Gene transfer of tissue inhibitor of metalloproteinase-2 ... - Nature

Gene Therapy (1998) 5, 1552–1560  1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt

Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins SJ George, AH Baker, GD Angelini and AC Newby Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, BS2 8HW, UK

Metalloproteinases (MMPs) are implicated in neointima formation and hence vein graft failure. Gene transfer to elevate local levels of tissue inhibitor of metalloproteinases (TIMPs) is therefore a potential treatment. In this study, we have used lumenal application of a replication-defective recombinant adenovirus to overexpress TIMP-2 and observe the effects on neointimal thickening in a well characterised human saphenous vein organ culture model. Increased TIMP-2 expression was localised to lumenal surface cells but nevertheless increased total functional TIMP-2 secretion after 14 days culture from 4.0 ± 2.0 to

21.8 ± 2.9 ng/mg wet weight/day (P ⬍ 0.05, n = 3). In situ zymography revealed a marked inhibition of gelatinolytic activity by TIMP-2 gene transfer throughout the vein segments. Neointima formation and neointimal cell numbers were reduced 79% and 71%, respectively (P ⬍ 0.05; n = 8). TIMP-2 overexpression had no effect on smooth muscle cell proliferation, secretion of pro-MMP-2 or -9 and did not inhibit the processing of pro-MMP-2 to its active form. Our data indicate that TIMP-2 overexpression reduces neointimal thickening, primarily by inhibiting MMP activity and hence smooth muscle cell migration.

Keywords: gene therapy; vein graft; tissue inhibitor of metalloproteinase; adenovirus; smooth muscle cell

Introduction Coronary artery bypass grafts are performed over 400 000 times in the USA each year1 with the saphenous vein being the most commonly used conduit.2 However, wall thickening occurs following vein grafting into the arterial circulation due to vascular smooth muscle cell (SMC) migration and proliferation. Lumenal narrowing and superimposition of atheroma on the thickened intima promote thrombotic occlusion and hence account for the poor patency rates observed.3–5 The matrix metalloproteinases (MMPs) have the ability to degrade all components of the basement membrane and the interstitial matrix.6 Increased proteolysis by MMPs has been associated with cancer cell invasion, rheumatoid arthritis and more recently with the neointima formation that characterises vascular diseases. In human saphenous vein (HSV) organ cultures, neointima formation is associated with increased secretion of MMP2 and -9 and activation of MMP-2.7 Moreover, synthetic inhibitors of MMPs reduce neointima formation in this model,8 as they do in animal models of vascular injury.9,10 This suggests the use of gene transfer of tissue inhibitors of metalloproteinases (TIMPs) as gene therapy for vascular disease. This possibility is further supported by recent experiments with reseeding of SMC stably expressing TIMP-1 in the rat balloon injury model.11 Our own previous studies have shown highly efficient transfer of the

Correspondence: AH Baker The first two authors contributed equally to this study Received 17 March 1998; accepted 30 June 1998

TIMP-1 gene to human saphenous veins using adenovirus-mediated gene delivery.12 TIMP-1 overexpression inhibited SMC migration and neointima formation while an adenovirus expressing ␤-galactosidase had no significant effect. The potential for using other TIMP genes for gene therapy has not, however, been investigated. The TIMPs are a family of four members, TIMPs-1, -2, -3 and the recently described and little characterised TIMP-4.13–16 All TIMPs share the ability to inhibit MMPs by binding to the active site in 1:1 stoichiometry.17 There are, however, important biological differences between the TIMPs that impact on their use for gene therapy. Unlike TIMPs-1 and -2, TIMP-3 is not freely secreted but remains associated with the extracellular matrix.18 Also uniquely, TIMP-3 promotes cell death by apoptosis.19 TIMPs-1 and -2 are secreted together from many cells, including SMC,20 but the primary structure of TIMP-2 is much more highly conserved than that of TIMP-1, which would reduce the species barrier to TIMP-2 gene transfer in experimental animal models.21 Functionally, TIMP-1, but not TIMP-2, can bind and slow activation of proMMP-9 by binding to a C-terminal domain.22 In contrast, TIMP-2 binds selectively to pro-MMP-2, an interaction that can serve to present the enzyme for activation by membrane type MMP-1 (MT1-MMP).22–25 Higher concentrations of TIMP-2, but not TIMP-1, inhibit MT1MMP.26,27 In a recent study of adenovirus-mediated TIMP gene transfer into isolated SMC,19 TIMP-2, but not TIMP1, was shown to inhibit proliferation, an effect not mediated by inhibition of MMPs. On the other hand, growth promoting effects of TIMP-2 on HT1080 fibrosarcoma, normal dermal fibroblasts and Raji lymphoma cells have been described.28,29 Hence, while TIMP-2 has poten-

TIMP-2 inhibits human vein graft neointima formation SJ George et al

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Figure 1 Immunocytochemical detection of TIMP-2. Immunoreactive TIMP-2 was localised in HSV segments by immunocytochemistry at days 7 and 14 after infection. (a–c) Uninfected HSV at days 1, 7 and 14 respectively; (d–f) RAdTIMP-2 infected HSV at days 1, 7 and 14, respectively. Small arrows in panels b and c indicate intimal medial boundary and large arrows in panels e and f indicate cells with high level TIMP-2 immunoreactivity. The scale bar represents 10 ␮m and is applicable to all panels.

tial advantages for use in gene therapy, its biological effects in vascular tissues have been much less studied than those of TIMP-1. This aim of this study was therefore to investigate directly the effects of TIMP-2 gene transfer in the human saphenous vein model. Moreover, we aimed to define the mechanisms responsible for any effect on smooth muscle cell proliferation, migration and neointima formation.

Results Evaluation of transgene production and effect on cell viability TIMP-2 was not detected by immunocytochemistry in uninfected cultures on day 1, although low levels of TIMP-2 were detected in neointimal cells on days 7 and 14 in culture (Figure 1). Veins infected with an adenovirus expressing ␤-galactosidase, RAd35, showed similar low levels of TIMP-2 immunoreactivity as uninfected cultures (not shown). In contrast, RAdTIMP-2 infected veins at day 7 and 14 showed cells with high levels of immunoreactivity for TIMP-2 (Figure 1). Given the limited power of immunocytochemistry to show quantitative changes, reverse zymography of conditioned media was used to quantify functional TIMP-2 secretion in uninfected and RAdTIMP-2-infected cultures. Secretion of endogenous TIMP-2 was increased in control uninfected cultures over the 14 day culture period, consistent with our previous findings30 (Figure 2, Table 1). However, conditioned media from RAdTIMP-2 infected cultures showed significantly greater elevations in TIMP-2 production at

Figure 2 Functional analysis of TIMP-2 secretion by reverse zymography. Twenty-four hour conditioned media samples from three independent vein infections were loaded equally for vein wet weight. Coomassie-stained functional MMP inhibitory bands (representing TIMP activity) appear dark against a paler background. Lanes 1, 3 and 5: days 1, 7, 14 RAdTIMP-2 infected samples respectively; lanes 2, 4 and 6: days 1, 7 and 14 uninfected controls. The running positions of TIMP-1 (28 kDa) and -2 (20.5 kDa) are indicated, as well as the recombinant TIMP-2 standard (ST) loaded on each gel (40 ng).

days 7 (three-fold) and 14 (five-fold) after infection (Figure 2, Table 1). Similar results were obtained by Western blot analysis of conditioned media (not shown). Although tissue levels of TIMP-2 were more difficult to measure by Western blotting, the same trend was seen as for levels of secreted TIMP-2. Values for infected and uninfected tissue (ng/mg wet weight) on day 1 were


1554 Table 1 Quantification of TIMP-2 secretion into conditioned media Sample

Collection time (day)

TIMP-2 level (ng/mg wet weight/24 h)

0–1 0–1 6–7 6–7 13–14 13–14

1.5 ± 0.6 2.4 ± 1.1 5.7 ± 1.1 16.7 ± 2.7a 4.0 ± 2.0 21.8 ± 2.9a

Control uninfected RAdTIMP-2 infected Control uninfected RAdTIMP-2 infected Control uninfected RAdTIMP-2 infected

Statistical significance versus uninfected control (P ⬍ 0.05, n = 3).

a

3 ± 2 versus 8 ± 5 (not significant) on day 7 were 51 ± 23 versus 8 ± 6 (P ⬍ 0.05) and on day 14 were 21 ± 10 versus 9 ± 8 (not significant). Analysis of lactate dehydrogenase (LDH) release into the conditioned media, the numbers of medial SMC/mm2 in transverse sections and the percentage of cells positive for in situ end labelling were used as markers of cell viability. These revealed that there was no toxic effect of RAdTIMP-2 gene transfer into HSV compared with paired uninfected controls (Table 2).

Effect of TIMP-2 overexpression on MMP secretion and activity To ascertain the effect of TIMP-2 overexpression on secretion and activation of MMPs-2 and -9 in vein segments, gelatin zymography of conditioned media and tissue extracts was performed. As previously demonstrated by immunoprecipitation,7 human saphenous veins secrete a 95 kDa pro-MMP-9, a 72 kDa pro-MMP2 and a 68 kDa active MMP-2. The secretion of each species is increased by culturing for 14 days.7 Secretion of the pro-MMP-2 and -9 into the conditioned media was unaffected by RAdTIMP-2 infection (Figure 3, Table 3). Moreover, TIMP-2 overexpression at the levels obtained did not either increase or decrease the levels of 68 kDa active MMP-2. Similar data were obtained with tissue extracts. No difference in the levels of MMP-2 (pro- or active) or MMP-9 were observed in tissue extracts in uninfected compared to RAdTIMP-2-infected veins (Table 3). Gelatin in situ zymography was used to investigate the balance between MMP and TIMP activity in sections of veins. This revealed that the TIMP-2 overexpression profoundly inhibited MMP activity in RAdTIMP-2 infected cultures at days 1, 7 and 14 after infection compared with

Figure 3 Zymographical analysis of MMP secretion. Conditioned media samples (loaded equally for weight wet) were analysed for gelatinolytic activity on 7.5% gelatin gels. Lanes 1, 3 and 5: RAdTIMP-2 infected samples from days 1, 7 and 14 respectively; Lanes 2, 4 and 6: uninfected controls at days 1, 7 and 14 respectively. Pro- and active MMP-2 and pro-MMP-9 bands are indicated. An aliquot of the same conditioned medium from HT1080 cells was electrophoresced on each gel as an internal standard (lane 7).

uninfected controls (Figure 4). Proteolytic inhibition occurred throughout the pre-existing neointima and medial layers of the vein implying that there was wide diffusion of the TIMP-2 derived from the transduced cells. Incubation of vein segments with standard MMP inhibitors (EDTA and Ro-31–9790) similarly inhibited the gelatinolytic activity (not shown).

Effect of TIMP-2 overexpression on proliferation, SMC migration and neointima formation RAdTIMP-2 infection significantly reduced neointima formation after 14 days in culture by 79% compared with uninfected controls (Table 4, Figure 5). The total number of neointimal cells was reduced 62% on day 7 and 71% on day 14 (Table 4). As a result the density of neointimal cells/mm2 was not affected by RAdTIMP-2 infection (Table 4). This implies that the major effect of RAdTIMP2 infection was on the cell population in the neointima rather than the amount of extracellular matrix. Infection of HSV with RAdTIMP-2 did not affect cell proliferation either in the medial layer or within the resulting neointima as analysed by continuous BrdU labelling (Table 4). However, the number of unlabelled neointimal cells (ie those that arose in the neointima by migration alone) was significantly reduced by RAdTIMP-2 infection. Hence the major effect of TIMP-2 overexpression was to inhibit migration of SMC.

Discussion Developments in gene transfer technology have enhanced the ability to overexpress specific genes in intact vascular tissue in the search for a suitable and effective gene therapy for vascular diseases. Thus far, attention has been focused on identifying suitable trans-

Table 2 Effect of TIMP-2 overexpression on cell viability Parameter

LDH (IU/1) Medial cell/mm2 Neointimal ISEL (%) Medial ISEL (%)

Day 1

Day 7

Day 14

Control

TIMP-2

Control

TIMP-2

Control

TIMP-2

12 ± 7 752 ± 45 — 12 ± 3

18 ± 7 720 ± 65 — 8±3

16 ± 4 667 ± 107 3±1 4±1

19 ± 7 613 ± 94 7±3 7±2

16 ± 7 579 ± 85 1±1 5±2

13 ± 6 541 ± 57 2±1 5±1


1555 Table 3 Effect of TIMP-2 overexpression on MMP-2 and -9 production Day 1

Pro-MMP-9 (95 kDa) Pro-MMP-2 (72 kDa) Active-MMP-2 (68 kDa)

Conditioned medium Tissue Conditioned medium Tissue Conditioned medium Tissue

Day 7

Day 14

Control

TIMP-2

Control

TIMP-2

Control

TIMP-2

4±1 53 ± 30 28 ± 10 71 ± 29 6±3 65 ± 22

5±2 66 ± 26 40 ± 22 95 ± 10 17 ± 9 94 ± 7

56 ± 19 78 ± 30 127 ± 26 329 ± 42 114 ± 42 490 ± 40

62 ± 18 97 ± 19 141 ± 32 283 ± 30 97 ± 40 401 ± 38

54 ± 18 80 ± 21 142 ± 30 224 ± 11 94 ± 33 410 ± 38

72 ± 15 87 ± 21 147 ± 30 273 ± 65 121 ± 50 383 ± 66

Zymograms were loaded with conditioned media (equivalent to 0.045 mg tissue per lane, n = 6) or tissue extracts (equivalent to 1.76 mg tissue per lane, n = 3). MMP activities were estimated by densitometric scanning of zymograms. Activities are expressed as a percentage of the HT1080 standard in optical density units. No statistically significant differences were observed between control and RAdTIMP2-infected cultures at any time-point.

Figure 4 Effect of overexpression of TIMP-2 on gelatinolytic MMP activity by in situ zymography. Frozen sections from uninfected and RAdTIMP2 infected HSV segments were analysed at days 1, 7 and 14 for gelatinolytic activity. Uninfected (a–c) at days 1, 7 and 14 days, respectively, and RAdTIMP-2 infected (d–f) at days 1, 7 and 14, respectively. Large arrows in panels (b) and (c) indicate neointimal-medial boundaries. Small arrows in (a) and (d–f) indicate luminal surface. White areas represent proteolytic degradation of substrate by MMP activity within the vein section. The scale bar in (a) represents 25 ␮m and is applicable to all panels.

genes and delivery systems. The present study is the first to identify TIMP-2 as a candidate for vascular gene therapy to reduce neointima formation, with particular relevance here to improving vein graft patency. Efficient overexpression of TIMP-2 was achieved at the lumenal surface of HSV by adenovirus-mediated delivery and this inhibited SMC migration and neointima formation in organ culture. No effect of TIMP-2 overexpression was

observed on pro-MMP-2 or -9 secretion or on activation of pro-MMP-2. Hence inhibition of the active MMPs underlies the effect on neointima formation. The infection protocol used for these studies produced a highly localised infection and subsequent production of TIMP-2 at the lumenal surface of HSV, as detected by immunocytochemistry. This is consistent with our previous findings for localised delivery of lacZ and TIMP-1 by


1556 Table 4 Effect of TIMP-2 overexpression on neointima formation and other morphometric parameters in HSV Day 1

Neointimal thickness (␮m) Neointimal cells/mm Neointimal cells/mm2 (×10) Medial BrdU index (%) Neointimal BrdU index (%) Unlabelled neointimal cells/mm a

Day 7

Day 14

Control

TIMP-2

Control

TIMP-2

Control

TIMP-2

0±0 0±0 — 0±0 0±0 0±0

0±0 0±0 — 0±0 0±0 0±0

4±1 13 ± 3 574 ± 122 17 ± 6 51 ± 7 6±2

1±1 5 ± 1a 584 ± 90 18 ± 5 47 ± 7 2 ± 1a

19 ± 6 24 ± 5 207 ± 34 19 ± 7 58 ± 6 10 ± 2

4 ± 1a 7 ± 2a 173 ± 56 20 ± 8 53 ± 10 3 ± 1a

Statistical significance versus uninfected control (P ⬍ 0.05, n = 8).

Figure 5 Effect of RAdTIMP-2 infection on neointima formation. Representative sections of HSV stained with EVG to highlight neointima formation at days 1 (a and d), 7 (b and e) and 14 (c and f) after infection. (a–c) Uninfected HSV; and (d–f) RAdTIMP-2 infected. The arrows indicate the neointima media boundary. The scale bar in (a) represents 25 ␮m and is applicable to all panels.

adenoviruses to veins.12 Given this localised infection, it is all the more remarkable that total levels of TIMP-2 secretion were elevated as shown by reverse zymography. This is undoubtedly due to the high efficiency of transgene transcription from the CMV IEP in HSV organ cultures. The feasibility of high efficiency adenovirus or adenovirus-facilitated gene transfer into vein grafts demonstrated here is supported by work from other groups.31–33 Organ culture of saphenous vein has provided a rapid

and highly reproducible human model of intimally directed smooth muscle cell proliferation. It has been used sucessfully for mechanistic,7,8 intervention34 and gene transfer12 studies. The contribution of cell proliferation to neointima formation has been quantified using continuous labelling with thymidine34 or BrdU (this work). Both methods yield the conclusion that approximately 50% of the new intimal cells remain unlabelled and must therefore have arisen solely by migration. This


is remarkably similar to the proportion of migrating cells shown by the same methods to contribute to the neointima of balloon injured rat carotid arteries.35 Extrapolation of any results from organ culture to the situation of vein grafting requires caution because the endogenous mitogens released in response grafting are substituted by the addition of serum. As with the rat studies, an effect on neointimal thickness and neointimal cell number without an effect on proliferation has been interpreted as showing an effect on migration.36 The effect of TIMP-2 to inhibit neointima formation seen here was mediated by these criteria through selective inhibition of SMC migration. This conclusion is consistent with work on isolated rat SMC19 and other work with synthetic MMP inhibitors.9,37 Overexpression had no effect on the overall neointimal or medial proliferation index. Previous data showed that adenovirus-mediated overexpression of TIMP-2 inhibits growth and BrdU incorporation in isolated rat SMC.19 However, this effect was confined to the transduced cells and was not mimicked by addition of TIMP-2.19 Since only a minority of the neointimal cells on day 14 were expressing TIMP-2, it is not surprising that the overall BrdU index was not affected. In other systems, adding recombinant TIMP-2 does inhibit basic fibroblast stimulated microvascular endothelial cell proliferation38 and retrovirus-mediated TIMP-2 overexpression inhibits both tumour growth and invasion of cancer cells.39,40 This may imply some cell type specific differences in sensitivity of proliferation to TIMP-2. Metalloproteinase activity has been shown to be tightly regulated at three levels, namely gene expression, control of activation of proforms and control of inhibition by varying the abundance of TIMPs.6 We therefore set out to determine at which level or levels gene transfer of TIMP-2 affected MMP activity. In our experiments, as expected, overexpression of TIMP-2 had no effect on the synthesis and secretion of MMP-2 or -9 measured by zymography. Unlike in the in situ zymography assay, in this method, any complexes between TIMPs and MMPs are dissociated by inclusion of SDS. More suprisingly TIMP-2 overexpression also had no effect on the activation of MMP-2. Although the presence of TIMP-2 is essential for the activation on MMP-2 by MT1-MMP,27 high levels of TIMP-2 are known to inhibit MT1MMP.27,41 Our results suggest either that TIMP-2 levels were insufficiently raised to inhibit MT1-MMP, which is present in HSV (A Kranzho¨fer and AC Newby, unpublished observations) or that there is an alternative pathway for activation of MMP-2 in HSV. Inhibition of MMP proteolytic activity was observed throughout the vein by in situ zymography, not only at the lumenal surface, hence indicating a substantial bystander effect from the transduced cells. Given the lack of effects on MMP expression and activation, inhibition of active MMPs appears to be the main factor underlying the inhibition of SMC migration by TIMP-2 overexpression. Data showing that TIMP-1 overexpression12 and a nonselective synthetic MMP inhibitor, Ro 31–9790, also reduce neointimal thickening to a similar extent in HSV organ cultures8 are consistent with this conclusion. In conclusion, TIMP-2 is an attractive candidate for gene therapy of vascular diseases such as vein graft stensosis. The secretion of TIMP-2 from infected cells not only increases locally available levels, but the 20.5 kDa

protein appears to diffuse widely throughout vein segments evoking a pronounced bystander effect. This would in turn enable lower titres of delivery vehicles to be used to achieve therapeutic responses. Due to diffusible nature of TIMP-2, and the pronounced bystander effect detected by MMP proteolytic inhibition, overexpression of TIMP-2 may be suitable for gene therapy of several vascular pathologies, including vein graft stenosis and restenosis following balloon angioplasty.

Materials and methods All reagents were purchased from Sigma (Poole, UK) except those below. LM-1 emulsion was obtained from Amersham International (Bucks, UK), l-glutamine, penicillin-streptomycin and culture media from Gibco BRL (Paisley, UK), amphotericin from McCarthy Medical (Wrexham, UK) and gentamycin from Roussel (Uxbridge, UK). D-19 developer and Unifix solution were from Kodak (Bridgend, UK). The polyclonal sheep anti-human TIMP-2 clone H225/542 was a generous gift from Professor G Murphy (University of East Anglia, UK) and biotinylated anti-sheep secondary antibodies were purchased from the Binding Site (Birmingham, UK). p-Aminophenylmercuricacetate (APMA)-activated rabbit fibroblast MMPs were used as the source of activated enzyme for reverse zymography (a generous gift from Dr A Kranzho¨fer, Bristol Heart Institute, UK). Ro 31–9790 was a generous gift from Dr Bradshaw (Roche, Welwyn, UK).

Recombinant adenovirus The replication-defective recombinant adenoviruses engineered to overexpress human TIMP-2 from the cytomegalovirus immediate–early promoter (CMV IEP), (RAdTIMP-2) and ␤-galactosidase (RAd35) have been described elsewhere.43,44 Stocks of adenovirus were banded on caesium chloride gradients, extensively dialysed and stored at −70°C before use. Lack of helper adenovirus was determined by titration on non-permissive Hela cells and lack of E1a by immunofluorescence using an anti-Ad5 E1a monoclonal antibody (Calbiochem, Nottingham, UK). Infection of human saphenous vein Surplus, surgically prepared human saphenous vein was collected from patients after coronary artery by-pass surgery as described previously.34,45 The vein was placed in wash medium (20 mm Hepes-buffered RPMI 1640 supplemented with 2 mm l-glutamine, 8 ␮g/ml gentamycin, 100 IU/ml penicillin and 100 ␮g/ml streptomycin). The adventitia was removed, the vein bisected transversely and one segment used as a paired uninfected control. The vein was infused with 100 ␮l of 1.2 × 1010 p.f.u./ml RAdTIMP-2 as described previously12 for a period of 1 h. Segments were then opened longitudinally and cut transversely into three 5–10-mm segments. The uninfected control was cut similarly. Vein segments were cultured separately endothelial surface uppermost using a modification of the method of Pederson and Bowyer46 as described previously34,45 in culture medium (RPMI 1640 supplemented with 30% foetal calf serum (FCS), 2 mm lglutamine, 100 IU/ml penicillin and 100 ␮g/ml streptomycin. Culture media were changed every 2 days. For collection of serum-free conditioned media, vein seg-

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ments were washed extensively in FCS free culture media and further incubated for 24 h before collection.

Immunocytochemical detection of TIMP-2 Paraffin-embedded sections were used for immunocytochemical analysis of TIMP-2 as described in detail elsewhere for TIMP-112 but using a sheep anti-human TIMP2 antibody. Sections were stained at days 1, 7 and 14 after infection for TIMP-2 protein. Protein extraction Uninfected and infected vein segments were snap frozen in liquid nitrogen and stored at −70°C until required. On the day of analysis, vein segments were crushed under liquid nitrogen and dissolved in lysis buffer (0.1 m NaCl, 10 mm Tris-HCl, 1 mg/ml aprotinin, 100 ␮g/ml phenylmethylsulphonylflouride (PMSF), pH 7.6). Zymography and reverse zymography Secretion of MMPs from vein segments was quantified using gelatin zymography as described.47,48 Briefly, unconcentrated conditioned media or tissue extract samples were loaded according to wet weight of vein on to 7.5% polyacrylamide gels containing 0.1% sodium dodecyl sulphate (SDS) and 2 mg/ml gelatin (prepared from calf skin type I collagen, Sigma C3511) in non-reducing buffer (0.0625 m Tris-HCl pH 6.8, 10% glycerol, 4% SDS and 0.005% bromophenol blue). Following electrophoresis, and removal of SDS, gels were incubated for 16 h in 50 mm Tris HCl pH 8.0, 50 mm NaCl, 10 mm CaCl2 and 0.05% (w/v) Brij-35 at 37°C. Gels were stained with 0.1% Coomassie brilliant blue and bands of lysis representing gelatinase activity were visualised against a blue background. The intensity of bands was quantified as justified in detail elswhere.7 To allow for standardisation, between gels, an aliquot of the same sample of HT1080 conditioned medium that contained proMMP-9, proMMP-2 and active MMP-2 was run on each gel. Values for each band were then expressed relative to the corresponding band in the HT1080 standard. For reverse zymograms,49 the same procedure was applied except the samples were electrophoresced through 11% gels and incubated for 2 h at 37°C in conditioned media from phorbol 12-myristate 13-acetate (PMA) activated rabbit skin fibroblasts, which provided a source of activated metalloproteinases. TIMP activity was visualised as zones of gelatinase inhibition against a partial gelatin digested background following Coomassie blue staining. Quantification was performed using 40 ng of recombinant human TIMP-2 as a standard for each gel. Bands were quantified using a BioRad GS 690 densitometer (BioRad, Hemel Hempstead, UK) and values are calculated to ng TIMP2/mg weight wet vein/24 h. Analysis of cell proliferation Vein segments were cultured continuously in the presence of 10 ␮M bromodeoxyuridine (BrdU). Paraffinembedded sections were analysed immunocytochemically for the presence of BrdU incorporation as described using a monoclonal anti-BrdU antibody (Europath, Bude, UK). Nuclei were counterstained with haematoxylin and the BrdU index quantified in five high power fields per section. Measurement of neointima formation Transverse sections (3 ␮m) were stained with Miller’s elastic van Gieson (EVG) and Mayer’s haematoxylin and

eosin staining and the neointima formation quantified using image analysis (Image ProPlus; Media Cybernetics, Maryland, USA). Briefly, neointimal thickness was measured by calculating the total neointimal area at four 100␮m spaced points per section (n = 8 veins per group) and dividing by the section length. The number of neointimal cells at 1, 7 and 14 days were counted on all pairs of sections and the mean calculated for each vein and divided by the length of the section (in mm) to normalise for vein diameter variations between patients (n = 8).

In situ zymography MMP inhibitory activity was determined by in situ zymography as described.50 Briefly, 8 ␮m frozen sections were applied to glass slides and coated with LM-1 photographic emulsion diluted 1:2 with incubation medium (50 mm Tris, 50 mm NaCl, 10 mm CaCl2, 0.05% (w/v) Brij 35, pH 7.6) and incubated overnight at 37°C. Sections were developed in the light with Kodak D-19 developer and fixed using Kodak Unifix solution. Gelatinolytic activity was identified as white holes of lysis on a black background. Controls included incubation with buffer supplemented with 20 mmol/l EDTA or 200 nmol/l MMP inhibitor Ro 31–9790 (kind gift of Dr Bradshaw, Roche Diagnotics, Welwyn, UK). Frozen sections were analysed at days 1, 7 and 14 after infection for both gelatinolytic and caseinolytic activity. Determination of cell viability Cell viability was determined by in situ end labelling as described.7,12 Briefly, sections were washed twice in 1 × TE (10 mm Tris HCl pH 8.0, 1 mm EDTA) and incubated in labelling mix (50 mm Tris HCl (pH 7.2), 10 mm MgSO4, 0.1 mm dithiothreitol, 0.01 mm dATP, dCTP, dGTP, 0.01 mm biotin-dUTP, and 8 units/ml of DNA polymerase I (Klenow; Promega, Southampton, UK) for 15 min at room temperature. Sections were rinsed in 1 × TE and endogenous peroxidase inhibited by incubation in 2% H2O2 for 5 min. Following further washing, biotin was labelled with Extravidin peroxidase (Sigma, Poole, UK) (1 in 200 dilution) in 10% FCS/PBS. Incubation in diaminobenzidine and subsequent counterstaining with haematoxylin were used to distinguish positive ‘nicked’ DNA from negative DNA. In addition, cell viability was also determined by measurement of SMC density in the medial layer and by release of lactate dehydrogenase (LDH) into the conditioned media (LDH assay kit; Boehringer Mannheim, UK). Statistical analysis Analysis was performed using two-way analysis of variance (ANOVA) for multiple comparisons between groups and where there was a significant difference between the groups Bonferroni tests were performed to test for significance between the two groups at each time-point.

Acknowledgements The authors wish to thank Professor G Murphy for the anti-TIMP-2 antibody, Melanie Smith, Jason Johnson and Catherine Keen for technical assistance. This work was funded by the Garfield Weston Foundation, the British Heart Foundation and the Medical Research Council for Great Britain.


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