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Cardiovascular Research 2003 57(2):572-585; doi:10.1016/S0008-6363(02)00710-1
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Copyright © 2003, European Society of Cardiology

Overexpression of matrix metalloproteinase-9 promotes intravascular thrombus formation in porcine coronary arteries in vivo

Kunio Morishigea, Hiroaki Shimokawaa,*, Yasuharu Matsumotoa, Yasuhiro Etoa, Toyokazu Uwatokua, Kohtaro Abea, Katsuo Sueishib and Akira Takeshitaa

aDepartment of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
bDepartment of Pathophysiological and Experimental Pathology, Kyushu University, Graduate School of Medical Sciences, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

shimo{at}cardiol.med.kyushu-u.ac.jp

* Corresponding author. Tel.: +81-92-642-5360; fax: +81-92-642-5374.

Received 22 May 2002; accepted 30 September 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Matrix metalloproteinases (MMPs) cause extracellular matrix degradation and may be involved in the rupture of atherosclerotic plaques by degrading fibrous cap, resulting in the intravascular thrombus formation. Here we examined whether local overexpression of MMP-9 alters the characteristics of arteriosclerotic vascular lesions and promotes thrombosis after balloon injury in porcine coronary arteries in vivo. Methods and results: Balloon angioplasty was performed in the left coronary arteries followed by injection of adenovirus vector solution encoding either MMP-9 or β-galactosidase (β-gal) gene into the injured coronary arteries. Three weeks after the gene transfer, histological examination demonstrated that macroscopic intravascular thrombus formation was noted at the MMP-9-transfected site but not at the β-gal-transfected site. Microscopic intramural thrombus area was significantly larger at the MMP-9-transfected site as compared to the β-gal-transfected site. Co-transfection of tissue inhibitor of metalloproteinase-1 (TIMP-1) with MMP-9 prevented the intravascular thrombus formation in vivo. Western blot analysis revealed the reduced expression of intact tissue factor pathway inhibitor-1 and the increased tissue factor (TF) expression at the MMP-9-transfected sites. Conclusion: These results provide the first in vivo evidence that overexpression of MMP-9 promotes intravascular thrombus formation after balloon injury due in part to the activation of TF-mediated coagulation cascade.

KEYWORDS Angioplasty; Atherosclerosis; Coronary disease; Extracellular matrix; Thrombosis/embolism


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Arteriosclerosis/atherosclerosis are inflammatory vascular diseases caused by a series of processes, including endothelial activation and dysfunction, migration of inflammatory cells and reactive proliferation of vascular smooth muscle cells (VSMC), in which process various extracellular molecules are involved [1]. Among those molecules, extracellular matrix (ECM) components play an important role in terms of lesion vulnerability and cell invasion [2]. ECM in the vessel wall is composed mainly of collagen and elastin, which play an important role in the integrity of three-dimensional structure of blood vessels and the cellular components (e.g., VSMC, fibroblasts and endothelial cells). Collagen and elastin fibers can be degraded by matrix metalloproteinases (MMPs) secreted from activated inflammatory cells, and their activity is tightly regulated by endogenous inhibitors, such as tissue inhibitors of metalloproteinases (TIMPs) [2].

Recent studies in vitro suggested that MMPs contribute to neointimal formation and vascular remodeling through ECM degradation [3]. It has recently been shown that long-term inhibition of MMP activity with the non-specific MMP inhibitor BB-94 (Batimastat) suppresses constrictive remodeling after balloon injury in iliac arteries in pigs in vivo [4]. By contrast, it was also demonstrated that activation of MMPs may cause positive remodeling in a different animal model [5]. MMPs may be involved in the molecular mechanism of rupture of atherosclerotic plaques by degrading fibrous cap, resulting in the intravascular thrombus formation [6–9]. Growth of intravascular thrombus may cause total occlusion of the artery, resulting in the occurrence of myocardial infarction. In addition to the effect on ECM components, MMPs have other substrates. MMPs (MMP-1, -7, -9, -12) degrade tissue factor pathway inhibitor (TFPI)-1 in vitro, and may promote coagulation cascade at the inflammatory lesions [10]. MMPs also promote cell migration through ECM degradation [11–13]. However, it remains to be elucidated whether local overexpression of MMPs alters the vascular lesion formation, such as neointimal formation, vascular remodeling and thrombus formation in vivo.

We have developed porcine models of arteriosclerotic and atherosclerotic coronary lesions in vivo by long-term treatment with inflammatory cytokines such as interleukin-1β and MCP-1, respectively [14,15]. Our porcine models are useful to examine the molecular mechanisms of coronary artery disease in humans [14–16].

In the present study, we thus examined whether overexpression of MMP-9, one of the major MMPs, alters the characteristics of arteriosclerotic coronary lesions and promotes thrombus formation after balloon injury in porcine coronary arteries in vivo.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
This experiment was reviewed by the Committee of the Ethics on Animal Experiments of the Kyushu University and was carried out under the control of the Guidelines for Animal Experiments of the Kyushu University.

2.1 Animal preparation
Male Yorkshire pigs (2–3 months old, weighing 25–30 kg) were sedated with intramuscular ketamine hydrochloride (1.5 mg/kg) and anesthetized with intravenous sodium pentobarbital (30 mg/kg). The animals were then intubated and ventilated with room air, and then a 10 F sheath was inserted through the carotid artery. After systemic heparinization (10 000 U/body), a preshaped 10F Judkins catheter was inserted into the carotid artery and coronary angiography (CAG) in a left oblique view was performed using the cineangiography system (Toshiba Medical, Tokyo). The animals were euthanized with an excess dose of intravenous pentobarbital (60–90 mg/kg) and coronary arteries were excised 1 and 3 weeks after the procedure, for immunostaining, immunoblot analysis, in situ zymography and histological analysis.

2.2 Construction of adenoviral vectors
Adenoviral vectors encoding human MMP-9 (AdMMP-9) or TIMP-1 (AdTIMP-1) (with the cytomegalovirus promoter) were used [17]. Efficient and selective overproduction of each recombinant protein was shown by immunofluorescence in either rabbit smooth muscle cells or human adenocarcinoma cells, and high-level secretion directly dependent on the multiplicity of infection (MOI) was observed for each functional transgene by gelatin zymography [17]. Adenovirus vector encoding β-galactosidase (Adβ-gal) was used as a control. The titer of the virus stock was assessed by a plaque formation assay that used the 293 cells and expressed as plaque forming unit (pfu).

2.3 Balloon injury followed by adenovirus-mediated in vivo gene transfer into the porcine coronary artery
The coronary artery was injured with a conventional balloon catheter with a diameter 1.5 times larger than coronary diameter by inflating it three times for 30 s at 8 atm in the left anterior descending (LAD) or the left circumflex (LCX) coronary arteries [18]. We have previously confirmed that the extent of coronary lesion induced by the balloon injury is comparable between LAD and LCX in pigs [18]. Then, adenoviral gene transfer was performed at the previously injured coronary segment after intracoronary administration of nitroglycerin (10 µg/kg) and heparin (3000 U/body). Infiltrator angioplasty balloon catheter (IABC) (Interventional Technologies, San Diego, CA) was advanced into the injured coronary artery followed by inflation of a 3.5-mm balloon at 2 atm [18,19]. This catheter is with 21 small nipples in three lines located on the surface of the balloon connected to the drug delivery port, filled with virus solution until droplets appeared through the needles before use. This catheter is useful to directly deliver fluid into the vessel wall in vivo with <90% efficiency with minimal vascular damage, and also for in vivo gene transfer into the coronary artery [18,19]. Adβ-gal (4x108 pfu in 0.4 ml sorbitol-added lactated Ringer's saline) and AdMMP-9 (4x108 pfu in 0.4 ml of the Ringer's saline) were randomly injected into the injured LAD or LCX. After the gene transfer, IABC was deflated and withdrawn, and the left carotid artery ligated. In some experiments, we co-transfected AdTIMP-1 (1.0x109 pfu in 0.4 ml of the Ringer's saline) with AdMMP-9 to examine the inhibitory effect of TIMP-1 on MMP-9 in vivo.

In this study, a total of 28 pigs (53 coronary artery segments) were used. Nine pigs (nine β-gal- and nine MMP-9-treated coronary segments) were sacrificed for histological examination 3 weeks after the operation. Four pigs (four β-gal- and four MMP-9-treated coronary segments) were also sacrificed for histological examination 1 week after the operation, and six pigs (six β-gal- and six MMP-9-treated coronary segments) were subjected to the Western blot analysis 1 week after the operation. In each animal, Adβ-gal and AdMMP-9 were randomly injected into the injured LAD or LCX. An additional nine pigs (seven LAD and eight LCX) were used for co-infection with MMP-9 and TIMP-1 protocol (histological examination and Western blot analysis).

2.4 Coronary angiography and coronary diameter measurement
CAG was performed before, 1 and 3 weeks after the in vivo gene transfer. The cineangiograms were projected on a screen using a cineprojector and an end-diastolic frame was selected. Coronary stenosis at the balloon-injured segments was expressed as a percent decrease in the luminal diameter compared to the mean diameter of adjacent proximal and distal normal coronary segments after intracoronary administration of nitroglycerin (10 µg/kg) [18,19]. Coronary diameter was automatically measured using densitometric analysis program in the cineangiography system [18,19].

2.5 Immunostaining for MMP-9, TIMP-1, TF and TFPI-1
Immunostaining for human MMP-9 and TIMP-1 was performed 1 week after the gene transfer to confirm the efficacy of the procedure and the in situ gene expression. After the coronary artery was excised, it was quickly frozen in OCT compound (Tissue-Tek, Sakura, Tokyo), sectioned at 5 µm by a cryostat, and subjected to immunostaining with polyclonal antibody for human MMP-9 and TIMP-1 (Fuji Yakuhin Kogyo, Toyama, Japan) at a 1:100 dilution. Intact and Adβ-gal-transfected coronary arteries and non-immune rabbit IgG were used as controls. Immunoreactive materials were visualized by use of a biotinylated anti-rabbit IgG antibody (Wako, Osaka, Japan), peroxidase-labeled streptavidin, and diaminobenzidine. Immunostaining for human tissue factor (TF) and TFPI-1 was also performed 1 week after the β-gal gene transfer to examine the in situ expression of these molecules. Polyclonal rabbit anti-human TF and mouse anti-human TFPI-1 (American Diagnostica, Greenwich, CT) were used at a 1:100 dilution.

2.6 Western blot analysis for tissue factor and tissue factor pathway inhibitor-1
Western blot analysis was performed to examine TF and TFPI-1 protein expression. One week after the gene transfer, isolated coronary artery without endothelium and adventitial tissue was frozen by immersion in acetone containing 10% trichloroacetic acid and 10 mmol/l dithiothreitol cooled with dry ice. Frozen tissues were washed twice with acetone containing 10 mmol/l dithiothreitol to remove trichloroacetic acid and then dried. The dried ring was cut into small pieces, exposed to 200 µl of SDS–PAGE sample buffer for the purpose of protein extraction. The supernatant of each extracted sample was subjected to SDS–PAGE at the same protein quantity, and transferred to nitrocellulose membrane for the immunoblot analysis. The primary antibodies were polyclonal rabbit anti-human TF and mouse anti-human TFPI-1 (American Diagnostica, Greenwich, CT) used at a 1:200 dilution. The secondary antibodies were anti-rabbit IgG for TF and anti-mouse IgG for TFPI-1 (Toyo Boseki, Osaka, Japan), respectively, conjugated to horseradish peroxidase (HRP) and were used at a 1:2000 dilution. Antigen detection was performed with Western blotting detection reagents (ECL plus) (Amersham–Pharmacia Biotech UK, UK). We have also performed quantitative densitometric analysis of the results obtained by the Western blot analysis.

2.7 In situ zymography
In situ zymography was performed 1 week after the gene transfer to confirm the enhanced gelatinolytic activity induced by the MMP-9 gene transfer. After the coronary artery was excised, it was quickly frozen in OCT compound, sectioned at 5 µm, and mounted onto the gelatin films that were coated with 7% gelatin solution (Fuji Photo Film, Tokyo, Japan). The films with sections were incubated for 12 h at 37°C in a moisture chamber and stained with Biebrich Scarlet (Aldrich, Milwaukee, WI) and hematoxylin. The gelatin in contact with the proteolytic areas of the sections was digested, and zones of enzymatic activity were indicated by negative staining as previously described [20]. Intact coronary arteries and Adβ-gal-transfected coronary arteries were also examined in the same manner.

2.8 Histological examination
One and 3 weeks after the gene transfer, the left coronary arteries were subjected to histological examinations. Three weeks after the gene transfer, the heart was removed and the left coronary artery was perfused with 6% formalin at a pressure of 120 mmHg and fixed with formalin for a week. For the light microscopic examination, tissue samples were embedded in paraffin, sectioned into slices 5-µm thick, mounted on glass slides, and stained with hematoxylin–eosin and van Gieson's methods. With a photomicroscopic photograph system (Microphot-FXA, Nikon, Tokyo), pictures were taken at x20 and x40 magnifications. In each specimen, the borders of vessel lumen, internal elastic lamina (IEL) and external elastic lamina (EEL) were traced, and each area was calculated with an automated computer-based image analyzer (Digitizer KD4600, Graphic, Yokohama, Japan) [18,19]. Neointimal area and percent intimal area (ratio of intimal area to IEL area) were calculated. Geometric remodeling of the coronary artery was assessed by measuring the ratio of the EEL, IEL, and lumen area at the balloon-injured coronary segments to those areas of adjacent proximal segments, respectively [18,19]. One week after the gene transfer, the left coronary artery was also perfused and excised in the same manner.

2.9 Data analysis
All results are expressed as the mean±S.E.M. Throughout the text, n represents the number of animals tested. Multiple comparisons were made by ANOVA for repeated examinations followed by Fisher's post-hoc test. Paired data were analyzed by Student's t-test. A P value of <0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Immunostaining for MMP-9, TF, and TFPI-1
One week after the gene transfer of MMP-9, its expression was noted mainly at the media and partially at the neointima and the adventitia at the AdMMP-9-transfected site (Fig. 1). The staining with non-immune IgG was negative, confirming the specificity of the immunostaining (Fig. 1). Three weeks after the gene transfer, the immunoreactivity of MMP-9 was markedly reduced. No significant immunoreactivity for the molecule was observed either at the coronary segment adjacent to the transfected site or at the Adβ-gal-transfected site (data not shown).


Figure 1
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  		Fig. 1 Expression of MMP-9 in a porcine coronary artery 1 week after in vivo gene transfer of MMP-9. The expression of MMP-9 was noted at the AdMMP-9-transfected site, mainly in the media (vascular smooth muscle cells) (M) and partially at the neointima (N) and the adventitia (A). Negative staining with non-immune IgG confirmed the specificity of the staining.

 
One week after the balloon injury, the expression of TF was noted throughout the vessel wall (mainly in the neointima and the media) (Fig. 2A), whereas no significant expression was detected at the normal coronary artery. The expression of TFPI-1 was limited in the endothelium in the normal coronary artery whereas 1 week after the balloon injury, it was abundantly expressed throughout the injured coronary artery and was co-localized with TF (Fig. 2B). These results indicate the importance of those molecules affecting various biological properties including thrombosis after vascular injury.


Figure 2
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  		Fig. 2 (A) Tissue factor (TF) expression in a porcine coronary artery 1 week after the in vivo gene transfer of β-galactosidase. The expression of TF was noted throughout the vessel wall at the transfected site, whereas no significant expression was noted at the normal coronary artery (data not shown). (B) Tissue factor pathway inhibitor (TFPI)-1 expression in a porcine coronary artery 1 week after the in vivo gene transfer. The expression of TFPI-1 was limited in the endothelium in the normal coronary artery (Normal) while it was abundantly noted throughout the vessel wall 1 week after the injury and gene transfer. I, intima; N, neointima; M, media; A, adventitia.

 
3.2 Coronary angiography
Immediately after the balloon injury followed by the in vivo gene transfer, coronary diameter was increased to the same extent at the AdMMP-9-transfected (151±4%) and the Adβ-gal-transfected site (147±3%). The coronary diameter was significantly reduced 3 weeks after the gene transfer at both sites and no significant difference in the restenotic change was observed between the AdMMP-9-transfected (79±5%) and the Adβ-gal-transfected site (74±4%) (Fig. 3).


Figure 3
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  		Fig. 3 Coronary angiograms before and 3 weeks after the in vivo gene transfer of β-galactosidase (β-gal) or MMP-9. Black arrows indicate the sites where balloon injury followed by gene transfer was performed. Coronary diameter was significantly reduced 3 weeks after the gene transfer in both groups.

 
3.3 Detection of gelatinolytic activity by in situ zymography
In situ zymography revealed that the gelatinolytic activity was significantly increased at the AdMMP-9-transfected site (as indicated by negative staining with Biebrich Scarlet) but not at the Adβ-gal-transfected site (Fig. 4). The activity was rarely observed in the normal coronary artery (Fig. 4). Intravascular thrombus formation was observed at the injured coronary artery with the increased gelatinolytic activity (Fig. 4).


Figure 4
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  		Fig. 4 Gelatinolytic activity of a porcine coronary artery evaluated by in situ zymography 1 week after the in vivo gene transfer. In situ zymography revealed that the gelatinolytic activity was increased at the AdMMP-9-transfected site (MMP-9) indicated by negative staining with Biebrich Scarlet as compared with the Adβ-gal-transfected site (β-gal). The activity was rarely observed in the normal coronary artery (Normal). Intravascular thrombus formation was observed at the injured and MMP-9-transfected coronary artery with the increased gelatinolytic activity. I, intima; N, neointima; M, media; A, adventitia.

 
3.4 Histological examinations
Three weeks after the in vivo gene transfer, histological examination demonstrated macroscopic intravascular thrombus formation developed in the injured coronary artery at the AdMMP-9-transfected site (four of nine) but not at the Adβ-gal-transfected site (none of nine) (Fig. 5). After balloon injury followed by the gene transfer, neointimal formation was developed and caused stenotic change of the coronary artery. However, no significant difference was observed in the development of neointiml formation or vascular geometric remodeling between the two groups (Fig. 6). In this study, the restenotic change of the coronary artery was primarily due to neointimal formation since vascular cross-sectional area was increased rather than decreased (Fig. 6).


Figure 5
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  		Fig. 5 Thrombus formation 3 weeks after the in vivo gene transfer of MMP-9. Intravascular thrombus formation developed at the injured coronary artery at the AdMMP-9-transfected site (MMP-9) but not at the Adβ-gal-transfected site (β-gal).

 

Figure 6
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  		Fig. 6 Quantitative analysis of neointimal formation and vascular remodeling of porcine coronary arteries 3 weeks after the gene transfer. No significant difference was noted in the extent of neointiml formation (intima, intima/EEL area) or vascular geometric remodeling (changes in EEL and EEL area) between the two groups.

 
Microscopic intramural thrombus area, evaluated by Masson's trichrome staining, was also significantly larger at the AdMMP-9-transfected site as compared to the Adβ-gal-transfected site (P<0.05) (Fig. 7). We also performed CAG and histological examination 1 week after the gene transfer, when overexpression of MMP-9 and the enhanced gelatinolytic activity were observed. Although CAG showed that the stenotic changes were comparable between the AdMMP-9-transfected and the Adβ-gal-transfected site, macroscopic intravascular thrombus formation was noted only at the former site (two of four) (Fig. 8) but not at the latter site (none of four).


Figure 7
Figure 7
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  		Fig. 7 (A) Intramural thrombus formation 3 weeks after the gene transfer of β-galactosidase (β-gal) and of MMP-9. Arrow indicates an intramural thrombus stained in red with Masson's trichrome staining. (B) Quantitative analysis of the intramural thrombus formation 3 weeks after the gene transfer of β-galactosidase (β-gal) and of MMP-9. Intramural thrombus area, evaluated by Masson's trichrome staining, was significantly larger at the AdMMP-9-transfected site compared to the Adβ-gal-transfected site.

 

Figure 8
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  		Fig. 8 Macroscopic (left) and microscopic (right) intravascular thrombus formation at the MMP-9-transfected site 1 week after the gene transfer. Right panel shows the intravascular thrombus adhering to the injured coronary artery excised after perfusion with saline. This intravascular thrombus formation was not noted in the β-gal-transfected site (data not shown).

 
3.5 Inhibition by TIMP-1 of MMP-9-induced intravascular thrombus formation
The expression of TIMP-1 was noted mainly in the media and partially in the neointima and the adventitia at the AdTIMP-1-transfected site (Fig. 9). Co-transfection of TIMP-1 with MMP-9 prevented intravascular thrombus formation, as illustrated by the absence of thrombosis in all of nine animals treated with MMP-9/TIMP-1 (Fig. 9).


Figure 9
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  		Fig. 9 Effect of in vivo co-transfection of MMP-9 and TIMP-1. The expression of TIMP-1 was noted at the AdTIMP-1-transfected site, mainly in the media (smooth muscle cells) (M) and partially in the neointima (N) and the adventitia (A) 1 week after the gene transfer. Co-transfection of TIMP-1 with MMP-9 prevented the intravascular thrombus formation 3 weeks after the gene transfer. I, intima; N, neointima; M, media; A, adventitia.

 
3.6 MMP-9 overexpression and the expression of TF and intact TFPI-1
Western blot analysis demonstrated that the expression of TF was moderately detected in the normal coronary artery, and was increased in the balloon-injured coronary artery. Quantitative densitometirc analysis demonstrated that the expression was significantly augmented at the AdMMP-9-transfected site as compared to the Adβ-gal-transfected site (Fig. 10). The expression of intact TFPI-1 was also detected in the normal coronary artery and was increased at the Adβ-gal-transfected site, whereas it was markedly decreased at the AdMMP-9-transfected site (Fig. 10). Considering that the anti-TFPI-1 antibody used in this experiment detects N-terminal of the TFPI-1, the reduction in the TFPI-1 expression might be due to the increased degradation of the molecule by MMP-9. In addition, the expression of fragmented TFPI-1 was also decreased at the MMP-9 site (data not shown), indicating that other MMPs may also be involved in the process of TFPI-1 degradation. It was previously demonstrated that the fragmented TFPI-1 has no inhibitory effect on tissue factor activity [10]. The MMP-9-mediated downregulation of TFPI-1 was ameliorated by the co-transfection of TIMP-1, and the MMP-9-mediated upregulation of MMP-9 tended to be so (Fig. 10).


Figure 10
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  		Fig. 10 (A) Western blot for the effects of MMP-9 and TIMP-1 on the expression of TF and TFPI in porcine coronary arteries 1 week after the in vivo gene transfer. (B) Quantitative densitometric analysis of the Western blot signals.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
To the best of our knowledge, this is the first report demonstrating that local MMP-9 overexpression promotes intravascular thrombus formation after balloon injury in coronary arteries in vivo. The finding that TFPI-1, but not TF, is susceptible to cleavage by MMPs was recently documented by Belaaouaj et al. [10]. Thus, MMPs may play an important role in the pathogenesis of thrombus formation at the arteriosclerotic coronary lesions. Thrombosis affects the severity of inflammatory vascular lesions, including arteriosclerosis, atherosclerosis and restenosis after balloon angioplasty and/or stent implantation. Recent pathological studies suggested that thrombus formation is a major complication at the ruptured plaque in the pathogenesis of acute coronary syndrome [21–23]. Taken together, MMP-9 and other MMPs that may be related to the TFPI-1 degradation could be regarded as novel therapeutic targets in the treatment of arteriosclerotic/atherosclerotic coronary lesions associated with thrombus formation.

In the present study, adenovirus-mediated MMP-9 overexpression did not significantly affect the severity of coronary lesions in terms of neointimal formation or vascular remodeling. It may be partly due to the short duration of the expression of the MMP-9 (the expression was not detected by immunostaining 3 weeks after the gene transfer) and its limited localization (mainly in the media). Bendeck et al. demonstrated that neointimal thickening after balloon injury in rat carotid arteries was not suppressed by a tentative inhibition of MMPs activity using MMP inhibitor GM 6001 (a peptide hydroxamic acid) [12]. As they discussed, ‘catch up growth’ of the neointima due to increased replication of VSMC is important in the long-term outcome of vascular lesions, and inhibition of VSMC migration may not be sufficient to inhibit the lesion growth [12]. Moreover, it is conceivable that circulating inflammatory cells (e.g., monocytes, neutrophils, lymphocytes, and other progenitor cells) and growth factors may be involved in the growth of vascular lesions after balloon injury [24,25]. Therefore, combination therapy with long-term inhibition on MMPs and cell replication may be more efficient to suppress vascular lesion formation.

The role of MMPs in the pathogenesis of vascular remodeling is still controversial. Recently, de Smet et al. demonstrated that constrictive remodeling after balloon injury in porcine iliac arteries was markedly suppressed by long-term inhibition of MMPs with synthetic and non-specific MMP inhibitor [4]. Their finding suggests that MMPs may promote constrictive remodeling in atherosclerotic vascular lesions. By contrast, other recent reports suggested that local expression of MMPs may be involved in the molecular mechanism of positive remodeling in animal models of balloon injury-induced vascular lesions [5] and aortic aneurysm [26]. Thus, differences apparently exist depending on animal models used, making the evaluation of the importance of MMPs in the pathogenesis of vascular remodeling more complicated. The present results suggest that MMP-9 overexpression may affect the biological properties of arteriosclerotic vascular lesions and enhance thrombus formation after vascular injury in vivo.

The present study also demonstrated that the expression of TFPI-1 was increased throughout the injured coronary arteries 1 week after balloon injury at the β-gal-transfected site. However, overexpression of MMP-9 reduced this endogenous protective system against TF activity and thus allowed the thrombus formation at the injured arteries in vivo. TF is one of the major procoagulant factors that mediate the extrinsic pathway of the coagulation cascade and also stimulates cell proliferation. It has been demonstrated that inhibition of TF activity could suppress the thrombus formation and neointimal formation in vivo [27]. The present results are consistent with those previous studies that suggested the importance of active TFPI in the attenuation of TF activity in vivo [27,28].

Recent pathological findings and biological examinations suggested that acute thrombosis after atherosclerotic plaque disruption in coronary arteries is a major complication of coronary artery diseases leading to acute coronary syndrome [21–23]. Thrombus formation is a major determinant factor for acute coronary syndrome as well as neointimal thickening. TF expression at the human atherosclerotic plaque may be an important determinant of thrombogenicity after plaque rupture [29,30]. Aikawa et al. recently demonstrated that hypercholesterolemia increases vascular activity of MMPs and of TF in rabbits in vivo and that lipid-lowering therapy with either diet [31] or HMG-CoA reductase inhibitors (statins) suppressed those activities [31,32]. Their findings suggest that lipid lowering therapy by either diet or statins may be useful in the prevention of plaque vulnerability and subsequent thrombogenicity. It has been recently revealed that expression of MMPs is upregulated by various growth factors/cytokines [33] and oxidative stress [34], all of which are increased in atherosclerotic/arteriosclerotic vascular lesions. Therefore, it may also be important to suppress those proinflammatory factors in order to inhibit MMPs activities in vivo. Taken together, MMPs could be regarded as a novel therapeutic target in the treatment of vulnerable and thrombotic coronary artery disease in humans.

Several limitations should be mentioned for this study. First, the experiments were performed in normal domestic pigs without any complications (e.g., hypercholesterolemia, diabetes mellitus, or hypertension). Thus, our findings need to be further examined in animal models with those coronary risk factors. Second, as previously described [18,19], adenovirus-mediated expression of MMP-9 was relatively transient and localized. For the evaluation of the long-term effect of MMP-9 overexpression on the development of arteriosclerotic/atherosclerotic vascular lesions, more efficient vector system is needed.

Time for primary review 27 days.


   Acknowledgements
 
The authors wish to thank Dr. A.C. Newby at Bristol Heart Institute, University of Bristol, for providing adenoviral vectors encoding MMP-9 and TIMP-1, Professor S. Mohri at the Center of Biomedical Research, Kyushu University Graduate School of Medical Sciences for cooperation in this study, and M. Sonoda and H. Kubota for excellent technical assistance. This work was supported in part by grants-in-aid from the Japanese Ministry of Education, Science, Sports, Culture and Technology, Tokyo, Japan (No. 09470169, 10177223, 10357006, 12032215, 12470158).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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