which occurs rapidly in the first 28 days and then abates up to 168 days.6

We have shown in the present study that pro-MMP-2 is constitutively expressed in the normal vein and artery, but there is little active MMP-2. Proteolysis of pro-MMP-2 to its active form by membrane-type metalloproteinases is believed to be of key importance for its regulation.34 35 36 In vein grafts, the increase in pro-MMP-2 and the appearance of active MMP-2 are not significant at 2 days, peak at 7 days, and then decline progressively after 28 and 168 days. Medial cell proliferation therefore both precedes and is significantly more prolonged than MMP-2 activation. MMP-2 is widespread in normal veins and 168-day grafts but is increased in the media 7 days after grafting. Prominent staining also occurs in the luminal endothelial cells and the most intimal SMCs, where PCNA-positive cells were most prevalent. There was, therefore, a partial correlation between MMP-2 activity and SMC proliferation. Constitutive expression of pro-MMP-2 has been observed in a variety of arterial and venous smooth muscle preparations, consistent with our findings here. Increased secretion of pro and active MMP-2 was observed after balloon injury to pig carotid arteries, in cholesterol-fed rabbits, in human atherosclerotic tissues, and in injured or cultured human saphenous veins, also consistent with the present data.25 26 33 37 Increased expression of MMP-2 has been previously reported in the regrowing endothelium of the pig carotid artery after balloon injury.25 Despite the wealth of evidence for upregulation of pro-MMP-2 secretion, the mechanisms responsible are an enigma, because the promoter lacks recognized transcription factor response elements and does not contain a TATA box.38

In previous studies, there has been uncertainty as to the state of MMP-9 activation in models of neointima formation. One study of rat carotid arteries appeared to show activity at 88-kDa, corresponding to the active form, whereas another showed activity at 92-kDa, more consistent with the pro form.22 24 Our previous studies in the pig carotid artery and in cholesterol-fed rabbits showed that the 95-kDa pro form of the enzyme overwhelmingly predominates.25 33 The 95-kDa molecular-weight form of MMP-9 secreted or extracted from vein grafts is identical to that secreted from the balloon-injured pig carotid artery and can therefore also be identified as the pro form.25 We have observed that activation of pro-MMP-9 can occur as an artifact of the extraction procedure, especially if protease inhibitors are omitted, and this may account for the discordant results from the 1 rat carotid study. Based on these findings, activation of pro-MMP-9 is likely to be a highly regulated and localized event. We found that pro-MMP-9 levels increased within 2 days after vein grafting and then progressively up to 28 days, after which they decline up to 168 days. The initial increase in MMP-9 levels occurs simultaneously with the increase in medial cell proliferation as measured by PCNA. MMP-9 levels peak during the maximum period of medial and neointimal proliferation. MMP-9 levels and neointimal proliferation both decline with a similar time course, although medial proliferation is significantly more prolonged. By immunocytochemistry, MMP-9 expression is absent from the media of ungrafted veins and from both the media and neointima of 168-day grafts. It is found to be widespread in the media and neointima of 2- to 28-day grafts and is most abundant in the SMCs at the luminal aspect of the neointima at 28 days, in close colocalization with PCNA staining. There is agreement that MMP-9 levels increase rapidly after balloon injury to rat and pig carotid arteries21 24 25 and after surgical preparative injury to human saphenous veins.26 Indeed, the increase in MMP-9 secretion precedes the increase in cell proliferation. Moreover, in saphenous veins, the increase in MMP-9 activity in the media of surgically prepared but not freshly isolated veins is correlated with the presence and location of subsequent SMC proliferation.26 Taken together, our data and previous studies show a close association between MMP-9 expression and SMC proliferation.

Studies with synthetic inhibitors and with the tissue inhibitor of metalloproteinases (TIMP) have consistently demonstrated an important role for MMPs in SMC migration both in vitro and in vivo.16 21 23 39 One study in vitro16 and 1 in vivo21 also demonstrated inhibition of proliferation, although this was not observed in another study in vivo.23 Stable transfection of baboon SMCs with TIMP-1 decreased cell proliferation.40 On the other hand, adenovirus-mediated overexpression of TIMP-1 had no effect on isolated rabbit SMC proliferation41 or in organ cultures of human saphenous vein.39 Hence, the importance of MMPs for SMC proliferation remains unclear. In the absence of selective inhibitors, the specific roles of MMP-2 and MMP-9 cannot be fully resolved. Nevertheless, experiments with synthetic inhibitors and inhibitory antibodies in rat SMCs, where MMP-2 was the predominant MMP expressed, confirmed its role in SMC proliferation.42 In addition, gene transfer studies of MMP-2 into cell lines increases their invasive capacity,43 a process that also involves invasion of extracellular matrixes. A specific role of MMP-2 activation in neointimal migration of SMCs is therefore an attractive hypothesis. The time course of activation of MMP-2 in the vein grafts observed here is consistent with a role in invasion of SMCs into the neointima.

Our studies extend to vein grafts the strong evidence for the collective involvement of MMP-2 and MMP-9 in neointima formation. A variety of metalloproteinase inhibitors has been developed for clinical application. Recently, the first results of clinical trials of rather nonselective metalloproteinase inhibitors (which therefore also target collagenase and stromelysin) have been published.44 45 46 47 Initial results suggest that the agents may be of value in slowing tumor progression, although nonselectivity may result in side effects during long-term administration. Selective inhibitors of gelatinases and collagenase are presently being tested for treatment of cancer and chronic rheumatoid arthritis, respectively.48 49 Our results suggest that MMP inhibitors, particularly gelatinase-selective agents, may be of value in suppressing vein graft intimal thickening and subsequent atheroma formation. This hypothesis now needs to be tested, initially in our animal model.


*    Acknowledgments
 
This work was supported by grants from the British Heart Foundation (RG/94007) and the Garfield Weston Trust of Great Britain (to G.D.A.). We thank M. Smith and D. Knight for their technical assistance and J. Johnson for helping with the histological analysis.

Received November 11, 1998; accepted November 17, 1998.


*    References
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 *References
 

  1. Izzat MB, Angelini GD. Arterial conduits in myocardial revascularization. Surgery.. 1994;12:37–41.
  2. Kraiss L, Clowes A. Vascular grafts and their sequelae. In: Fuster VRR, Topol EJ, eds. Atherosclerosis and Coronary Artery Disease. Philadelphia, Pa: Lippincott-Raven; 1996;1:727–737.
  3. Angelini GD, Newby AC. The future of saphenous vein as a coronary artery bypass conduit. Eur Heart J. 1989;10:273–280.[Abstract/Free Full Text]
  4. Cox J, Chiasson D, Gotlieb A. Stranger in a strange land: the pathogenesis of saphenous vein graft stenosis. Prog Cardiovasc Dis. 1991;34:45–68.[Medline] [Order article via Infotrieve]
  5. Fremes S, Levinton C, Naylor C, Chen E, Christakis G, Goldman B. Optimal antithrombotic therapy following aortocoronary bypass: a meta-analyses. Eur J Cardiothorac Surg. 1993;7:169–180.[Abstract]
  6. Angelini GD, Bryan AJ, Williams HMJ, Soyombo AA, Williams A, Tovey J, Newby AC. Time-course of medial and intimal thickening in pig venous arterial grafts: relationship to endothelial injury and cholesterol accumulation. J Thorac Cardiovasc Surg. 1992;103:1093–1103.[Abstract]
  7. Newby AC, George SJ. Proposed roles for growth factors in mediating smooth muscle proliferation in vascular pathologies. Cardiovasc Res. 1993;27:1173–1183.[Free Full Text]
  8. Francis SE, Hunter S, Holt CM, Gadsdon PA, Rogers S, Duff GW, Newby AC, Angelini GD. Release of platelet-derived growth factor activity from pig venous arterial grafts. J Thorac Cardiovasc Surg. 1994;108:540–548.[Abstract/Free Full Text]
  9. George SJ, Williams A, Newby AC. An essential role for platelet-derived growth factor in neointima formation in human saphenous vein in vitro. Atherosclerosis. 1996;120:227–240.[Medline] [Order article via Infotrieve]
  10. Majesky MW, Reidy MA, Bowen-Pope DF, Hart CE, Wilcox JN, Schwartz SM. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol. 1990;111:2149–2158.[Abstract/Free Full Text]
  11. Ferns GAA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science. 1991;253:1129–1132.[Abstract/Free Full Text]
  12. Jackson CL, Raines EW, Ross R, Reidy MA. Role of endogenous platelet-derived growth factor in arterial smooth muscle cell migration after balloon catheter injury. Arterioscler Thromb. 1993;13:1218–1226.[Abstract/Free Full Text]
  13. Soyombo AA, Angelini GD, Bryan AJ, Newby AC. Surgical preparation induces injury and promotes smooth muscle cell proliferation in a culture of human saphenous vein. Cardiovasc Res. 1993;27:1961–1967.[Abstract/Free Full Text]
  14. Campbell JH, Black MJ, Campbell GR. Replication of smooth muscle cells in atherosclerosis and hypertension. In: Meyer P, Marche P, eds. Blood Cells and Arteries in Hypertension and Atherosclerosis. New York, NY: Raven Press; 1989:15–33.
  15. Hedin U, Bottger BA, Forsberg E, Johansson S, Thyberg J. Diverse effects of fibronectin and laminin on phenotypic properties of cultured smooth muscle cells. J Cell Biol. 1988;107:307–319.[Abstract/Free Full Text]
  16. Southgate KM, Davies M, Booth RFG, Newby AC. Involvement of extracellular-matrix-degrading metalloproteinases in rabbit aortic smooth muscle cell proliferation. Biochem J. 1992;288:93–99.
  17. Newby AC, Fabunmi RP, George SJ, Southgate KM, Banning AP, Thurston VJ, Williams A. Neointimal fibrosis in vascular pathologies: role of growth factors and metalloproteinases in vascular smooth muscle proliferation. Exp Nephrol. 1995;3:108–113.[Medline] [Order article via Infotrieve]
  18. Matrisian LM. Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet. 1990;6:121–125.[Medline] [Order article via Infotrieve]
  19. Galis ZS, Muszynski M, Sukhova GK, Simon-Morissey E, Unemori E, Lark MW, Amento E, Libby P. Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of extracellular enzymes required for extracellular matrix degradation. Circ Res. 1994;75:181–189.[Abstract/Free Full Text]
  20. Fabunmi RP, Baker AH, Murray EJ, Booth RFG, Newby AC. Divergent regulation by growth factors and cytokines of 95-kDa and 72-kDa gelatinases and tissue inhibitors of metalloproteinases-1, -2 and -3 in rabbit aortic smooth muscle cells. Biochem J. 1996;315:335–342.
  21. Zempo N, Koyama N, Kenagy R, Lea H, Clowes AW. Regulation of vascular smooth muscle cell migration and proliferation in vitro and in injured rat arteries by a synthetic matrix metalloproteinase inhibitor. Arterioscler Thromb Vasc Biol. 1996;16:28–33.[Abstract/Free Full Text]
  22. Zempo N, Kenagy RD, Au T, Bendeck M, Clowes MM, Reidy MA, Clowes AW. Matrix metalloproteinases of vascular wall cells are increased in balloon-injured rat carotid artery. J Vasc Surg. 1994;20:209–217.[Medline] [Order article via Infotrieve]
  23. Bendeck MP, Irvin C, Reidy MA. Inhibition of matrix metalloproteinase activity inhibits smooth muscle cell migration but not neointimal thickening after arterial injury. Circ Res. 1996;78:38–43.[Abstract/Free Full Text]
  24. Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res. 1994;75:539–545.[Abstract/Free Full Text]
  25. Southgate KM, Fisher M, Banning AP, Thurston VJ, Baker AH, Fabunmi RP, Groves PH, Davies M, Newby AC. Upregulation of basement membrane–degrading metalloproteinase secretion after balloon injury of pig carotid arteries. Circ Res. 1996;79:1177–1187.[Abstract/Free Full Text]
  26. George SJ, Zaltsman AB, Newby AC. Surgical preparative injury and neointima formation increase MMP-9 expression and MMP-2 activation in human saphenous vein. Cardiovasc Res. 1997;33:447–459.[Abstract/Free Full Text]
  27. Angelini GD, Bryan AJ, Williams HMJ, Morgan R, Newby AC. Distention promotes platelet and leukocyte adhesion and reduces short-term patency in pig arteriovenous bypass grafts. J Thorac Cardiovasc Surg. 1990;99:433–439.[Abstract]
  28. Galand P, Degraef C. Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S-phase cells in paraffin sections from animal and human tissues. Cell Tissue Kinet. 1989;22:383–392.[Medline] [Order article via Infotrieve]
  29. Izzat MB, Mehta D, Bryan AJ, Reeves B, Newby AC, Angelini GD. Influence of external stent size on early medial and neointimal thickening in a pig model of saphenous vein bypass grafting. Circulation. 1996;94:1741–1745.[Abstract/Free Full Text]
  30. Gown AM, Tsukada T, Ross R. Human atherosclerosis: immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am J Pathol. 1986;125:191–207.[Abstract]
  31. Roussel F, Dalion J. Lectins as markers of endothelial cells: comparative study between human and animal cells. Lab Anim. 1988;22:135–140.[Abstract/Free Full Text]
  32. Mehta D, George SJ, Jeremy JY, Izzat MB, Southgate KM, Bryan AJ, Newby AC, Angelini GD. External stenting reduces long-term medial and neointimal thickening and platelet derived growth factor expression in a pig model of arteriovenous bypass grafting. Nature. 1998;4:235–239.[Medline] [Order article via Infotrieve]
  33. Zaltsman AB, Newby AC. Increased secretion of gelatinase A and B from the aortas of cholesterol fed rabbits: relationship to lesion severity. Atherosclerosis. 1997;130:61–70.[Medline] [Order article via Infotrieve]
  34. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature. 1994;370:61–65.[Medline] [Order article via Infotrieve]
  35. Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, Goldberg GI. Mechanism of cell surface activation of 72-kD type IV collagenase: isolation of the activated form of the membrane metalloproteinase. J Biol Chem. 1995;270:5331–5338.[Abstract/Free Full Text]
  36. Lichte A, Kolkenbrock H, Tschesche H. The recombinant catalytic domain of membrane-type matrix metalloproteinase-1 (MT-MMP) induces activation of progelatinase A and progelatinase A complexed with TIMP-2. FEBS Lett. 1996;397:277–282.[Medline] [Order article via Infotrieve]
  37. Galis Z, Sukhova G, Lark M, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994;94:2493–2503.
  38. Huhtala P, Tuuttila A, Chow LT, Lohi J, Keski-Oja J, Tryggvason K. Complete structure of human gene for 92-kDa type IV collagenase: divergent regulation of expression for the 92- and 72-kilodalton enzyme genes in HT-1080 cells. J Biol Chem. 1991;266:16485–16490.[Abstract/Free Full Text]
  39. George SJ, Johnson JL, Angelini GD, Newby AC, Baker AH. Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits smooth muscle cell migration and neointimal formation in human saphenous vein. Hum Gene Ther. 1998;9:867–877.[Medline] [Order article via Infotrieve]
  40. Forough R, Koyama N, Hasenstab D, Lea H, Clowes M, Nikkari ST, Clowes AW. Overexpression of tissue inhibitor of matrix metalloproteinase-1 inhibits vascular smooth muscle cell functions in vitro and in vivo. Circ Res. 1996;79:812–820.[Abstract/Free Full Text]
  41. Baker AH, Zaltsman AB, George SJ, Newby AC. Divergent effects of tissue inhibitor of matrix metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation and death in vitro. J Clin Invest. 1998;101:1478–1487.[Medline] [Order article via Infotrieve]
  42. Pauly PR, Passaniti A, Bilato C, Monticone R, Cheng L, Papadopoulos N, Gluzband YA, Smith L, Weinstein C, Lakatta EG, Crow MT. Migration of cultured vascular smooth muscle cells through basement membrane barrier requires type IV collagenase activity and is inhibited by cellular differentiation. Circ Res. 1994;75:41–54.[Abstract/Free Full Text]
  43. Kawamata H, Kameyama S, Kawai K, Tanaka Y, Nan L, Barch DH, Stetler-Stevenson WG, Oyasu R. Marked acceleration of the metastatic phenotype of a rat bladder carcinoma cell line by the expression of human gelatinase A. Int J Cancer. 1995;63:568–575.[Medline] [Order article via Infotrieve]
  44. Talbot D, Brown P. Experimental and clinical studies on the use of matrix metalloproteinase inhibitors for the treatment of cancer. Eur J Cancer. 1996;32:2528–2533.
  45. Wojtowicz-Praga S, Low J, Marshall J, Ness E, Dickson R, Barter J, Sale M, McCann P, Moore J, Cole A, Hawkins MJ. Phase I trial of a novel matrix metalloproteinase inhibitor batimastat (BB94) in patients with advanced cancer. Invest New Drugs. 1996;14:193–202.[Medline] [Order article via Infotrieve]
  46. Wojtowicz-Praga S, Torri J, Johnson M, Steen V, Marshall J, Ness E, Dickson R, Sale M, Rasmussen HS, Chiodo TA, Hawkins MJ. Phase I trial of marimastat, a novel matrix metalloproteinase inhibitor, administered orally to patients with advanced lung cancer. J Clin Oncol. 1998;16:2150–2156.[Abstract]
  47. Millar AW, Brown PD, Moore J, Galloway WA, Cornish AG, Lenehan TJ, Lynch KP. Results of single and repeat dose studies of the oral matrix metalloproteinase inhibitor marimastat in healthy male volunteers. Br J Clin Pharmacol. 1998;45:21–26.[Medline] [Order article via Infotrieve]
  48. Tamura Y, Watanabe F, Nakatani T, Yasui K, Fuji M, Komurasaki T, Tsuzuki H, Maekawa R, Yoshioka T, Kawada K, Sugita K, Ohtani M. Highly selective and orally active inhibitors of type IV collagenase (MMP-9 and MMP-2): N-sulfonylamino acid derivatives. J Med Chem. 1998;41:640–649.[Medline] [Order article via Infotrieve]
  49. Levy DE, Ezrin AM. Matrix metalloproteinase inhibitor drugs. Emerg Drugs. 1997;2:205–230.



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