Published online before print August 30, 2007, doi: 10.1161/ATVBAHA.107.151282
© 2007 American Heart Association, Inc.
Vascular Biology |
Thrombospondin-1 Activates Medial Smooth Muscle Cells and Triggers Neointima Formation Upon Mouse Carotid Artery Ligation
From the Center for Molecular and Vascular Biology (R.M., P.V., K.C., M.F.H.) and the Center for Transgene Technology and Gene Therapy (M.T.), University of Leuven, Belgium.
Correspondence to Marc Hoylaerts, PhD, Center for Molecular and Vascular Biology, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium. E-mail Marc.Hoylaerts{at}med.kuleuven.be
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Abstract |
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Objective— Thrombospondin-1 (TSP1) is described as a positive regulator of vascular smooth muscle growth in cell culture. However, insight into the in vivo effects of TSP1 on smooth muscle cell (SMC) function is lacking.
Methods and Results— We analyzed wild-type (WT) and TSP1-deficient (Tsp1–/–) mice in a carotid artery ligation model, in which neointimal lesions form without overt mechanical damage to the endothelium. On ligation, the expression of TSP1 increased strongly in the matrix of neointima and adventitia. In the early phase after ligation (day 3 to 7), activation, proliferation, and migration of medial SMCs were delayed and impaired in Tsp1–/– mice, in parallel with defective upregulation of metalloproteinase (MMP)-2 activity. As a result, Tsp1–/– arteries developed smaller neointimal lesions, a thicker media but comparably attenuated patency as in WT arteries, 28 days after ligation. Furthermore, medial and neointimal SMCs in Tsp1–/– mice produced more collagen, more osteopontin, and displayed weaker smooth muscle actin staining than WT SMCs, indicative of a modified SMC phenotype in Tsp1–/– mice.
Conclusions— Arterial SMC activation in the absence of TSP1 is delayed and dysregulated, reducing neointima formation, on mild vascular injury.
We analyzed WT and Tsp1–/– mice in a neointima model without endothelial denudation. Early proliferation and migration of medial SMCs were delayed and impaired. As a result, Tsp1–/– arteries developed smaller neointimas, thicker media, but comparably attenuated patency as in WT arteries. Tsp1–/– SMCs had a modified profibrotic phenotype.
Key Words: carotid artery • matricellular proteins • neointima • smooth muscle cells
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Introduction |
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Thrombospondin-1 (TSP1) is a matricellular protein, with a complex multidomain structure which can interact with a great variety of receptors, ligands, and matrix components (reviewed in1,2). It is highly expressed during development, but present at a low level in adult tissues and upregulated in response to injury.3–5 TSP1 is present in several cell types, including vascular cells, from which it is secreted on cell activation to participate in fine modulation of cell–cell and cell–matrix interactions and regulation of cell function.6–8 TSP1 gene-deficient (Tsp1–/–) mice are viable, and apparently normal, but challenge of these mice in various models has shown that TSP1 inhibits angiogenesis,9,10 promotes wound healing in an excisional wound healing model,11 and contributes to pulmonary homeostasis.12 We have recently shown an in vivo role for TSP1 in platelet and thrombus adhesion to the injured vessel wall.13
Percutaneous coronary interventions often result in the occurrence of intimal hyperplasia and restenosis.14 They are triggered by endothelial injury, and involve proliferation and migration of smooth muscle cells (SMCs) from the media into the intima. A role for TSP1 in vascular injury has been suggested by the strong TSP1 upregulation in rat carotid arteries after balloon-angioplasty15 and at sites of hyperplasia, in hypercholesterolemic lesions, and in the adventitia of diabetic rats.5,16,17
Although TSP1 is not essential for SMC growth in vitro, the treatment of SMCs with growth factors results in strong TSP1 expression, which further amplifies SMC proliferation.18–21 The inhibition of TSP1 by specific antibodies reduced SMC proliferation.21 TSP1 also acts as a chemoattractant for SMCs via interactions between its carboxyl terminus and CD47, and hence integrins.22–26 In addition, Tsp1–/– SMCs show selective defects in proliferation and chemotaxis responses to PDGF.27 The TSP1-induced SMC migration on gelatin appears to rely on metalloproteinase (MMP)-2 activation.28 Indeed, endogenous TSP1 increases MMP2 levels and limits collagen production in the vascular outgrowth of muscle explants.29
In vivo, TSP1 neutralization by antibodies resulted in less neointima development, after carotid artery denudation,30 but it remains unclear to which extent this was attributable to accelerated reendothelialization or decreased vascular SMC function. Nevertheless, the study in Tsp1–/– mice of healing myocardial infarcts has revealed a role for TSP1 in protecting the noninfarcted myocardium from fibrotic remodeling.31 In experimental models of ischemic injury, Tsp1–/– mice show an increased nitric oxide (NO)-mediated tissue perfusion, implicating TSP1 as a physiological antagonist of NO-mediated vasodilatation and tissue perfusion. Elegant work has shown that CD47 is the TSP1 receptor, limiting the NO-mediated vascular smooth muscle relaxation.32
In this study, by comparing WT and Tsp1–/– mice, we aimed at studying the specific role of TSP1 in SMC activation and in phenotype switching from contractile to synthetic SMCs, after mild vascular injury. Therefore, we have used an established model of carotid artery ligation,33 in which, after an early phase of inflammatory cell recruitment, medial SMCs rapidly proliferate and migrate toward the lumen, leading to extensive neointima formation after 4 weeks, without inflicting direct damage to the endothelium.
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Methods |
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Model of Mouse Carotid Artery Ligation
All animal experiments were reviewed and approved by the Institutional Review Board of the University of Leuven and were performed in compliance with the guidelines of the International Society on Thrombosis and Hemostasis.34 Tsp1–/– mice12 were kindly provided by Dr J. Lawler (Harvard, Boston, Mass). Male Tsp1–/– and WT littermate mice, aged 12 to 24 weeks and bred for more than 8 generations into the C57Bl/6 background were used in this study. The carotid artery ligation model has been previously described.33 Briefly, the left common carotid artery was dissected free of connective tissue and permanently ligated proximally to the bifurcation. At specified time points after surgery, animals were euthanized, perfused, and paraffin serial 7-µm-thick sections were prepared, covering the area 5 mm proximal to the ligation site. The area analyzed comprised a minimum of 4 cross-sections in the zone of maximal response, between 1 mm and 3 mm, distally from the ligation site. For more details on the animal model, histology, and morphometry, please see the supplemental materials (available online at http://atvb.ahajournals.org).
SMC Assessment and Proliferation
The number of nuclear profiles per power field in the media was counted on H&E stainings to assess loss and recovery of medial SMCs after ligation. Results were expressed as cell densities. Because smooth muscle actin (SMA) is a marker for contractile SMCs and immunoreactivity to SMA is reduced in replicating, synthetic SMCs, SMA staining was used to evaluate the SMC phenotype. Cell proliferation was measured by immunostaining for proliferating cell nuclear antigen (PCNA). Proliferation rates were expressed as a percentage of PCNA-stained nuclei/total nuclei.
Immunoblot Analysis
Vascular extracts of ligated carotid artery (0.5 to 10 µg of total protein) were subjected to SDS-PAGE. Blots were revealed via indirect staining, using antibodies against SMC actin (1/1000 Dako SMA Mo851), OPN (1/1000 Santa Cruz Biotechnology, sc-10593), and TSP1 (50 µg/mL home-made Rabbit-anti-human TSP1). Staining was performed with the Pierce Western blotting enhanced chemiluminescence (ECL) detection reagent (Perbio).
Gelatin Zymography
Gelatin zymography was performed as described.35 Protein extracts (5 µg) of carotid arteries mixed with SDS sample buffer were loaded onto 10% gels containing 0.1% gelatin (Invitrogen, NOVEX EC6175BOX). Digestion bands were quantified using the Zeiss KS300 software. Cell extracts of HT1080 cells served as an internal reference for the identification of MMP2 and MMP9 activity bands.
Statistical Analysis
Data are represented as the mean±SD. Significance of differences was analyzed using a 2-tailed unpaired t test with Welch correction, allowing for populations with different SD. A value of P<0.05 was considered statistically significant. Time course experiments were analyzed by 1-way analysis of variance (ANOVA).
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Vascular Tsp1 Expression After Carotid Artery Ligation in Mice
In native carotid arteries of WT mice, TSP1 was faintly expressed in the adventitia, and in some areas of the endothelial lining and media (Fig 1A). At day 3 after ligation, TSP1 was upregulated in the adventitia (Figure 1B and 1C) and at day 28, the developed neointima stained strongly for TSP1 (Figure 1D and 1E). Detailed analysis revealed that TSP1 expression was mostly localized in the extracellular matrix (ECM), but was also cell-associated in SMCs and inflammatory cells (Figure 1C and 1E). TSP1 staining was specific, as evidenced by negative or very faint staining of Tsp1–/– arteries (data not shown). Western Blots of carotid artery extracts confirmed that TSP1 was upregulated as soon as 3 days after ligation (Figure 1F).
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Carotid Artery Morphology and Neointima Development After Ligation
No morphological differences were observed in resting carotid artery cross-sections of WT and Tsp1–/– mice (Figure 2, day 0). Three days after ligation, medial SMCs in WT sections had lost their elongated profile, leading to medial thickening. This was not observed in Tsp1–/– sections (Figure 2, day 3). At day 7, medial thickening in the absence of neointima was observed in both genotypes (Figure 2, day 7), whereas at day 28, neointima had developed in both genotypes, 1.7-fold (supplemental Figure Id) more extensively in WT than Tsp1–/– mice. These data indicated impaired neointima development and suggested delayed SMC activation, in the absence of TSP1.
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Geometric Remodeling and Vascular Patency After Carotid Artery Ligation
Morphometric analysis of the ligated carotid artery revealed that the external elastic lamina area (EEL) increases beyond day 7 in WT but not in Tsp1–/– mice (supplemental Figure Ia). In WT but not in Tsp1–/– mice, medial thickening early after ligation (Figure 2), was accompanied by an increase in medial area from day 3 to 28 (supplemental Figure Ib). This occurred despite a reduction in the medial area index (medial area/ EEL area), a parameter of medial thickening, at day 28 (supplemental Figure Ic). In contrast, medial thickness was still observed in Tsp1–/– mice on day 28 (Figure 2 day 28, supplemental Figure Ic), suggestive of long-lasting impaired SMC migration.
We wished to investigate whether the larger vessel size with a thin media in WT and smaller vessel size with a thick media in Tsp1–/– mice, resulted from different eutrophic wall remodeling. We therefore studied arterial remodeling in both genotypes by plotting the EEL at day 28 as a function of the neointima formed. This analysis revealed a linear relationship in WT cross-sections (R2=0.87; P<0.0001; slope=0.81; y intersection=41 790) and Tsp1–/– cross-sections (R2=0.47; P=0.0002; slope=0.83; y intersection=35 050), with comparable slopes and intersections in both genotypes (supplemental Figure Ie and If). These data confirm that the relative outward eutrophic geometric remodeling in WT and Tsp1–/– mice is identical, its extent in Tsp1–/– mice being determined by the smaller neointima on day 28, in these mice (supplemental Figure Id).
The comparable luminal cross-sectional area on day 28 in WT and Tsp1–/– mice (supplemental Figure Id), is therefore explained by less neointima formation, less outward remodeling, and a thicker media in Tsp1–/– mice.
Responses of Medial SMCs After Ligation
Ligation of the WT murine carotid artery leads to an initial loss of medial SMCs followed by SMC proliferation and recovery.33 We therefore quantified, by counting the nuclei per high power field on H&E stained sections, the medial SMC density of WT and Tsp1–/– mice at different time points after ligation. At baseline, no genotypic differences were found in WT and Tsp1–/– mice (Figure 3A). At day 3 after ligation, WT and Tsp1–/– mice underwent comparable and significant reduction in medial SMC density. In WT mice, this drop was followed at day 7 by a 1.5-fold increase of the SMC density, and at day 28 by a secondary drop (Figure 3A). In contrast, Tsp1–/– mice seemed to fail in responding to the initial loss of SMCs up to 28 days after ligation. More detailed analysis in both genotypes of SMC proliferation by quantifying PCNA-stained nuclei in the media after ligation, showed that medial SMC proliferation was initiated around day 3, increased at day 7, and was almost back to baseline levels at day 28 (Figure 3B). Both at day 3 and day 7, the percentage of PCNA-stained cells was drastically reduced in Tsp1–/–, compared with WT mice. Thus, SMC proliferation was impaired in Tsp1–/– mice, on carotid artery ligation.
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MMP2 is a marker of migrating and activated SMCs during vasculopathy.36,37 We therefore analyzed its expression by zymography using arterial extracts (supplemental Figure II). At baseline, MMP2 was hardly detectable in both genotypes (Figure 3C). In WT mice, vessel occlusion resulted in greatly increased MMP2 levels, 3 and 7 days after ligation (Figure 3C). In contrast, the increase of MMP2 levels was significantly reduced in Tsp1–/– mice (Figure 3C). Notably, these results might further corroborate the impaired migration of Tsp1–/– SMCs (see above). The smaller increase of MMP2 levels in Tsp1–/– mice was specific as the expression of MMP9, predominantly derived from infiltrated inflammatory cells,37 was not different between WT and Tsp1–/– mice (Figure 3D). Indeed, MMP9 levels increased after ligation in both genotypes, consistent with enhanced and similar infiltration in WT and Tsp1–/– vessels of leukocytes and macrophages, identified by CD45 and Mac3 staining, respectively (supplemental Table I).
Loss of Contractile SMC Phenotype and Collagen Deposition in the Media
The presence of SMA is characteristic of contractile nonactivated SMCs.33 We therefore used the absence of SMA as a parameter of SMC differentiation. At baseline, medial SMA staining was comparable between genotypes (supplemental Figure III). In WT mice, medial SMA staining was strongly reduced 3 days after ligation compared with baseline and remained strongly negative 7 days after ligation. In contrast, SMA staining in Tsp1–/– mice was only minimally affected 3 days after ligation, and decreased significantly only at day 7 (supplemental Figure III). This finding was in agreement with the lack of medial thickening at day 3, which only occurred at day 7 (see Figure 2). These results suggest that loss of the contractile SMC phenotype in Tsp1–/– mice was delayed. Consistently, medial SMA staining had returned to normal levels in WT cross-sections at day 28, but remained attenuated in Tsp1–/– sections, in agreement with a different medial SMC phenotype at day 28 in Tsp1–/– carotid arteries (supplemental Figure III).
Because SMCs are a predominant source of collagen in blood vessels,36 we analyzed the collagen content in the vessels by Sirius Red (SR) staining. At baseline, the medial SR-positive area was approximately 40% in WT and Tsp1–/– mice, and only insignificantly increased 3 days after ligation (supplemental Figure IVa). At day 28 after ligation, the medial collagen content was not altered in WT mice, but was elevated 1.5-fold in the Tsp1–/– media (supplemental Figure IVa). The higher collagen deposition was not evenly distributed, but rather appeared as patches throughout the Tsp1–/– media (supplemental Figure IVc and IVd). Adventitial collagen analysis confirmed that TSP1 specifically controlled collagen deposition of SMCs, but not of adventitial fibroblasts, because adventitial collagen did not differ over time between genotypes (supplemental Figure IVb).
Different Neointima Phenotype in Tsp1–/– Mice
The different WT and Tsp1–/– media composition at day 28, prompted us to analyze the SMC-rich neointima in more detail. In WT mice, the mature neointima contained predominantly SMA-positive cells, with a SMA-positive area equivalent to 57±21% (n=19; Figure 4A). These data suggest that the vast majority of neointimal SMCs 28 days after ligation had reacquired the contractile nonactivated phenotype. Surprisingly, the SMA-positive area within the neointima was significantly smaller in Tsp1–/– mice (22±23%; n=19; P<0.0001 versus WT; Figure 4B), with a larger proportion of cells not staining for SMA, suggesting late dedifferentiated neointimal SMCs. The smaller SMA content was also detectable by Western blotting (Figure 4C), when equal amounts of vascular extracted proteins were compared. As for the media, the collagen content was increased in the neointimal lesion of Tsp1–/– mice (SR+ area was 28±20% in WT versus 53±27% in Tsp1–/– mice; n=12; P<0.05; Figure 4D and 4E).
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Because the medial SMC recovery was defective in Tsp1–/– ligated arteries (see above), we also quantified nuclei numbers per high-power field in the neointima. In WT mice, nuclear density was 5916±1963 nuclei/µm2 (n=24; Figure 4F). Consistent with less proliferation and smaller lesions (see above), Tsp1–/– neointimas showed a slightly lower nuclear density (4842±1781; n=24; P=0.05 versus WT; Figure 4G), though barely significantly different. The comparably low proliferation index of 10.4±9.2% in WT versus 9.2±5.9% in Tsp1–/– neointima, confirmed that SMCs had halted to proliferate to the same degree in both genotypes.
Collagen-producing SMCs synthesize more osteopontin (OPN).38 Therefore, we quantified OPN immunohistochemically in the neointima of WT and Tsp1–/– mice. OPN was mostly found in the adventitia and neointima, although some SMCs in the media were also strongly positive (Figure 4H and 4I). In WT mice, the OPN-positive area in the neointima equalled 18±15% (n=16; Figure 4H). Consistent with increased collagen deposition, the OPN positivity was significantly increased in the neointima of Tsp1–/– mice (32±20%; n=16; P<0.05 versus WT, Figure 4I). Western blots of vascular extracts confirmed that OPN expression was induced by the carotid artery ligation both in WT and in Tsp1–/– vessels (not shown), but was upregulated to higher levels in Tsp1–/– mice at day 28 (Figure 4J). Hence, Tsp1–/– neointimas have less cells, less contractile SMA-positive cells, more collagen, and accordingly, more OPN, a marker for collagen-producing cells.
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Discussion |
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This study provides functional evidence for a role of TSP1 in activation, proliferation, and migration of SMCs in the vessel wall after vascular injury. The key findings of this study are: (1) loss of TSP1 delays SMC activation; (2) loss of TSP1 reduces SMC proliferation and MMP2 expression; (3) loss of TSP1 impairs SMC migration resulting in smaller neointimas and thicker media 4 weeks after carotid artery ligation; (4) loss of TSP1 promotes collagen and osteopontin production in the late phase after ligation, altering the SMC phenotype.
Ligation of the carotid artery causes medial cell death the first 2 days. Cell death results in the concomitant release of growth factors and is followed by medial SMC activation and proliferation.33,39 This response was strongly attenuated in Tsp1–/– media. Indeed, whereas WT medial SMC numbers rose between day 3 and 7 after ligation, Tsp1–/– medial SMC densities hardly changed. These differences could be attributed to a 2-fold reduction in the proliferation rate of Tsp1–/– medial SMCs compared with WT, throughout the early recovery phase, possibly in conjunction with enhanced apoptosis of medial SMCs. Furthermore, the SMC response to the ligation between day 3 and 7, was delayed, noticeable from retarded medial thickening in Tsp1–/– mice and from retarded reduction of SMA expression, characteristic of noncontractile SMC phenotype. The reduced MMP2 levels in Tsp1–/– mice at day 3 and 7 after ligation, are in agreement with defective SMC migration, because MMP2 expression is associated with SMC migration. Indeed, MMP2–/– mice develop significantly less neointima after carotid artery ligation than WT controls, because of defective degradation of ECM proteins surrounding migrating SMCs.37 Furthermore, in vitro, TSP1 upregulates MMP2 by transcriptional and nontranscriptional mechanisms, and MMP2 activation is relevant for TSP1-induced vascular SMC migration.28 Our findings therefore indicate that vascular TSP1 participates in early SMC activation after vessel occlusion, affecting morphological features of medial SMC and triggering SMC proliferation and migration.
In WT mice, the early proliferation and migration of medial SMCs after carotid artery ligation are followed by a reduction of SMC numbers in the media and the development of a neointima, as SMCs migrate into the lumen.33 In contrast, in Tsp1–/– mice, as SMC numbers in the media did not change beyond day 3, Tsp1–/– vessels persisted with a thick media and a 2-fold smaller neointima at day 28. Detailed morphological analysis of the outward remodeling, accompanying neointima formation in WT and Tsp1–/– mice, revealed the same relationship in both genotypes between the extent of neointima formation and vascular enlargement, as measured via the external elastic lamina area (EEL). This analysis underscores that the smaller remodeling in Tsp1–/– mice is secondary to the defective smooth muscle cell phenotype and does not stem from a more direct role for TSP1 in vascular remodeling. The less neointima, thicker media, and attenuated vessel enlargement in Tsp1–/– vessels resulted in comparable luminal patency in WT and in Tsp1–/– vessels.
Nevertheless, the deficiency of TSP1 modified the SMC phenotype in the media and in the neointima, and altered the neointima composition into a more fibrotic phenotype. In WT mice, mature neointimal lesions are composed mostly of SMA-positive cells.33 At completion of the neointimal proliferation, these cells return to a resting and contractile state, characterized by SMA expression.33 In the present study, we found that more than half of the WT neointimal area at day 28 consisted of SMA-positive cells, the remainder being covered by either collagen or osteopontin positive areas (marker for collagen-producing SMCs). In contrast, collagen was the main component of Tsp1–/– neointima (more than half of the area) and the amount of osteopontin was significantly increased, with only a small area consisting of SMA-positive cells (Figure 4K and 4L). These findings indicate that in the absence of TSP1, SMCs differentiate into a more fibrotic collagen-producing SMC type. In view of the similar relationship between EEL and neointima in WT and Tsp1–/– mice, the enhanced fibrosis is not greatly affecting eutrophic outward remodeling.
Because our data suggest that TSP1 modulates SMC function in the early phase (3 to 7 days) after ligation, knowledge of the SMC receptor(s) involved is highly relevant. Both CD47–/–40 and β3–/–41 mice show reduced neointima formation after carotid artery ligation, primarily because of defective SMC proliferation and migration, respectively. Several recent studies have identified CD47 as the major TSP1 receptor on SMCs, mediating the activity of TSP1 at physiological concentrations. Hence, whereas the neutralization of either CD36 or CD47 is sufficient to inhibit NO-stimulated vascular cell responses, only CD47 is necessary for this activity.42 Likewise, CD47 is the necessary receptor in vivo to explain the limiting NO-mediated vascular smooth muscle relaxation and tissue survival after ischemic injury in skin flaps and hindlimbs.32 These findings are in agreement with defective SMC proliferation and neointima formation in models of carotid artery ligation in CD47–/–40 and Tsp1–/– mice. Taken together, these results strongly suggest that CD47 is the receptor responsible for the presently described SMC phenotype.
In agreement with many in vitro studies, the present study points to a stimulatory effect for TSP1 on SMCs. Yet, TSP1 can behave as a strong SMC inhibitor, eg, antagonizing nitric oxide stimulated vascular SMC responses, in vitro and in vivo43 and endothelial cell activation (ECs).44 Although TSP1 is generally a positive regulator for SMCs and an inhibitor for ECs, these opposite roles are not surprising taking in consideration the multidomain structure of TSP1. Furthermore, the function of TSP1 in a specific environment is determined by receptor availability, molecular orientation, state of cell activation, and TSP1 concentration. Therefore, considering both the high TSP1 expression in WT neointima and the persistent noncontractile SMC phenotype in the neointima of Tsp1–/– mice, TSP1 may actively contribute to arresting SMC proliferation in the matured neointima, ie, facilitate their return to the dedifferented contractile state.
In summary, we provided evidence for a role for TSP1 in SMC activation after mild vascular injury. TSP1 facilitates SMC proliferation and migration after injury and the development of a neointima. In the absence of TSP1, proliferation and migration of SMCs are reduced and a more fibrotic neointima develops.
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Acknowledgments |
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We thank M. Van Hul for the help with the zymograms, L. Frederix and S. Terclavers for excellent technical assistance, and Prof J. Lawler and Prof Em J. Vermylen for critically reading the manuscript.
Sources of Funding
This work was supported by K.U. Leuven grant GOA/2004/09 and by FWO Vlaanderen (project no. G.0569.05). The CMVB is supported by the "Excellentie financiering KULeuven". M.T. is a research fellow of the Belgian IWT.
Disclosures
None.
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Footnotes |
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Original received March 8, 2007; final version accepted July 27, 2007.
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