This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lijnen, H. R. Articles by Collen, D. Search for Related Content PubMed PubMed Citation Articles by Lijnen, H. R. Articles by Collen, D. Related Collections Genetically altered mice Quantitative modeling Other Vascular biology

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2863.)
© 1999 American Heart Association, Inc.

Vascular Biology

Accelerated Neointima Formation After Vascular Injury in Mice With Stromelysin-3 (MMP-11) Gene Inactivation

H. Roger Lijnen; Berthe Van Hoef; Ingrid Vanlinthout; Maria Verstreken; Marie-Christine Rio; Désiré Collen

From the Center for Molecular and Vascular Biology (H.R.L., B.V.H., I.V., M.V., D.C.), University of Leuven, Leuven, Belgium; and CNRS-INSERM-ULP (M.-C.R.), Illkirch, France.

Correspondence to H. R. Lijnen, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven, Belgium. E-mail: roger.lijnen{at}med.kuleuven.ac.be

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—The hypothesis that stromelysin-3 (MMP-11), a unique member of the matrix metalloproteinase (MMP) family, plays a role in neointima formation was tested with the use of a vascular injury model in wild-type (MMP-11^+/+) and MMP-11–deficient (MMP-11^-/-) mice. Neointima formation 2 to 3 weeks after electric injury of the femoral artery was significantly enhanced in MMP-11^-/- as compared with MMP-11^+/+ mice, in both mice of a pure 129SV genetic background (0.014 versus 0.0010 mm² at 2 weeks, P<0.001) and those of a 50/50 mixed 129SV/BL6 background (0.030 versus 0.013 mm² at 3 weeks, P<0.05). The medial areas were comparable, resulting in intima/media ratios that were significantly increased in MMP-11^-/- as compared with MMP-11^+/+ arteries, in mice of both the 129SV (1.0 versus 0.18, P<0.001) and mixed (1.5 versus 0.70, P<0.05) backgrounds. Nuclear cell counts in cross-sectional areas of the intima of the injured region were higher in arteries from MMP-11^-/- mice than in those from MMP-11^+/+ mice (210 versus 48, P<0.001, in pure 129SV mice and 290 versus 150, P<0.01, in mice of the mixed genetic background). Immunocytochemical analysis revealed that -actin–positive and CD45-positive cells were more abundant in intimal sections of MMP-11^-/- mice. Degradation of the internal elastic lamina was more extensive in arteries of MMP-11^-/- mice than in those of MMP-11^+/+ mice (39% versus 6.8% at 3 weeks, P<0.005). The mechanisms by which MMP-11 could impair elastin degradation and cellular migration in this model remain, however, unknown.

Key Words: neointima • restenosis • transgenic mice • stromelysin-3

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Neointima formation is a wound-healing process that occurs in response to vascular injury. Degradation of the extracellular matrix with migration of smooth muscle cells (SMCs) from the media to the intima, followed by deposition of extracellular matrix, leads to this intimal hyperplasia.^{1 2} The plasminogen/plasmin and matrix metalloproteinase (MMP) systems are believed to play a role in this phenomenon by degrading the extracellular matrix and thereby allowing SMC migration.^{3 4 5 6 7 8} In a balloon-injured rat carotid artery model, a correlation was observed between changes in plasminogen activators³ or MMPs^{5 6} and SMC migration. This was supported by the observation that in vivo SMC migration in the rat was inhibited by the plasmin inhibitor tranexamic acid⁹ and by MMP inhibitors.^{5 10} The finding that arterial neointima formation after vascular injury was significantly impaired in urokinase-deficient and in plasminogen-deficient mice provided more direct evidence of a significant role of the plasminogen/plasmin system.^{11 12} Enhanced expression of MMP-2 and MMP-9 has been observed after perivascular electric injury in the mouse.¹³ In baboon aortic explants, SMC migration requires plasminogen activators and both MMP-2 and MMP-9.^{8 14} Little is known, however, about the potential role of other MMPs in vascular remodeling.

Most MMPs are produced in a latent (pro) form that may be activated by different mechanisms.¹⁵ MMP-11 (stromelysin-3) is a unique MMP because it is not secreted as a zymogen but is processed by furin within the constitutive secretory pathway.¹⁶ The mature form of MMP-11 is unable to hydrolyze the major extracellular matrix components, but it hydrolyzes ₁-proteinase inhibitor.¹⁷ However, the 28-kDa NH₂-terminal domain of mouse MMP-11 has the properties of a weak metalloproteinase,¹⁸ and on deletion of 175 COOH-terminal amino acids, it acquires enzymatic activity against casein, laminin, and type IV collagen.¹⁹ In addition, the catalytic domain of murine MMP-11 was found to degrade the A-chain of fibrinogen.²⁰ MMP-11 is expressed at a high level in most invasive carcinomas.^{21 22 23} Tissues that normally undergo extensive remodeling, such as the placenta, uterus, and postlactation mammary glands, also express MMP-11.^{21 22 24} These observations suggest that it may play a role in extracellular matrix remodeling.

In the study described here, we investigated the potential role of MMP-11 in neointima formation after vascular injury in mice. Surprisingly, we found more pronounced neointima formation in mice with MMP-11 gene deficiency.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals
MMP-11^-/- and wild-type mice (CNRS-INSERM-ULP, Illkirch, France) of the same genetic background (129SV or 129SV/BL6, 50/50) were obtained and characterized as described elsewhere.²⁵ Homozygosity of offspring was confirmed by genotyping of tail-tip DNA (digested with SpeI) by using as probe a 0.8-kb SpeI-EcoRI restriction fragment derived from intron 7 and Southern blotting (data not shown). Mice were kept in microisolation cages on a 12-hour-day/12-hour-night cycle and fed standard chow. Animals were anesthetized by intraperitoneal injection of 60 mg/kg Nembutal (Abbott Laboratories), and all experiments were performed in accordance with guidelines of the American Physiological Society and the International Society on Thrombosis and Hemostasis.²⁶ Mice (males and females) were 12 to 15 weeks old with a body weight (mean±SEM) of 26±0.9 g (n= 23) or 25±0.6 g (n= 21) for 129SV/BL6 MMP-11^+/+ or MMP-11^-/- mice, respectively, and 28±0.9 g (n= 10) or 28±1.2 g (n= 10) for 129SV MMP-11^+/+ or MMP-11^-/- mice, respectively. Statistical analysis was performed by using a 2-tailed t test (nonparametric Mann-Whitney U test).

Vascular Injury Model
Perivascular electric injury to the femoral artery of mice was performed essentially as described elsewhere.²⁷ In brief, arteries were exposed by blunt-end dissection and injured by electric current at distances of 1 mm over a total length of 2 to 3 mm. One to three weeks after injury, the animals were killed and vessel segments were fixed in 1% paraformaldehyde or directly embedded in OCT (Tissue-Tek), snap-frozen in 2-methyl butane, and stored at -80°C. Seven-micron-thick sections were made throughout the whole artery and stained with hematoxylin and eosin, Verhoeff’s and von Gieson’s stains, or the appropriate antiserum or were used for fibrin overlay as described below. Longitudinally alongside the artery, different positions were identified corresponding to noninjured sections (positions 1 and 5), to the borders of the injury (positions 2 and 4), and to the center of the injury (position 3).²⁷

In Situ Zymography on Fibrin Overlay
In situ zymography on 7-µm cryostat sections of arteries was performed by fibrin overlay at 37°C for 2 hours, without or with addition to the gel of antibodies against murine tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA) (final concentration, 40 µg/mL), or was performed for 48 hours in the presence of anti-tPA and anti-uPA antibodies to determine PA-independent activity.^{11 13} For comparison, the lysis area (mm²) was normalized to the total area of the section. Data are reported as mean±SEM of 6 experiments (using different animals); within each experiment, 6 to 14 sections equally spaced throughout uninjured arteries or throughout the center of the injury (position 3) were analyzed.

Histological and Immunocytochemical Testing
Morphometric measurements of cross-sectional areas and cell counts were made in a blinded manner on transverse arterial sections (stained with hematoxylin and eosin) using a computer-assisted image analysis system as described elsewhere.²⁷ Measurements were made at equally spaced positions (80 to 100 µm apart) across the artery. After staining for elastin with Verhoeff’s and von Gieson’s stains, the circumference of the internal elastic lamina was measured by computer-assisted image analysis.

For immunostaining, the primary monoclonal antibodies used were rat anti-mouse macrophage-specific Mac3 (clone M3/84, Pharmingen), biotinylated mouse anti-human smooth muscle -actin (clone 1A4, Sigma Chemical Co), biotinylated rat anti-mouse pan-leukocyte antigen CD45 (clone 30F11.1, Pharmingen), and biotinylated rat anti-mouse polymorphic neutrophils (clone 7/4, Biosource). Immunostaining for Mac3 was performed by using biotinylated rabbit anti-rat immunoglobulins (Dakopatts) and the Tyramide signal amplification kit (Dupont-NEN), whereas for -actin, CD45, and neutrophils, biotinylated primary antibodies were used in combination with the Vectastain system (ABC Elite kit, Vector Laboratories). Peroxidase activity was determined by incubating sections in 0.05 mol/L Tris-HCl buffer, pH 7.0, containing 0.06% 3,3'-diaminobenzidine and 0.01% H₂O₂, followed by counterstaining with Harris’ hematoxylin. Specificity of the staining was confirmed by omission of the primary antibody or by replacing it with equivalent amounts of isotype-matched, nonimmune IgG or serum. The number of positive cells was semiquantitatively graded on a scale of - (no positive cells) to ++++ (>75% cells positive), with +++, ++, and + representing 50% to 75%, 25% to 50%, and <25% of positively stained cells, respectively.

Proliferating cells were identified by immunostaining with monoclonal anti–proliferating cell nuclear antigen (PCNA; clone PC10, Sigma) as recommended by the manufacturer and counted microscopically. According to the manufacturer, anti-PCNA recognizes the acidic nonhistone auxiliary protein of DNA polymerase.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Histological Testing
Hematoxylin and eosin staining of sections of noninjured femoral arteries (from control animals) showed similar findings for MMP-11^+/+ and MMP-11^-/- mice; the adventitial and medial areas were comparable, whereas no significant neointima was detectable (Table 1 and Figure 1a). Nuclear cell counts (mainly endothelial cells) did not reveal differences between MMP-11^+/+ and MMP-11^-/- arteries (23±2 versus 28±2 in 129SV/BL6 mice and 25±1 versus 22±1 in 129SV mice, mean±SEM, n= 6).

View this table: [in this window] [in a new window]   Table 1. Morphometric Quantitation of Cross-Sectional Areas of Adventitia, Media, and Intima After Injury of the Femoral Artery in MMP-11+/+ or MMP-11-/- Mice of a 129SV/BL6 or 129SV Genetic Background

View larger version (156K): [in this window] [in a new window]   Figure 1. Light microscopic analysis of femoral arterial sections of noninjured controls (a and b) or from the center of injury after 3 weeks in MMP-11+/+ (c and d) and MMP-11-/- (e and f) 129SV/BL6 mice. Sections were stained with hematoxylin and eosin (a, c, and e) or antiserum against -actin (b, d, and f). Arrows and arrowheads indicate the internal and external elastic lamina, respectively. Scale bars indicate 50 µm.

Staining of sections obtained 1 to 3 weeks after injury at equally spaced locations (positions 2 to 4) throughout the damaged artery showed neointima formation in both MMP-11^+/+ and MMP-11^-/- mice (Figure 1c and 1e). In MMP-11^+/+ mice, the intimal area was always smaller than at corresponding time points in MMP-11^-/- mice, whereas the medial areas were comparable, with the exception of a larger medial area at 2 weeks after injury in 129SV MMP-11^-/- mice (P<0.01). This results in significantly lower intima/media ratios in MMP-11^+/+ mice. Also, the adventitial areas of injured arteries were smaller in MMP-11^+/+ mice than in MMP-11^-/- mice (Table 1). This observation was confirmed in mice of 2 different genetic backgrounds, although intima formation in 129SV MMP-11^+/+ mice was significantly lower than that in 129SV/BL6 MMP-11^+/+ mice. In the different experimental groups, no significant differences in neointimal area were observed between male and female mice (P>0.1).

Nuclear cell counts revealed comparable cell populations in media of control (noninjured) arteries and in normal sections (positions 1 and 5) of injured arteries (not shown). In the injured area (positions 2 to 4), cell counts were overall comparable over time in the media of both genotypes, whereas cell counts in the intima were higher in MMP-11^-/- than in MMP-11^+/+ mice (Table 2). Separate analysis of the data at the borders of injury (positions 2 and 4) revealed somewhat, but not statistically significant, higher nuclear cell counts at 2 weeks after injury in the intima of MMP-11^-/- than in MMP-11^+/+ mice: 61±9 versus 48±7 (mean±SEM, n=11 to 12, P=0.32) in 129SV/BL6 mice and 106±26 versus 43±9 (mean±SEM, n=5, P=0.056) in 129SV mice. anti-PCNA staining did not reveal cell proliferation in the media or intima at 1 week after injury in MMP-11^+/+ or MMP-11^-/- 129SV/BL6 mice; at 2 weeks, the rate of cell proliferation was also very low (0.9±0.5% and 2.8±1.7% of the total nuclear cell counts in intima and media, respectively, of MMP-11^+/+ arteries, with corresponding values of 1.5±0.7% and 2.3±0.6% for MMP-11^-/- arteries). At 2 weeks after injury, virtually no cell proliferation occurred in the intima or media of 129SV MMP-11^+/+ mice; in 129SV MMP-11^-/- mice, the rate of cell proliferation was also very low (1.5±0.7% and 1.2±0.8% of the total nuclear cell counts in the intima and media, respectively).

View this table: [in this window] [in a new window]   Table 2. Cell Accumulation in Media and Intima After Injury of the Femoral Artery in MMP-11+/+ or MMP-11-/- Mice of a 129SV/BL6 or 129SV Genetic Background

Immunocytochemical Testing
Noninjured control arteries of MMP-11^+/+ and MMP-11^-/- mice were very similar, with 2 or 3 layers of -actin–immunoreactive smooth muscle cells in the media but none in the intima or adventitia (Figure 1b).

The cell population in the injured arteries was heterogeneous, as shown by immunostaining for smooth muscle cells (-actin), leukocytes (CD45), polymorphic neutrophils, and macrophages (Mac3) (Figures 1 and 2). In 129SV/BL6 MMP-11^+/+ and MMP-11^-/- mice, at 1 week after injury, the neointima in the injured region contained mainly CD45-positive cells, only occasionally Mac3-positive cells, and no -actin–positive cells. At 2 and 3 weeks after injury, fewer CD45-positive cells (<25%) but more -actin–positive cells (25% to 50% at 2 weeks and 50% to 75% at 3 weeks) were observed in the neointima of both genotypes. In the media of MMP-11^+/+ and MMP-11^-/- mice, comparable amounts of -actin–positive cells were seen at 1 and 2 weeks (25%), whereas at 3 weeks, these were more abundant in injured arteries of MMP-11^-/- mice (50% versus <25%). It should be kept in mind, however, that the number of SMCs at 1 week after injury may be underestimated because proliferating/migrating SMCs are not efficiently stained for -actin.²⁸ In the adventitia of MMP-11^+/+ or MMP-11^-/- arteries, no -actin–positive cells were detected at any time point, and most cells were CD45- or Mac3-positive (Table 3).

View larger version (109K): [in this window] [in a new window]   Figure 2. Light microscopic analysis of femoral arterial sections from MMP-11+/+ (a–d) and MMP-11-/- (e–h) mice. Sections were obtained from the center of injury after 3 weeks in 129SV/BL6 mice (a, b, e, and f) and after 2 weeks in 129SV mice (c, d, g, and h). Sections were stained with antiserum against Mac-3 (a and e), CD45 (c and g), or polymorphic neutrophils (d and h). Elastin staining (b and f) was performed using Verhoeff’s and von Gieson’s stains. Scale bars indicate 50 µm.

View this table: [in this window] [in a new window]   Table 3. Topographic Pattern of Cell Localization in Cross-Sectional Areas of Injured Femoral Arteries of 129SV/BL6 MMP-11+/+ or MMP-11-/- Mice, as Determined by Immunostaining With Antisera Specific for -Actin or CD45

In 129SV mice, at 2 weeks after injury (largest difference in neointima between MMP-11^+/+ and MMP-11^-/- mice), virtually no -actin– or CD45-positive cells or polymorphic neutrophils were detected in the neointima of arteries from MMP-11^+/+ mice. In the neointima of arteries from MMP-11^-/-mice, -actin–positive cells (<25%), CD45-positive cells (50%), and polymorphic neutrophils (<25%) were more abundant. In the media, less -actin–positive cells (<25% versus 50% to 75%) and more CD45-positive cells (50% to 75% versus <25%) were seen in arteries from MMP-11^-/- mice than in arteries from MMP-11^+/+ mice. Diffuse staining of neutrophils was observed in media of MMP-11^-/- but not MMP11^+/+ arteries. In the adventitia, no -actin–positive cells were observed in MMP-11^+/+ or MMP-11^-/- arteries, but more CD45-positive cells (100% versus <25%) and neutrophils (<25% versus 0%) were observed in sections from MMP-11^-/- mice than MMP-11^+/+ mice. Thus, in absolute numbers, the amount of -actin– and CD45-positive cells and of neutrophils in the neointima of arteries from MMP-11^-/- mice was higher than in arteries from MMP-11^+/+ mice.

Verhoeff and von Gieson staining of elastin indicated that in both genotypes, the external elastic lamina was strongly degraded, whereas the internal elastic lamina remained essentially intact up to 2 weeks. Determination of the circumference by computer-assisted image analysis (6 experiments each with MMP-11^+/+ and MMP-11^-/- control, noninjured arteries and 6 experiments each at 1 and at 2 weeks after injury; 8 sections analyzed per artery) did not reveal significant differences between the 2 genotypes (95% of the internal elastic lamina was intact). Visual inspection of the autofluorescence of the elastic lamina on hematoxylin and eosin–stained sections confirmed this observation (not shown). Three weeks after injury, however, degradation of the internal elastic lamina was significantly more pronounced in arteries from MMP-11^-/- mice than in those from MMP-11^+/+ mice: 39±4.1% versus 6.8±3.1% (mean±SEM, n= 6, P=0.0022) (Figure 2b and 2f). At the sites of extensive elastic lamina degradation, macrophages (Mac-3–positive cells) were abundantly present in the media (Figure 2e).

Zymographic Analysis
In situ zymographic analysis of fibrinolytic activity was performed by fibrin overlay of arterial sections. This assay detects primarily tPA activity, as shown by the finding that lysis of the fibrin gel (after 2 hours at 37°C) with femoral arterial sections obtained 3 weeks after injury in wild-type mice was virtually abolished on addition of anti-tPA antibodies (residual lysis, 1%) but was not significantly affected by addition of anti-uPA antibodies (residual lysis, 90±4%; mean±SEM, n= 12). Fibrinolytic activity (lysis zone) in arterial sections obtained 3 weeks after injury in 129SV/BL6 mice was increased 3- to 10-fold as compared with that in noninjured arteries. After normalization for the section areas, however, the activities (mean±SEM, n= 6) were comparable both in noninjured arteries (2.2±0.56 versus 3.3±1.1 for arteries from MMP-11^+/+ and MMP-11^-/- mice, respectively) and at the center of injury after 3 weeks (2.2±0.16 versus 2.9±0.50 for arteries from MMP-11^+/+ and MMP-11^-/- mice, respectively).

Prolonged overlay at 37°C in the presence of anti-tPA antibodies did not reveal a significant difference in lysis of the fibrin gel between injured sections of MMP-11^+/+ and MMP-11^-/- arteries (lysis zone, mean±SEM, 0.042±0.007 mm², n=18 versus 0.064±0.010 mm², n=15; P=0.053). On addition of both anti-tPA and anti-uPA antibodies, the residual lysis zones were also comparable for sections from MMP-11^+/+ and MMP-11^-/- mice (0.018±0.003 mm², n=18 versus 0.017± 0.002 mm², n=15).

These data indicate that tPA- or uPA-mediated lysis of the fibrin gel, as well as tPA- and uPA-independent lysis (eg, MMP-2–dependent¹³ ), was not significantly different between sections obtained at the center of injury in MMP-11^+/+ and MMP-11^-/- mice.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Remodeling after vessel wall injury may contribute to luminal stenosis.²⁹ It involves migration of SMCs from the media across the internal elastic lamina underneath the endothelium, proliferation in the neointima, and deposition of extracellular matrix.^{1 2 30} Proteinases of the plasminogen/plasmin and MMP systems may play a role in SMC migration by degrading the extracellular matrix that prevents migration.^{3 4 5 6 7 8} Within the MMP family, MMP-11 (stromelysin-3) is an unusual member, because its mature form is unable to hydrolyze the major components of the extracellular matrix, whereas a fragment containing the catalytic domain acquires some enzymatic activity.^{17 18 19} The biological role of MMP-11 is not fully understood. MMP-11^-/- mice are viable, fertile, and normal in appearance and behavior.²⁵ This is compatible with our finding that vessel formation is normal in MMP-11^-/- mice, because no differences were observed in normal, uninjured femoral arteries from wild-type mice. Recently, it was shown that MMP-11, in a paracrine manner, promotes homing of malignant epithelial cells, a key process for primary tumors and metastases; this function requires extracellular matrix–associated growth factors.²⁵ Furthermore, it was reported that MMP-11 hydrolyzes the insulin-like growth factor–binding protein-1 and may thus regulate bioavailability of insulin-like growth factor-I, favoring cell survival and proliferation.³¹

To assess a potential role of MMP-11 in neointima formation, a perivascular electric injury model was applied to mice with targeted inactivation of MMP-11. In this model, wound healing initiates from the adjacent uninjured borders and progresses into the necrotic center and is associated with migration of SMCs and leukocytes.²⁷ Surprisingly, we found accelerated neointima formation in MMP-11^-/- mice. Because of previous observations of pronounced differences in atherosclerotic lesion formation between mice of different genetic backgrounds^{32 33 34} and in neointima formation after vascular injury,^{11 12 35} we confirmed our finding in mice of 2 different backgrounds. Neointima formation in wild-type mice with a pure 129SV background up to 3 weeks after injury was more than 5-fold lower than that in mice with the mixed 129SV/BL6 (50/50) background; however, in mice of both genetic backgrounds, neointima formation after vascular injury was significantly enhanced with deficiency of MMP-11.

Because no specific antiserum was available, we were not able to study the temporal and topographic expression pattern of MMP-11 in wild-type mice after vascular injury. However, several previous studies have demonstrated local expression of MMP-11 during remodeling processes, such as tissue involution,²⁴ tissue repair,³⁶ and human carcinoma development,²¹ but systemic effects were not reported. During the reparation process after skin wound healing, MMP-11 is temporally and locally expressed,³⁶ and its expression has also been observed in human atherosclerotic lesions but not in normal arteries.³⁷ Strong expression has been observed in fibroblasts.²² In MMP-11^-/- mice, the absence of MMP-11 mRNA has been clearly demonstrated. Thus, although we cannot conclusively show whether the observed effects in this study are direct or indirect, the presence or absence of MMP-11 is the main difference between both genotypes, suggesting that the absence of MMP-11 contributes to neointimal thickening.

This unexpected observation probably cannot be explained by direct effects of MMP-11 on matrix degradation, as suggested by our previous finding that lysis of a ³H-proline–labeled subendothelial matrix by in vivo thioglycollate-stimulated macrophages was similar for wild-type and MMP-11^-/- mice.²⁰ We have also shown that MMP-11 deficiency does not affect expression and activation of MMP-2 or MMP-9 in segments of aorta.²⁰ Furthermore, it is unlikely that degradation of soluble serpins by MMP-11 plays a role in this model, because lower proteolytic activity in MMP-11^-/- mice would be expected to result in reduced neointima formation.

We explored several other potential mechanisms that may be involved in the accelerated neointima formation observed in MMP-11^-/- mice. The overall fibrinolytic activity in arterial sections was enhanced after vascular injury, but after normalization for the section area, there was no difference between wild-type and MMP-11^-/- mice. Additional quantitative analysis did not reveal significant differences in the medial areas, whereas the adventitial areas after injury were larger in the arteries from MMP-11^-/- mice than in those from wild-type mice. Nuclear cell counts in the intima were higher after injury in arteries from MMP-11^-/- mice. Anti-PCNA staining revealed virtually no proliferating cells at 1 or 2 weeks after injury at the center of injury in media or intima of arteries from MMP-11^+/+ mice, and the cell proliferation rate was also very low in arteries from MMP-11^-/- mice, suggesting that mainly invading cells are present. Although anti-PCNA staining on arterial sections may underestimate the actual number of proliferating cells, it is unlikely that the large difference in nuclear cell counts between both genotypes (Table 2) is caused by different proliferation rates. The cell population in the intima was heterogeneous, consisting mainly of -actin–positive and CD45-positive cells, of which a significant fraction was identified as polymorphic neutrophils in the arteries of MMP-11^-/- mice, possibly related to an inflammatory response. In mice of the pure 129SV background, in which the largest increase in intimal area in MMP-11^-/- mice was observed, these cells appeared to be more abundant than in wild-type mice. It cannot be excluded that proteinases produced by inflammatory cells contribute to degradation of matrix and other tissue components. It is, however, unclear why a stronger inflammatory response would occur in MMP-11^-/- than in wild-type animals. In 129SV/BL6 mice, the relative contribution of different cell types in the total population seemed more comparable in arteries of wild-type and MMP-11^-/- mice, but the absolute numbers were significantly higher in arteries of MMP-11^-/- mice. Furthermore, degradation of the internal elastic lamina at 3 weeks after injury was significantly more pronounced in arteries from MMP-11^-/- mice than in those from MMP-11^+/+ mice. Extensive degradation was seen at sites (Figure 2f) where Mac-3–positive cells were abundant in the media (Figure 2e), compatible with a role of macrophage-secreted proteinases in degradation of the elastica lamina.³⁸

Together, these findings suggest that the enhanced neointima formation occurring after vascular injury in MMP-11^-/- mice is a complex process in which enhanced elastin degradation and enhanced cellular migration, with participation of inflammatory cells, play a role. The mechanisms by which MMP-11 may impair these processes remain unknown and require further investigation.

	Acknowledgments

 
This study was supported by grants from the Flemish Fund for Scientific Research (contract G.0293.98) and from the Interuniversitaire Attractiepolen (contract P4/34). CNRS-INSERM-ULP was supported by INSERM, CNRS, the Bristol Myers Squibb Pharmaceutical Research Institute, the Association pour la Recherche sur le Cancer, the Ligue Nationale contre le Cancer, and BIOMED2 (contract BMH 4CT 96-0017). The skillful technical assistance of A. Dewulf is gratefully acknowledged.

Received December 29, 1998; accepted April 12, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Libby P, Schwartz D, Brogi E, Tanaka H, Clinton SK. A cascade model for restenosis: a special case of atherosclerosis progression. Circulation. 1992;86(suppl III):III47–III52.
2. Reidy MA, Jackson D, Lindner V. Neointimal proliferation: control of vascular smooth muscle cell growth. Vasc Med Rev. 1992;3:156–167.
3. Reidy MA, Irvin C, Lindner V. Migration of arterial wall cells: expression of plasminogen activators and inhibitors in injured rat arteries. Circ Res. 1996;78:405–414.[Abstract/Free Full Text]
4. Dollery CM, McEwan JR, Henney AM. Matrix metalloproteinases and cardiovascular disease. Circ Res. 1995;77:863–868.[Free Full Text]
5. 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]
6. Zempo N, Kenagy RD, Au YPT, 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]
7. Webb KE, Henney AM, Anglin S, Humphries SE, McEwan JR. Expression of matrix metalloproteinases and their inhibitor TIMP-1 in the rat carotid artery after balloon injury. Arterioscler Thromb Vasc Biol. 1997;17:1837–1844.[Abstract/Free Full Text]
8. Kenagy RD, Hart CE, Stetler-Stevenson WG, Clowes AW. Primate smooth muscle cell migration from aortic explants is mediated by endogenous platelet-derived growth factor and basic fibroblast growth factor acting through matrix metalloproteinases 2 and 9. Circulation. 1997;96:3555–3560.[Abstract/Free Full Text]
9. 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]
10. Zempo N, Koyama N, Kenagy RD, Lea HJ, 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]
11. Carmeliet P, Moons L, Herbert J-M, Crawley J, Lupu F, Lijnen HR, Collen D. Urokinase-type but not tissue-type plasminogen activator mediates arterial neointima formation in mice. Circ Res. 1997;81:829–839.[Abstract/Free Full Text]
12. Carmeliet P, Moons L, Ploplis V, Plow E, Collen D. Impaired arterial neointima formation in mice with disruption of the plasminogen gene. J Clin Invest. 1997;99:200–208.[Medline] [Order article via Infotrieve]
13. Lijnen HR, Van Hoef B, Lupu F, Moons L, Carmeliet P, Collen D. Function of plasminogen/plasmin and matrix metalloproteinase systems after vascular injury in mice with targeted inactivation of fibrinolytic system genes. Arterioscler Thromb Vasc Biol. 1998;18:1035–1045.[Abstract/Free Full Text]
14. Kenagy RD, Vergel S, Mattsson E, Bendeck M, Reidy MA, Clowes AW. The role of plasminogen, plasminogen activators, and matrix metalloproteinases in primate arterial smooth muscle cell migration. Arterioscler Thromb Vasc Biol. 1996;16:1373–1382.[Abstract/Free Full Text]
15. Birkedal-Hansen H. Proteolytic remodeling of extracellular matrix. Curr Opin Cell Biol. 1995;7:728–735.[Medline] [Order article via Infotrieve]
16. Pei D, Weiss SJ. Furin-dependent intracellular activation of the human stromelysin-3 zymogen. Nature. 1995;375:244–247.[Medline] [Order article via Infotrieve]
17. Pei D, Majmudar G, Weiss SJ. Hydrolytic activation of a breast carcinoma cell-derived serpin by human stromelysin-3. J Biol Chem. 1994;269:25849–25855.[Abstract/Free Full Text]
18. Murphy G, Segain J-P, O’Shea M, Cockett M, Iannou C, Lefebvre O, Chambon P, Basset P. The 28-kDa N-terminal domain of mouse stromelysin-3 has the general properties of a weak metalloproteinase. J Biol Chem. 1993;269:15435–15441.
19. Noël A, Santavicca M, Stoll I, L’Hoir C, Staub A, Murphy G, Rio M-C, Basset P. Identification of structural determinants controlling human and mouse stromelysin-3 proteolytic activities. J Biol Chem. 1995;270:22866–22872.[Abstract/Free Full Text]
20. Lijnen HR, Ugwu F, Rio M-C, Collen D. Plasminogen/plasmin and matrix metalloproteinase system function in mice with targeted inactivation of stromelysin-3 (MMP-11). Fibrinolysis Proteolysis. 1998;12:155–164.
21. Basset P, Bellocq JP, Wolf C, Stoll I, Hutin P, Limacher JM, Podhajcer CL, Chenard MP, Rio M-C, Chambon P. A novel matrix metalloprotease gene specifically expressed in stromal cells of breast carcinomas. Nature. 1990;348:699–704.[Medline] [Order article via Infotrieve]
22. Basset P, Wolf C, Chambon P. Expression of the stromelysin-3 gene in fibroblastic cells of invasive carcinomas of the breast and other human tissues: a review. Breast Cancer Res Treat. 1993;24:185–193.[Medline] [Order article via Infotrieve]
23. Basset P, Bellocq J-P, Lefebvre O, Noël A, Chenard M-P, Wolf C, Anglard P, Rio M-C. Stromelysin-3: a paradigm for stroma-derived factors implicated in carcinoma progression. Crit Rev Oncol Hematol. 1997;26:43–53.[Medline] [Order article via Infotrieve]
24. Lefebvre O, Wolf C, Limacher J-M, Hutin P, Wendling C, LeMeur M, Basset P, Rio M-C. The breast cancer-associated stromelysin-3 gene is expressed during mouse mammary gland apoptosis. J Cell Biol. 1992;119:997–1002.[Abstract/Free Full Text]
25. Masson R, Lefebvre O, Noël A, El Fahime M, Chenard MP, Wendling C, Kebers F, LeMeur M, Dierich A, Foidart JM, Basset P, Rio M-C. In vivo evidence that the stromelysin-3 metalloproteinase contributes in a paracrine manner to epithelial cell malignancy. J Cell Biol. 1998;140:1535–1541.[Abstract/Free Full Text]
26. Giles AR. Guidelines for the use of animals in biomedical research. Thromb Haemost. 1987;58:1078–1084.[Medline] [Order article via Infotrieve]
27. Carmeliet P, Moons L, Stassen JM, Van Vlaenderen I, Declercq C, Kockx M, Collen D. Vascular wound healing and neointima formation induced by perivascular injury in mice. Am J Pathol. 1997;150:761–766.[Abstract]
28. Kocher O, Gabbiani F, Gabbiani G, Reidy MA, Cokay MS, Peters H, Hüttner I. Phenotypic features of smooth muscle cells during the evolution of experimental carotid artery intimal thickening. Lab Invest. 1991;65:459–470.[Medline] [Order article via Infotrieve]
29. Schwartz SM, Reidy MA, O’Brien ER. Assessment of factors important in atherosclerotic occlusion and restenosis. Thromb Haemost. 1995;74:541–551.[Medline] [Order article via Infotrieve]
30. Clowes AW, Reidy MA. Prevention of stenosis after vascular reconstruction: pharmacologic control of intimal hyperplasia: a review. J Vasc Surg. 1991;13:885–891.[Medline] [Order article via Infotrieve]
31. Mañez S, Mira E, del Mar Barbacid M, Ciprés A, Fernández-Resa P, Buesa JM, Mérida I, Aracil M, Márquez G, Martínez-A C. Identification of insulin-like growth factor-binding protein-1 as a potential physiological substrate for human stromelysin-3. J Biol Chem. 1997;272:25706–25712.[Abstract/Free Full Text]
32. Paigen B. Genetics of responsiveness to high-fat and high-cholesterol diets in the mouse. Am J Clin Nutr. 1995;62:458S–462S.[Abstract/Free Full Text]
33. Paigen B, Ishida BY, Verstuyft J, Winters RB, Albee D. Atherosclerosis susceptibility differences among progenitors of recombinant inbred strains of mice. Arteriosclerosis. 1990;10:316–323.[Abstract/Free Full Text]
34. Qiao J-H, Xie P-Z, Fishbein MC, Kreuzer J, Drake TA, Demer LL, Lusis AJ. Pathology of atheromatous lesions in inbred and genetically engineered mice: genetic determination of arterial calcification. Arterioscler Thromb. 1994;14:1480–1497.[Abstract/Free Full Text]
35. Carmeliet P, Moons L, Dewerchin M, Rosenberg S, Herbert J-M, Lupu F, Collen D. Receptor-independent role of urokinase-type plasminogen activator in pericellular plasmin and matrix metalloproteinase proteolysis during vascular wound healing in mice. J Cell Biol. 1998;140:233–245.[Abstract/Free Full Text]
36. Okada A, Tomasetto C, Lutz Y, Bellocq JP, Rio M-C, Basset P. Expression of matrix metalloproteinases during skin wound healing: evidence that membrane type-1 matrix metalloproteinase is a stromal activator of pro-gelatinase A. J Cell Biol. 1997;137:67–77.[Abstract/Free Full Text]
37. Schönbeck U, Mach F, Sukhova GK, Atkinson E, Levesque E, Herman M, Graber P, Basset P, Libby P. Expression of stromelysin-3 in atherosclerotic lesions: regulation via CD40/CD40 ligand signaling in vitro and in vivo. J Exp Med. 1999;189:843–853.[Abstract/Free Full Text]
38. Carmeliet P, Moons L, Lijnen HR, Baes M, Lemaître V, Tipping P, Drew A, Eeckhout Y, Shapiro S, Lupu F, Collen D. Urokinase-generated plasmin is a candidate activator of matrix metalloproteinases during atherosclerotic aneurysm formation. Nature Genet. 1997;17:439–444.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				K. Imai, S. S. Dalal, J. Hambor, P. Mitchell, Y. Okada, W. C. Horton, and J. D'Armiento Bone growth retardation in mouse embryos expressing human collagenase 1 Am J Physiol Cell Physiol, October 1, 2007; 293(4): C1209 - C1215. [Abstract] [Full Text] [PDF]

				L. Nakopoulou, A. C. Lazaris, I. Boletis, S. Michail, C. Iatrou, G. Papadakis, S. Athanassiadou, and C. Stathakis The expression of matrix metalloproteinase-11 protein in various types of glomerulonephritis Nephrol. Dial. Transplant., January 1, 2007; 22(1): 109 - 117. [Abstract] [Full Text] [PDF]

				S. Janssens and H. R. Lijnen What has been learned about the cardiovascular effects of matrix metalloproteinases from mouse models? Cardiovasc Res, February 15, 2006; 69(3): 585 - 594. [Abstract] [Full Text] [PDF]

				J. P.G. Sluijter, D. P.V. de Kleijn, and G. Pasterkamp Vascular remodeling and protease inhibition-bench to bedside Cardiovasc Res, February 15, 2006; 69(3): 595 - 603. [Abstract] [Full Text] [PDF]

				J. A. Spencer, S. L. Hacker, E. C. Davis, R. P. Mecham, R. H. Knutsen, D. Y. Li, R. D. Gerard, J. A. Richardson, E. N. Olson, and H. Yanagisawa Altered vascular remodeling in fibulin-5-deficient mice reveals a role of fibulin-5 in smooth muscle cell proliferation and migration PNAS, February 22, 2005; 102(8): 2946 - 2951. [Abstract] [Full Text] [PDF]

				A. C. Newby Dual Role of Matrix Metalloproteinases (Matrixins) in Intimal Thickening and Atherosclerotic Plaque Rupture Physiol Rev, January 1, 2005; 85(1): 1 - 31. [Abstract] [Full Text] [PDF]

				D. L. Myers and L. Liaw Improved Analysis of the Vascular Response to Arterial Ligation Using a Multivariate Approach Am. J. Pathol., January 1, 2004; 164(1): 43 - 48. [Abstract] [Full Text] [PDF]

				A. Cho and M. A. Reidy Matrix Metalloproteinase-9 Is Necessary for the Regulation of Smooth Muscle Cell Replication and Migration After Arterial Injury Circ. Res., November 1, 2002; 91(9): 845 - 851. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lijnen, H. R. Articles by Collen, D. Search for Related Content PubMed PubMed Citation Articles by Lijnen, H. R. Articles by Collen, D. Related Collections Genetically altered mice Quantitative modeling Other Vascular biology