© 2003 by European Society of Cardiology
Copyright © 2003, European Society of Cardiology
Relationship between type IV collagen degradation, metalloproteinase activity and smooth muscle cell migration and proliferation in cultured human saphenous vein
Bristol Heart Institute, University of Bristol, Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK
a.newby{at}bris.ac.uk
* Corresponding author. Tel.: +44-117-928-3582; fax: +44-117-928-3581.
Received 12 June 2002; accepted 23 January 2003
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Abstract |
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The relationship between degradation of basement membranes, metalloproteinase (MMP) activity and smooth muscle cell (SMC) migration and proliferation has not been previously investigated in any intervention study. We used adenoviral overexpression of tissue inhibitors of metalloproteinases (TIMPs) in cultured human saphenous veins. By immunocytochemistry, the percentage of medial SMC surrounded by basement membrane type IV collagen (Coll-IV) decreased from 93±1 to 77±4% and 82±1% (n = 18, both P<0.001) after 7 and 14 days of culture, respectively, while all SMC that migrated to the neointima lacked Coll-IV. Overexpression of TIMP-1 or TIMP-3 significantly increased the percentage of medial SMC surrounded by Coll-IV (94±2 or 98±2%, respectively, both P<0.01 vs. no treatment) and decreased the number of neointimal SMC. Some 44±18 and 30±6%, respectively, of BrdU or PCNA labeled medial SMC remained surrounded by type IV collagen and this was not affected by overexpression of TIMP-1 or TIMP-3. We conclude that MMPs mediate loss of basement membrane and this is closely related to SMC migration. SMC proliferation does not require complete basement membrane degradation, which itself does not require MMPs in proliferating SMC.
KEYWORDS Atherosclerosis; Extracellular matrix; Vein; Smooth muscle; Remodeling
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1 Introduction |
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The blood vessel wall consists of two major types of extracellular matrix, namely basement membranes and interstitial matrix. Smooth muscle cells (SMC) are surrounded by a basement membrane composed of collagen IV, laminin and heparan sulphate proteoglycans [1]. The interstitial matrix contains types I and III collagen, elastin, fibronectin and both chondroitin and dermatan sulphate proteoglycans [2]. Proteolysis is required for turnover of matrix components. Matrix degrading metalloproteinases (MMPs) capable of degrading basement membrane components are upregulated in atherosclerosis [3–5], balloon injury [6–8] and vein grafts [9,10]. However, the ability of MMPs to degrade basement membrane has not been directly demonstrated in any of these studies.
Three inter-related functions have been proposed for basement membrane degradation in SMC. Classic in vitro experiments conducted by Thyberg and colleagues demonstrated that phenotypic change of SMC is inhibited by the basement membrane proteins, collagen IV and laminin, and is promoted by the interstitial matrix protein, fibronectin [11–13]. Indeed, a decrease in expression of laminin and increase in fibronectin is reversibly associated with phenotypic modulation of SMC in balloon injured rat carotid arteries [14].
Migration of SMC out of rabbit, rat and baboon explant cultures was inhibited using synthetic MMP inhibitors [15–17]. SMC migration and neointima formation is also inhibited by overexpression of tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2 or TIMP-3 [18–20] in organ culture of human saphenous vein (HSV). SMC migration and neointima formation in balloon injured rat carotid arteries is reduced by MMP inhibitors [21,22] and seeding of SMC transduced with TIMP-1 or adenovirus infection with either TIMP-1 or TIMP-2 also reduced neointima formation [23,24]. Based on this extensive literature a relationship between basement membrane degradation and migration of SMC has been implied, but has never been directly demonstrated.
Three reports found that MMP inhibitors also decreased SMC proliferation [15,22,25], but most studies found that MMP inhibitors and TIMPs did not [18–21,23,24]. The relationship between basement membrane degradation and SMC proliferation is less clear therefore and has also never been directly studied.
Human saphenous veins cultured in foetal calf serum reproducibly develop a neointima, in which 
50% of the cells arise by migration alone [26]. Neointima formation depends on the presence of factors secreted from the endothelium [27], and on endogenous release of platelet-derived growth factor [28]. The injury caused during surgical preparation of veins doubles the extent of neointima formation and increases by 10-fold the amount of medial SMC proliferation [29,30]. We therefore used quantitative immunocytochemistry in cultures of surgically prepared human saphenous veins to investigate the relationship between the presence of type IV collagen, as a marker for basement membranes, and neointima formation or medial SMC proliferation. We previously demonstrated that increased MMP expression and activation accompanies neointima formation and medial SMC proliferation in human saphenous veins [9]. Moreover, adenovirus-mediated gene transfer of TIMP-1 and TIMP-3 leads to overexpression of TIMPs and inhibition of MMP activity [18,20]. Paired human saphenous veins with and without adenovirus-mediated gene transfer of TIMP-1 and TIMP-3 were used therefore to study the influence of MMP activity on basement membrane degradation. Dual immunocytochemistry was used to investigate the presence of type IV collagen and its relationship to MMP activity in proliferating SMC.
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2 Methods |
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2.1 Material
All chemicals, unless stated otherwise, were obtained from Sigma (Poole, Dorset, UK). Media, antibiotics, and FCS were purchased from Gibco/BRL, nucleotides from Boehringer Mannheim, and enzymes from Promega. Replication-defective recombinant adenoviruses RadlacZ (reported gene), RAdTIMP-1 (human TIMP-1), and RAdTIMP-3 (human TIMP-3) have been described elsewhere [31].
2.2 Vein collection
HSV segments (2–5 mm diameter, 6–15 mm circumference) were collected and cultured as detailed previously [29]. Vein segments were obtained from patients undergoing coronary artery bypass grafting, who gave informed consent. The investigation conforms with the principles outlined in the Declaration of Helsinki. Surgically prepared segments were obtained after adventitial stripping, side branch ligation, gentle manual distension, and storage in heparinized blood for 60–120 min. Ethical permission was obtained from the United Bristol Healthcare Ethics Committee (reference E2847). Veins were collected in prewarmed sterile 20 mM HEPES-buffered RPMI 1640 medium containing papaverine hydrochloride (0.225 mg/ml) as a vasorelaxant, amphotericin B (5 µg/ml), and sodium heparin (20 IU/ml).
2.3 Adenovirus-mediated infection of human saphenous vein and subsequent organ culture
Luminal delivery of adenoviruses to HSV was performed as described [18,20]. Briefly, 4–6-cm segments were cannulated, and the luminal surface was exposed to adenovirus at 1.2x1010 p.f.u./ml at physiological pressure for 1 h. Segments of vein were cultured for 14 days in RPMI 1640 medium containing 30% FCS and antibiotics.
2.4 Immunocytochemistry and in situ zymography
Serial 3-µm paraffin sections were dewaxed and rehydrated (n = 6 paired segments). Endogenous peroxidase activity was inhibited with hydrogen peroxide. Antigen retrieval was carried out by using trypsin digestion. After the sections were blocked with 20% (v/v) goat serum in PBS, sections were incubated for 1 h at room temperature with primary antibodies diluted in 1.5% (w/v) BSA in PBS (mouse anti-human type IV collagen 1:50 (Sigma, clone CIV 22), anti-
-smooth muscle actin 1:400 and anti-smoothelin 1:5). Sections were incubated for 30 min with goat anti mouse biotinylated antibody and then with horseradish peroxidase labeled extravidin diluted by 1:400 and 1:200, respectively, in 1.5% BSA in PBS. Colour was developed with 0.5% (w/v) 3,3-diaminobenzidine (DAB), 0.03% (v/v) hydrogen peroxide for 10 min and followed by counterstaining with Mayer's hematoxylin. A negative control, for which the primary antibody was replaced with mouse IgG at the same dilution, was always included.
For dual immunostaining, slides were prepared and incubated with primary antibodies diluted in 1.5% (w/v) BSA in PBS (1:10 anti-bromodeoxyuridine (BrdU, ICN Pharmaceuticals) or 1:100 mouse monoclonal anti-proliferating cell nuclear antigen (PCNA; DAKO)) followed by a treatment with biotinylated secondary antibody, avidin–peroxidase and then developed with DAB as above. They were subsequently treated with avidin and biotin blocking reagent in 20% (v/v) goat serum (avidin–biotin blocking kit, Vector Laboratories) and incubated overnight with 1:50 anti-human type IV collagen, 1:400 anti--smooth muscle actin or 1:25 anti-CD-68 (Dako) followed by incubation with biotin-labeled secondary antibody, then avidin-alkaline-phosphatase and developed with Fast Red (Sigma).
In situ zymography was conducted on 8-µm frozen sections, as previously described [32]. Briefly, sections were coated with LM-1 photographic emulsion diluted 1:1 with 50 mmol/l Tris–HCl, pH 7.6, 50 mmol/l NaCl, 10 mmol/l CaCl2 and 0.05% Brij 35, with and without 20 mmol/l of EDTA to control for specificity, and incubated for 18 h at 37°C before air dying and photographic development. Metalloproteinase activity appeared as white areas on a black background and was quantified by pixel counting.
2.5 Quantification of type IV collagen expression
To quantify the number of SMC surrounded by type IV collagen in the media, each tissue section (n = 6 per group) was divided in six areas and positive and negative cells were quantified by use of image analysis (Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA). The results were expressed as number of type IV collagen positive SMC as a percentage of the total.
2.6 Statistical analysis
Data were analyzed using analysis of variance (ANOVA) for multiple comparisons. Paired analysis between two groups was performed using Student's t-test. Statistical significance was accepted when P<0.05.
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3 Results |
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3.1 Expression of type IV collagen during neointima formation in organ culture and its relationship to MMP activity
By immunocytochemistry, type IV collagen (Coll-IV) surrounded almost 95% of medial cells at the beginning of the experiment (Figs. 1A and 2
). A similar pattern was seen for laminin, although the intensity of staining was much less with the available antibody (results not shown). These data confirm that Coll-IV marks the presence of basement membranes. All medial cells were positive for -actin and almost all for smoothelin (Fig. 1E,F), consistent with their identification as SMC. As previously reported [26], adventitial cells were mainly -actin negative fibroblasts, which did not stain for type IV collagen (not shown). Culturing for 7 or 14 days led to the appearance of Coll-IV negative cells (Fig. 1B,C), which were located both close to the neointima and in the deeper layers of the media. During culturing there was a significant decrease in the percentage of medial SMC that were surrounded by Coll-IV after either 7 or 14 days (Fig. 2C). Culturing also led to the development of a neointima, which contained -actin and smoothelin positive SMC (Fig. 1E,F). All neointimal cells lacked Coll-IV (Fig. 1D).
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P<0.001 TIMPs or β-galactosidase vs. control veins.To investigate the role of MMPs in the loss of Coll-IV staining during culture, we used adenovirus-mediated gene transfer of TIMP-1 and TIMP-3. Gene transfer of TIMPs was confirmed by immunocytochemistry, as previously described in detail [18,33]. The percentage of TIMP-1 positive cells in the most luminal SMC layer of uninfected veins was 13±4, 21±7 and 23±2% after 1, 7 and 14 days culture (n = 4), respectively. This was not affected by transfer of the marker gene β-galactosidase (11±3, 22±3 and 25±1%, respectively), but was significantly increased to 57±3, 74±3 and 63±6% (P<0.05) after TIMP-1 gene transfer. TIMP-3 was localized bound to the extracellular matrix as expected. Transfer of TIMP-3 increased the percentage of the section stained from 10±1, 10±3 and 9±3% on days 1, 7 and 14, respectively, in uninfected veins to 17±3, 26±2 and 23±2% (P<0.05 for days 7 and 14), respectively. As previously documented in detail, infection with RAdTIMP-1 significantly reduced the number of neointimal cells per millimetre of vein circumference after 14 days of culture from 24±2 to 5±3; the corresponding reduction for RAdTIMP-3 was from 29±4 to 10±1 (both n = 6, P<0.05) [18,33]. The density of the cells per mm2 in the neointima (1800±600) was not different after TIMP-1 (800±600) or TIMP-3 (3000±1000) gene transfer. Medial cell density was also unaffected [18,33].
The few remaining neointimal cells in the presence of TIMP gene transfer were negative for Coll-IV (Fig. 2A,B). Adenovirus-mediated overexpression of TIMP-1 or TIMP-3 significantly reversed the decline in the percentage of medial SMC surrounded by Coll-IV seen during culture (Fig. 2A–C). This did not occur when veins were infected under the same conditions with adenovirus which drives expression of the marker gene, β-galactosidase (Fig. 2C). Overexpression of TIMP-1 or TIMP-3 also dramatically reduced MMP activity, as measured by in situ zymography (Fig. 3), as previously reported [18,20]. When quantified by morphometry of the white area as a percentage of the total section area, MMP activity after 14 days of culture was 49±6% (n = 7) in control veins, 45±5% in β-galactosidase transduced veins and significantly less at 15±3 and 12±2%, respectively, in TIMP-1 and TIMP-3 transduced veins. MMP activity was suppressed throughout the media in both superficial and deep layers, and even in the adventitia (Fig. 3). Taken together these experiments demonstrated that MMPs are essential for basement membrane degradation in at least some medial SMC.
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3.2 Expression of type IV collagen and its relationship to MMP activity in proliferating SMC
To investigate the relationship between basement membrane degradation and SMC proliferation, dual immunocytochemistry was carried out in tissues cultured for 14 days (Fig. 4). Dual staining for PCNA and Coll-IV was conducted in both series of veins, although only the TIMP-3 series of veins had been treated with (and hence were stained for) BrdU. PCNA gives an ‘instantaneous’ measure of proliferation, whereas BrdU was present continuously throughout the culture period and so gave a measure of total cell proliferation. As expected from Fig. 1D, all neointimal SMC were negative for Coll-IV, whether or not they had proliferated (Fig. 4A,C). Proliferating neointimal cells were positive for
-actin, consistent with their identification as SMC (Fig. 4E). To examine the contribution of monocytes to neointimal proliferation, we dual stained for CD-68 and BrdU, but found no CD-68 positive cells (not shown). In the media, most proliferating cells were found in the outer media (Fig. 4B,D), although a few proliferating cells were found in the layer close to the neointima (Fig. 4A,C). Medial proliferating cells were also -actin positive (Fig. 1F) and CD-68 negative (not shown), consistent with their identification as SMC. Among the BrdU (Fig. 4A,B) and PCNA (Fig. 4C,D) labeled medial SMC, some were partially or totally surrounded by Coll-IV (small arrows) and some were Coll-IV negative (arrowheads). Indeed, 44±18% of BrdU labeled cells and 30±6% of PCNA positive cells remained partially or completely surrounded by type IV collagen (Fig. 5). This was significantly (P<0.001) less than for the whole cell population (Fig. 2), which demonstrates an association between loss of basement membrane and SMC proliferation. However, the fact that 30–40% of proliferating cells remained surrounded by Coll-IV implies that complete basement membrane degradation is not necessary for SMC proliferation.
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TIMP-1 or TIMP-3 gene transfer did not significantly affect the percentage of proliferating medial SMC (9±2 vs. 10±1% for control vs. TIMP-1; 13±3 vs. 16±4% for control vs. TIMP-3; both n = 6) [18,33]. TIMP gene transfer also had no effect on the percentage of PCNA or BrdU positive proliferating medial SMC that were surrounded by Coll-IV after 14 days (Fig. 5). This implies that basement membrane degradation in proliferating SMC did not require MMPs.
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4 Discussion |
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Our results clearly demonstrate that loss of Coll-IV staining occurs during neointima formation in human saphenous veins. We focused on Coll-IV because suitable antibodies against the human protein are available and the staining was more intense than for laminin. However, the similar distribution of Coll-IV and laminin indicates that Coll-IV staining is a good marker for the presence of basement membranes. Given the integral nature of the basement membrane, complete loss of Coll-IV most probably indicates basement membrane disassembly and degradation. The human saphenous vein organ culture maintains a high degree of cell viability and has a predictable response to injury in terms of both neointimal and medial SMC proliferation [29]. It has been reported that some of the neointimal cells are adventitial myofibroblasts [34]. However, we showed that medial cells labelled on day 0 by gene transfer of β-galactosidase make up a significant part of the neointima formed after 14 days [18]. In addition, we show here that most of the neointimal cells express smoothelin, a marker for differentiated SMC [35]. Clearly many of the neointimal cells in our present experiments are Coll-IV negative SMC.
Human saphenous veins secrete potential basement membrane-degrading MMPs, particularly the pro- and active forms of MMP-2 and at least the pro-form of MMP-9 [9]. Other MMPs, including MMPs-1 and -3 are also secreted [36] and TIMPs-1 and -3 have the ability to inhibit all these enzymes. Adenovirus-mediated transfer of TIMP-1 and TIMP-3 genes occurs into 50% of surface cells and these cells survive to constitute a significant proportion of the neointimal cells [18,20]. In agreement with previous studies [37], human saphenous veins display gelatinolytic activity measured by in situ zymography (Fig. 3) and adenovirus-mediated gene transfer of TIMPs to the luminal surface of veins profoundly inhibits MMP activity in all cell layers [18,20]. This is because MMPs and TIMP-1 are largely secreted [9,37] and can therefore neutralize each other. Even in the case of TIMP-3, which remains matrix-bound [37], diffusion of MMPs apparently permits inhibition to occur. Inhibition of MMP activity by TIMP overexpression inhibited the decline in Coll-IV positive SMC, which is the first direct demonstration that MMPs mediate basement membrane degradation in any vascular tissue.
Cells that migrated to the neointima were devoid of type IV collagen, in agreement with data at early timepoints from the rat balloon injury model [14]. As previously reported [26] and confirmed here (Fig. 3A,C), 50% of neointimal cells were unlabelled with BrdU or PCNA and so arose in the neointima purely by migration. All these cells were negative for type IV collagen (Figs. 1D and 4A,C
) but positive for -actin (Fig. 4E), which implies that SMC that migrated to the neointima lost type IV collagen whether or not they subsequently divided. Moreover, a significant proportion of medial cells, including those in the inner part of the media, also became devoid of basement membrane, consistent with the idea that SMC lose their basement membrane before migrating to the neointima. Previous studies [18,20], confirmed here, showed that that TIMP-1 or -3 overexpression reduced the total number of SMC that migrated to the neointima. Other work showed that adenovirus-mediated overexpression of TIMPs decreased the migration of isolated SMC across artificial basement membranes [31]. From these observations and similar data in other models, it has been implied that MMP-mediated basement membrane degradation is necessary for SMC migration [15–22]. This is the first intervention study to directly demonstrate, however, that inhibition of MMPs does indeed decrease the loss of basement membrane around SMC. The requirement for basement membrane degradation could be either because the extracellular matrix is a physical brake on movement of SMC or because the signaling pathways mediating SMC migration are inhibited by SMC–basement membrane interactions (reviewed in Ref. [38]). The residual SMC that enter the neointima in the presence of TIMPs must do so either because inhibition of MMPs is incomplete or because of the presence of other proteases.
The relationship between basement membrane degradation and SMC proliferation has also not been examined directly until now. We found that medial SMC proliferation, at least in human saphenous vein, does not require complete loss of basement membrane since 40% of BrdU positive SMC and 30% of PCNA positive SMC remained partly or completely surrounded by Coll-IV. The small discrepancy between the PCNA and BrdU results was not statistically significant. Of course our data do not rule out some degree of matrix turnover in all proliferating SMC but this appears to be less than in SMC that migrated to the neointima. We found, furthermore, that MMPs were not required for basement membrane degradation in proliferating SMC, since neither TIMP-1 nor TIMP-3 overexpression reversed the breakdown of type IV collagen in medial proliferating SMC. Initially, this conclusion might seem to disagree with the overall effect of TIMP-1 and TIMP-3 on Coll-IV positivity. However, given that proliferating cells represented 10% of the total cell population, their contribution was too small to be reflected in the overall values, which are dominated by non-proliferating SMC. Our results help to explain why most studies, including all those using TIMPs, showed no effect on SMC proliferation [18–21,23,24]. The nature of the additional classes of protease present selectively in proliferating SMC, perhaps plasminogen activators, cathepsins or other undefined proteases, is beyond the scope of this study. Either these extra proteolytic activities are acquired during cell cycle progression or they exist in a pre-existing subpopulation of SMC. Indeed, there is direct evidence for a rat SMC population that is predominantly involved in neointima formation and overexpresses tissue type plasminogen activators [39]. Whether similar populations of cells exist in human saphenous vein is yet to be determined.
A limitation of our study is the identification of medial proliferating cells as SMC. Since all the medial cells in human saphenous vein stained for -actin and almost all for smoothelin before culture (Fig. 1E,F), it is logical to conclude that the proliferating medial cells are indeed SMC. Proliferating medial cells continued to be stained with -actin, consistent with this conclusion (Fig. 4E). However, proliferating SMC generally show reduced levels of -actin and lose late differentiation markers such as smoothelin [35], which makes it difficult to rule out that some proliferating medial cells might be myofibroblasts. Indeed a contribution from myofibroblasts has been proposed in vein-graft medial and neointimal thickening in vivo [40]. However, since proliferating myofibroblasts would be Coll-IV negative, their contribution does not affect our major conclusion that a proportion of proliferating SMC remained Coll-IV positive. Neither could it obscure the observation that TIMPs did not increase the proportion of Coll-IV positive proliferating SMC.
In summary, we demonstrate directly for the first time in vascular tissue that loss of basement membrane type IV collagen requires endogenously produced MMPs. Neointimal migration of SMC is invariably associated with loss of basement membranes. However SMC proliferation does not require complete basement membrane degradation, which itself does not require MMPs in proliferating SMC.
Time for primary review 28 days.
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Acknowledgements |
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This study was supported by the British Heart Foundation, the University of Granada and the Ministry of Education of Spain.
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