doi: 10.1161/hq0302.104529
© 2002 American Heart Association, Inc.
Thrombosis |
Lack of Plasminogen Activator Inhibitor-1 Promotes Growth and Abnormal Matrix Remodeling of Advanced Atherosclerotic Plaques in Apolipoprotein E–Deficient Mice
From the Center for Transgene Technology and Gene Therapy (A.L., E.S., M.F.H., L.M., D.C., P.C.), Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium; Cardiovascular Biology Research Program (F.L., J.C.), Oklahoma Medical Research Foundation, Oklahoma City; the Cardiovascular/Thrombosis Research Department (F.B., J.-M.H.), Sanofi-Synthélabo, Toulouse, France; Joint Diseases Laboratory (A.R.P.), Shriners Hospitals for Children, McGill University, Montreal, Quebec, Canada; and the Center for Inflammatory Diseases (P.T.), Monash University Medical Center, Clayton, Victoria, Australia.
Correspondence to P. Carmeliet, MD, PhD, Center for Transgene Technology and Gene Therapy, Campus Gasthuisberg, Herestraat 49, University of Leuven, Leuven, B-3000, Belgium. E-mail peter.carmeliet{at}med.kuleuven.ac.be
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Abstract |
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Epidemiological studies suggest that elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) predispose an individual to ischemic heart disease or promote plaque progression by inhibiting fibrinolysis. In the present study, loss of PAI-1 in apolipoprotein E (apoE)-deficient (apoE-/-:PAI-1-/-) mice promoted the growth of advanced atherosclerotic plaques, which was due to enhanced extracellular matrix deposition. ApoE-/-:PAI-1-/- plaques also exhibited collagen fiber disorganization and degradation. Immunostaining and bone marrow transplantation revealed that smooth muscle cells, not macrophages, primarily expressed PAI-1 in plaques. Thus, although PAI-1 may promote plaque growth because of its antifibrinolytic properties, the present study reveals a protective role for PAI-1 by limiting plaque growth and preventing abnormal matrix remodeling.
Key Words: plasminogen activator inhibitor-1 • atherosclerosis • collagen • matrix • transforming growth factor-ß1
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Introduction |
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Epidemiological studies indicate that elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) constitute a risk factor for ischemic heart disease that is due to impaired thrombolysis after plaque rupture.1 Elevated PAI-1 plasma levels have also been associated with the progression of atherosclerosis, supposedly by inhibiting the clearance of fibrin incorporated into atherosclerotic plaques.2 Fibrin in plaques may contribute to their growth by increasing lesion mass or attracting inflammatory cells, endothelial cells, or smooth muscle cells (SMCs; see Salomaa et al2 and the references therein). Independent of its antifibrinolytic effect, PAI-1 may also affect cellular migration, matrix remodeling, and the activation of growth factors.3,4 How and to what extent these mechanisms influence atherosclerotic plaque progression remain poorly understood.
The availability of PAI-1–deficient (PAI-1-/-) mice5,6 allows for the study of the effects of PAI-1 on lesion progression. Loss of PAI-1 in apoE-deficient (apoE-/-) mice has been reported to have no significant influence on plaque growth in the proximal aorta but to reduce it at the carotid bifurcation.7,8 The mechanisms for this apparently divergent role of PAI-1 in different vascular beds remain undefined.
The plasminogen system plays a complex role in atherosclerosis. The loss of plasminogen has been shown to accelerate plaque growth,9 whereas the loss of tissue-type plasminogen activator or urokinase-type plasminogen activator has been shown to have no effect on plaque growth.10 Furthermore, fibrinogen deficiency has been reported to have no influence on lesion development in apoE-/- mice but to reduce plaque growth in apo(a) transgenic mice (see Xiao et al11 and references therein). These apparently contradictory reports suggest that atherosclerosis in gene-inactivated mice is influenced by differences in genetic background, diet, or atherosclerotic mouse model.
In the present study, we reevaluated the effect of PAI-1 deficiency on atherosclerosis in apoE-/- mice. Surprisingly, the loss of PAI-1 did not reduce but stimulated plaque growth at advanced stages of atherosclerosis because of the increased matrix deposition. Therefore, the present genetic study unveiled an atheroprotective role for PAI-1 by limiting plaque growth.
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Methods |
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An extended Methods section is available in the online data supplement (which can be accessed at http://atvb.ahajournals.org).
Mice, Gene Expression, and Cell Culture
PAI-1-/- mice were intercrossed with apoE-/- mice to generate breeding pairs with heterozygous deficiency of apoE and PAI-1 (apoE+/-:PAI-1+/-), which sired apoE-/-:PAI-1-/- mice and apoE-/-:PAI-1+/+ littermate offspring with a mixed genetic background of 87.5% C57Bl/6 and 12.5% 129/SvJ. Mice were maintained on a chow diet for 5 weeks, when they were fed a cholesterol/cholate diet (see online supplement I at http://atvb.ahajournals.org) or chow diet. Blood from mice that fasted overnight was drawn from the vena cava and centrifuged; plasma was used for lipid analysis with commercially available reagents in the University Hospital of Leuven. PAI-1 levels were determined by ELISA. Unfixed tissues were frozen immediately after dissection. Transforming growth factor (TGF)-ß1 levels were determined on whole protein extracts by using commercially available assays (R&D Systems). Plasmin levels were determined on whole protein extracts by immunoblotting under reducing conditions.12 Gelatin zymography on whole protein extracts from plaques was performed, and preparation and culture of peritoneal macrophages and vascular SMCs were performed as described.10,12
Morphological Analysis
For matrix metalloproteinase (MMP) staining, dissected tissues were embedded in OCT compound (Tissue-Tek) and snap-frozen in precooled 2-methyl butane; for all other stainings, tissues were postfixed in 4% formaldehyde, transferred to PBS containing 20% sucrose, embedded in OCT compound, and stored at -80°C. For antibodies, see online supplement II (which can be accessed at http://atvb.ahajournals.org). Staining for hematoxylin-eosin and Sirius red, double labeling for PAI-1 and smooth muscle 
-actin,12 in situ hybridization,13 and electron microscopy14 were performed as described. For determination of cross-sectional plaque area, morphometric measurements were performed on 5 to 20 sections (each 70 or 80 µm apart) from different locations throughout the arterial tree (see online supplement III at http://atvb.ahajournals.org). Morphometric analyses after immunostaining and Sirius red staining or densitometric analyses of plasmin and MMP levels were performed with a Leitz DMRXE microscope (Leica Imaging Systems Ltd), a 3CCD color video camera (DXC-93OP, Sony), and a Leica Qwin software system by persons unaware of the genotype.
Bone Marrow Transplantation and Gene Transfer
Bone marrow transplantation was performed as previously described,12 and plaque analysis in the aortic root was performed after 20 weeks of diet (see online supplement IV at http://atvb.ahajournals.org). For adenoviral gene transfer, apoE-/-:PAI-1-/- mice were injected in the tail vein after 16 weeks of diet with 1.3x109 plaque-forming units of AdCMVPAI or AdRR5 control virus.12 Animals were euthanized 6 days after injection, and the plasma was then prepared for measurement of PAI-1. Aorta and liver were processed for TGF-ß1 measurements or PAI-1 immunostaining (see online supplement V at http://atvb.ahajournals.org).
Thrombosis
Thrombosis was photochemically induced by endothelial denudation of the carotid artery, and thrombus size was quantified by the amount of transilluminated light (see online supplement VI at http://atvb.ahajournals.org).
Statistical Analysis
The data are represented as mean±SEM. ApoE-/-:PAI-1-/- mice were compared with apoE-/-:PAI-1+/+ mice by the unpaired Student t test or by the Mann-Whitney U test. Data were considered statistically significant at P<0.05.
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Results |
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Plasma Cholesterol, PAI-1 Expression, and Plasmin
ApoE-/-:PAI-1-/- mice and apoE-/-:PAI-1+/+ littermates, fed a cholesterol diet for 25 weeks, displayed comparable weight gain and plasma lipid levels (not shown). Plasma PAI-1 levels in apoE-/-:PAI-1+/+ mice were 2±0.5 ng/mL on a normal diet and 12±2 and 25±5 ng/mL after 10 and 25 weeks of the cholesterol diet, respectively (P<0.05, n=7). In situ hybridization and immunostaining revealed increased PAI-1 expression throughout the plaque and the fibrous cap, mainly in SMC
-actin (SMA)-positive myofibroblasts (not shown). By immunoblotting of plaque extracts, plasmin levels were 8±3 arbitrary densitometric units/µg protein in apoE-/-:PAI-1+/+ plaques and 53±7 arbitrary densitometric units/µg protein in apoE-/-:PAI-1-/- plaques (P<0.05, n=4). Thus, as in patients,2,13 plasma PAI-1 levels and expression of PAI-1 in plaques increase during atherosclerosis.
Increased Fibrinolysis in ApoE-/-:PAI-1-/- Mice
Loss of PAI-1 accelerated fibrinolysis, as revealed by the following: First, mural thrombi were induced by endothelial denudation, and their sizes were measured in arbitrary light transmission units (AU). After 40 minutes, thrombi in apoE-/-:PAI-1-/- mice were smaller (70±20 AUx106) than thrombi in apoE-/-:PAI-1+/+ mice (140±16 AUx106, P<0.05; n=10). Second, apoE-/-:PAI-1-/- plaques contained 3-fold less fibrin(ogen) than did apoE-/-:PAI-1+/+ plaques (fibrin(ogen)-immunoreactive area as percentage of total plaque area 1.9±0.6% versus 5.7±0.9%, respectively, P<0.05; n=6).
Increased Plaque Growth in ApoE-/-:PAI-1-/- Mice
Plaque size in thoracic and abdominal aortas was comparable in both genotypes after 5 and 10 weeks of the cholesterol diet (not shown), but it was significantly larger in apoE-/-:PAI-1-/- mice than in apoE-/-:PAI-1+/+ mice after 25 weeks of cholesterol feeding (Figure 1A through 1D, Table 1). Notably, plaques in thoracic and abdominal aortas were also larger in apoE-/-:PAI-1-/- mice than in apoE-/-:PAI-1+/+ mice when they were fed a normal chow diet for 25 weeks, indicating that the effect of PAI-1 was independent of the diet used (Table 1). To study the mechanisms of plaque enlargement in apoE-/-:PAI-1-/- mice, the accumulation of macrophages and myofibroblasts was evaluated by measuring the plaque area immunoreactive for Mac-3 and SMA, respectively, and expressing it as a percentage of the total plaque area (density).
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After 25 weeks of the cholesterol diet, apoE-/-:PAI-1-/- plaques, compared with apoE-/-:PAI-1+/+ plaques, contained similar numbers of SMA-immunoreactive myofibroblasts (SMA-positive area 34 000±18 000 versus 31 500±6000 µm2, respectively, P=NS; n=5) but more macrophages (Mac-3–positive area 27 000±3000 versus 4100±700 µm2, respectively, P<0.05; n=5). When compared with the density in apoE-/-:PAI-1+/+ plaques, the density in advanced apoE-/-:PAI-1-/- plaques was increased by 360% for macrophages (3±1% versus 11±2%, respectively, P<0.05; n=5) and reduced by 44% for myofibroblasts (23±2% versus 13±1%, respectively, P<0.05; n=5).
Matrix components were more abundantly deposited in apoE-/-:PAI-1-/- plaques. Plaque areas, stained for Sirius red (collagen) or immunoreactive for tenascin-C, versican, or fibronectin, were 2-fold larger in apoE-/-:PAI-1-/- plaques than in apoE-/-:PAI-1+/+ plaques (Figure 1C through 1E). Tenascin-C and versican densities in advanced apoE-/-:PAI-1-/- plaques were increased by 25% and 55%, respectively (P<0.05, n=5), whereas densities of collagen and fibronectin were comparable (P=NS, n=5). Ultrastructural analysis confirmed the increased matrix deposition (not shown). Thus, loss of PAI-1 resulted in an increased accumulation of macrophages and extracellular matrix in advanced atherosclerotic lesions.
Collagen Disorganization and Degradation in ApoE-/-:PAI-1-/- Plaques
Microscopic examination revealed that collagen fibers were aligned in thick bundles, primarily in fibrous caps in apoE-/-:PAI-1+/+ plaques (Figures 1C and 2A). In contrast, in apoE-/-:PAI-1-/- plaques, collagen fibers were disorderly deposited in a fine meshwork (Figures 1D and 2C). As a result, apoE-/-:PAI-1-/- plaques contained numerous small necrotic cores, whereas apoE-/-:PAI-1+/+ plaques contained fewer but large necrotic cores (core size 1200±100 versus 4800±1000 µm2, respectively, P<0.05 [n=13], and number of cores/mm2 250±20 versus 120±13, respectively, P<0.05 [n=13]; Figures 1C, 1D, 2A, and 2C).
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On Sirius red–polarization microscopy, thick tightly packed collagen fibers appear red, whereas thin loosely assembled fibers appear green.15 No genotypic differences in collagen characteristics were detected in early lesions (not shown). In contrast, after 25 weeks of the cholesterol diet, the majority of collagen fibers were thin and dispersed, and they exhibited a fine yellow-green birefringence in apoE-/-:PAI-1-/- plaques, whereas most fibers were thick, densely packed, and orange-red in apoE-/-:PAI-1+/+ plaques. The ratio of orange-red/yellow-green birefringence was 0.4±0.01 in apoE-/-:PAI-1-/- mice versus 3.7±0.4 in apoE-/-:PAI-1+/+ mice (P<0.05, n=5; Figure 2B and 2D). Ultrastructural analysis revealed tightly aligned thick collagen bundles in apoE-/-:PAI-1+/+ plaques and disorderly scattered bundles in apoE-/-:PAI-1-/- plaques separated by granular extracellular material, likely representing macromolecules such as versican (Figure 2E and 2F).
Direct evidence of collagen degradation was obtained by immunostaining for COL2-3/4cshort, which is a neoepitope generated after collagenase-mediated cleavage of mainly type I collagen fibers.16 The COL2-3/4cshort-positive area was 19 000±2000 µm2 in apoE-/-:PAI-1-/- plaques versus 9000±1600 µm2 in apoE-/-:PAI-1+/+ plaques (P<0.05, n=6; Figure 3A and 3B) and mostly present in plaque shoulders. Moreover, ultrastructural analysis demonstrated that collagen fibers were fragmented and thin in apoE-/-:PAI-1-/- plaques (Figure 3D), as typically found after collagenolysis,17 whereas the fibers had uniform diameters and were sharply delineated in apoE-/-:PAI-1+/+ mice (Figure 3C).
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We also found that levels of MMP, able to degrade collagen on activation of their latent proform, were increased in apoE-/-:PAI-1-/- plaques. Plaque extracts were analyzed by gelatin zymography, and the lysis zones were quantified by densitometry in arbitrary units per 10 µg protein. MMP levels in apoE-/-:PAI-1-/- versus apoE-/-:PAI-1+/+ plaques were, respectively, as follows (in arbitrary units/10 µg protein): 107±7 versus 78±7 for latent MMP-2, 9±1 versus 6±1 for active MMP-2, and 27±4 versus 9±4 for latent MMP-9 (P<0.05, n=4). Immunostaining confirmed the upregulated MMP expression in apoE-/-:PAI-1-/- plaques. Plaque areas immunoreactive for MMP-9 were 26 000±7000 µm2 versus 5500±1500 µm2 in apoE-/-:PAI-1-/- versus apoE-/-:PAI-1+/+ mice (corresponding to an increase in MMP-9 density by 77%, P<0.05 [n=5]; Figure 3E and 3F). MMP-13–positive areas were 28 000±9500 versus 5500±1800 µm2 in apoE-/-:PAI-1-/- versus apoE-/-:PAI-1+/+ mice (or an increase in MMP-13 density by 86%, P<0.05 [n=5]; Figure 3G and 3H). Thus, in the absence of PAI-1, there were signs of extracellular matrix disorganization and degradation, likely attributable to increased MMP levels.
Distinct Role of PAI-1 From Myofibroblasts and Macrophages
By double immunostaining, PAI-1 was found primarily in plaque myofibroblasts and minimally in macrophages (not shown). Consistent herewith, 100 000 cultured SMCs produced over 24 hours 350±35 pg PAI-1, whereas activated macrophages produced only 10±2 pg PAI-1 (P<0.05, n=3). In addition, because macrophages and myofibroblasts in plaques are primarily derived from the bone marrow and vessel wall, respectively, we transplanted apoE-/-:PAI-1+/+ or apoE-/-:PAI-1-/- bone marrow into apoE-/-:PAI-1+/+–recipient or apoE-/-:PAI-1-/-–recipient mice and analyzed advanced plaques in the aortic root. Plaques were significantly larger (Figure 4) and contained more collagen (not shown) when the recipient (irrespective of the donor marrow genotype) lacked PAI-1, suggesting that myofibroblasts, not macrophages, played an essential role in controlling plaque growth and collagen content by the production of PAI-1.
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Increased TGF-ß1 Levels in Advanced ApoE-/-:PAI-1-/- Plaques
Because plasmin is known to activate latent TGF-ß1,4 levels of latent and active TGF-ß1 in the vessel wall were determined. Normal aorta contained minimal levels of latent or active TGF-ß1 (Table 2). The total amount (latent and active) of TGF-ß1 minimally changed in atherosclerotic plaques of apoE-/-:PAI-1+/+ mice but increased 
20-fold in apoE-/-:PAI-1-/- lesions after 25 weeks of the cholesterol diet (Table 2). Approximately half the amount of TGF-ß1 was active in early and advanced apoE-/-:PAI-1+/+ plaques. This was also true for early apoE-/-:PAI-1-/- plaques, but the levels of active TGF-ß1 were 2-fold higher than those of latent TGF-ß1 in advanced apoE-/-:PAI-1-/- plaques (Table 2).
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To confirm that the increased levels of active TGF-ß1 in apoE-/-:PAI-1-/- plaques were related to the PAI-1 genotype and could be restored by elevated plasma PAI-1 levels, we intravenously injected the PAI-1–expressing adenovirus AdCMVPAI-1 or a control AdRR5 virus in apoE-/-:PAI-1-/- mice. This resulted in PAI-1 production by the liver and increased the plasma PAI-1 levels to 1.1±0.7 µg/mL after 6 days. Plaque levels of active TGF-ß1 were 25±3 pg/µg protein after AdRR5 versus 6±1 pg/µg protein after AdCMVPAI-1 (P<0.05, n=10). Because adenoviral gene transfer only transiently increased plasma PAI-1 levels, long-term effects on plaque growth and matrix deposition could not be addressed.
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Discussion |
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PAI-1 is generally believed to enhance atherosclerotic plaque growth in humans by reducing fibrin clearance in plaques. Eitzman et al7 observed that plaque growth was not affected by PAI-1 deficiency in the aortic root and was reduced in the carotid bifurcation. Unexpectedly, we observed that PAI-1 deficiency resulted in larger plaques at all sites of the vasculature, but only at advanced stages of atherosclerosis. Notably, loss of PAI-1 increased plaque growth in mice fed a normal chow or cholesterol-rich diet. Because Eitzman et al used congenic C57Bl/6 PAI-1-/- mice and we used PAI-1-/- mice with a mixed C57Bl/6x129Sv/J genetic background, the differences between the previous and present findings are likely attributable to differences in genetic background. Every gene, also a disease candidate gene such as PAI-1, is potentially influenced by modifier genes. In a congenic background, a particular set of modifier genes is present. Depending on which modifier genes are present and how strongly the gene candidate is influenced by these modifier genes, the phenotype of a mutant mouse can significantly vary (and even exhibit opposite features) in different congenic backgrounds. Several examples of such influences have been previously reported.18 Conversely, in a mixed genetic background, modifier genes of either background will codetermine the phenotype of a gene-inactivated mouse. Therefore, the phenotypic differences of PAI-1 deficiency in distinct genetic backgrounds suggest that the role of PAI-1 is influenced by other modifier genes. This has important consequences for our understanding of the atherosclerotic risk associated with PAI-1 in humans, because humans have mixed genetic backgrounds. In fact, the observation that PAI-1 is not always associated with the extent of atherosclerosis19 suggests that its role in plaque growth is influenced by other modifier genes. Future genetic profiling in mice or humans might aid in identifying such PAI-1 modifier genes.
How would genetic differences cause different PAI-1-/- phenotypes? Although a final answer remains to be determined, our findings suggest some possible mechanisms. In humans and in some atherosclerotic mouse models, impaired fibrinolysis may promote fibrin accumulation in plaques or vascular lesions, such as those found in plasminogen-deficient mice.9 Conversely, increased fibrinolysis reduced the growth of fibrin-rich vascular lesions in PAI-1-/- mice.7,20,21 Because fibrin content was minimal in lesions of either genotype in the present study, PAI-1 likely suppressed plaque growth via mechanisms unrelated to its antifibrinolytic properties. Possible mechanisms contributing to the increased plaque growth in PAI-1-/- mice may relate to increased deposition of collagen and other matrix components and the accumulation of macrophages. We speculate that the involvement of fibrin-related or -unrelated mechanisms may be determined by the genetic background.
One reason why plaque growth was increased in PAI-1-/- mice relates to the increased accumulation of macrophages, which might be attributable to several mechanisms. For instance, the resultant increase in plasmin and MMP-dependent proteolysis could cause the breakdown of matrix components and pave a way for infiltrating cells. Alternatively, in the absence of PAI-1, integrin-dependent cellular migration might be enhanced.3 Increased levels of TGF-ß1 could also enhance macrophage infiltration by its chemoattractive properties, but its possible (dual) role is further discussed below. Whatever the mechanism, a role for the plasminogen system in cellular migration has been observed in numerous previous gene-targeting studies.22
A second mechanism contributing to the increased plaque growth in PAI-1-/- mice relates to the more abundant deposition of collagen, versican, fibronectin, and tenascin-C. Increased deposition of matrix components is difficult to reconcile with the elevated plasmin and MMP levels, because increased proteolysis would cause breakdown, not deposition, of matrix. This suggests that indirect mechanisms are operational and responsible for matrix deposition. Because (myo)fibroblasts produce matrix components, their increased accumulation could be responsible for the increased matrix deposition. However, the density of SMA-positive cells was reduced in the absence of PAI-1, which could result from the inhibitory effect of TGF-ß1 on SMC proliferation.23 Nonetheless, we cannot exclude the possibility that we might have underestimated their accumulation, because not all (myo)fibroblasts express this marker. Another possibility is that the increased matrix deposition in the absence of PAI-1 might be attributable to increased TGF-ß1 levels, because this growth factor is a known stimulator of matrix deposition by (myo)fi-broblasts and can be activated and liberated from matrix stores by plasmin (besides thrombospondin and acidic microenvironments).4,24 Remarkably, levels of active TGF-ß1 were elevated at advanced stages of plaque progression (eg, when the genotypic differences in matrix deposition became apparent). Because active TGF-ß1 levels were restored by adenoviral PAI-1 gene transfer in PAI-1-/- mice, the PAI-1 genotype appears closely linked to active TGF-ß1 levels. Several plaque cell types could produce TGF-ß1, and the increased accumulation of macrophages likely contributed to the increased TGF-ß1 levels in PAI-1-/- plaques.24 TGF-ß1, once active, could further upregulate its own expression in an autocrine feedback loop (see Lawrence24 and references therein). Nonetheless, even though our findings are not inconsistent with an increased profibrotic effect of TGF-ß1 in the absence of PAI-1, this hypothesis remains hypothetical because TGF-ß1 has pleiotropic effects on matrix remodeling (see below). If TGF-ß1 would predominantly stimulate matrix deposition, PAI-1 might constitute a negative-feedback pathway to control plaque growth by preventing excessive generation of TGF-ß1 and matrix deposition.
Although matrix was more abundant in the absence of PAI-1, ultrastructural and immunocytochemical signs of collagen degradation were also observed in apoE-/-:PAI-1-/- plaques. Although at first glance inconsistent, these findings stress the complex and often opposite role proteinases may have in biological processes in vivo. We recently documented an even more striking example of the dual role of proteinases in tissue degradation (cardiac rupture) and building (infarct healing) after coronary ligation.12 Increased MMP-dependent proteolysis was likely responsible for collagen degradation, as evidenced by the presence of the COL2-3/4cshort epitope, which is generated only by active collagenases. Because plasmin levels were increased in PAI-1–deficient plaques, they could also have contributed by activating the proform of these MMPs.10 The marked infiltration of macrophages is consistent with matrix destruction, because these cells are known sources of proteinases.10 An intriguing question is whether the abundant amounts of fibronectin, tenascin-C, and versican (bulky matrix components that are more abundant in remodeling wounds than in stable scars)25 contributed to the formation of a loose and disorganized collagen meshwork in PAI-1-/- mice by random interposition between collagen fibrils (see Sayani et al26 and references therein).
Although active TGF-ß1 levels were significantly increased in the absence of PAI-1, it should be stressed that TGF-ß1 is a pleiotropic growth factor with dual (even opposite) effects on matrix remodeling and macrophage infiltration, which are dependent on its concentration and the activation differentiation status, passage, and density of target cells, along with a number of additional parameters.27–30 For instance, TGF-ß1 enhances matrix production and suppresses matrix degradation, but this growth factor has also been implicated in matrix degradation by upregulating MMPs.29,30 Therefore, the precise role of this growth factor in the complex environment of an atherosclerotic plaque in PAI-1-/- mice remains to be determined.
PAI-1 was primarily expressed in myofibroblasts and, minimally, in macrophages (in plaques in vivo and in cultured cells in vitro). The significant contribution of PAI-1 production by myofibroblasts was further revealed by bone marrow transplantation studies indicating that PAI-1 deficiency in host-derived myofibroblasts but not in donor-derived macrophages enlarged plaque size and collagen deposition. These findings suggest that plasminogen activators and their inhibitors have cell type–specific effects on plaque growth.
In conclusion, in addition to its positive effects on plaque growth by impairing fibrin degradation, PAI-1 may also suppress advanced atherosclerosis by affecting cellular infiltration and matrix accumulation.
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Acknowledgments |
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This work was supported by the British Heart Foundation (grant PG/99019) and by an Institute voor de aanmocdiging van innovatic door Wetenschap en technologie Vlaanderen (grant VLAB/COT-008). A.L. and E.S. are Fonds voor Wetenschappelijk Onderzoek, Vlaanderen research assistants. The authors thank M. De Mol, B. Hermans, S. Jansen, A. Manderveld, K. Maris, B. Vanwetswinkel, S. Wyns, A. Vandenhoeck (Center for Transgene Technology and Gene Therapy, KU, Leuven) and W. Landuyt (Radiobiology, KU Leuven) for technical support.
Received October 16, 2001; accepted October 31, 2001.
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