Local Plasminogen Activator Inhibitor Type 1 Overexpression in Rat Carotid Artery Enhances Thrombosis and Endothelial Regeneration While Inhibiting Intimal Thickening
Abstract—Elevated levels of plasminogen activator inhibitor type 1 (PAI-1) are found in advanced atherosclerotic plaque compared with normal vessel and may contribute to plaque progression and complications associated with plaque rupture. Increased expression of PAI-1 probably contributes to the thrombotic properties of advanced atherosclerotic plaque by impeding plasmin generation and degradation of fibrin. To test this hypothesis, we have deliberately created synthetic neointimas by seeding onto the denuded luminal surface of rat carotid arteries smooth muscle cells transduced with replication-defective retrovirus encoding rat PAI-1. This cell-based gene transfer method results in stable, long-term, and localized gene expression. PAI-1 overexpression increases mural thrombus accumulation at 4 days but decreases neointimal area by 30% and 25% at 1 week and 2 weeks, respectively. PAI-1 overexpression accelerates reendothelialization of injured arteries compared with control arteries at 1 week, 2 weeks, and 1 month. PAI-1 overexpression does not alter matrix accumulation at 1 week. Increased PAI-1 expression in the rat carotid artery enhances thrombosis and endothelial regeneration while inhibiting intimal thickening. These results suggest that PAI-1 could play a direct role in the development of advanced atherosclerotic plaque and in the repair of the diseased vessel after fibrous cap disruption.
- Received August 25, 1999.
- Accepted October 7, 1999.
Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiological inhibitor of the plasminogen activator system and thereby blocks the conversion of plasminogen to plasmin. The plasminogen activator (PA) system is composed of urokinase PA (uPA) and tissue PA that proteolytically convert plasminogen to the serine protease plasmin. Components of the PA system are localized to the cell surface by receptors or extracellular matrix binding sites.
Increased expression of PAI-1 has been demonstrated in atherosclerotic arteries.1 2 PAI-1 is elevated in mesenchymal-appearing intimal cells at the base of the plaque and in the necrotic core. Its function in the advanced atherosclerotic lesion is not known. PAI-1 may limit the fibrinolytic capacity of the plaque.3 It also might modulate cellular proliferation or migration in the lesion through changes in matrix composition and growth factor release. The effects of PAI-1 are likely to be exerted locally in view of the fact that a majority of patients with generalized atherosclerosis have normal plasma fibrinolytic profiles.4 5
The various biological effects of PAI-1 generate a dilemma.6 On the one hand, local PAI-1 overexpression should enhance fibrin accumulation and thereby contribute to the growth of atherosclerotic lesions.3 7 On the other hand, increased PAI-1 inhibits smooth muscle cell (SMC) migration and the formation of neointima in injured mouse vessels; this result supports the hypothesis that PAI-1 overexpression retards the growth of atherosclerotic lesions.8 9
In the following experiments, we have attempted to define the role of PAI-1 in the intima to resolve this dilemma and to understand how PAI-1 may influence the biology of the advanced atherosclerotic lesion. We have developed a rat model for this purpose. This model makes use of a novel approach that involves localized gene overexpression and is well suited for the study of genes that may be involved in atherosclerotic progression. Localized gene overexpression is achieved by constructing a synthetic neointima in the carotid artery by seeding rat SMCs transduced in vitro with a retroviral vector containing the gene of interest. We have previously demonstrated that this cell-seeding technique gives long-term and biologically significant gene expression in the intima.10
In the present study, we demonstrate that increased PAI-1 expression in the rat carotid artery inhibits neointimal formation, increases platelet accumulation, and accelerates endothelialization of the injured carotid artery. Our findings demonstrate that PAI-1 plays an important role in neointimal formation in the rat and suggests that PAI-1 may influence the biology of the atherosclerotic lesion in humans.
Construction of Rat PAI-1 Retroviral Vector
Rat PAI-1 cDNA, supplied by T. Gelherter (University of Michigan, Ann Arbor),11 was modified to have a Kozak12 consensus sequence (ACCATGG) by polymerase chain reaction with appropriate primers. The PAI-1 coding sequence was introduced into the unique HpaI site of the retroviral vector (LXSN)13 to construct the recombinant PAI-1 vector (LPAISN, Figure 1⇓). The LXSN vector without the PAI-1 coding region was used as a control. DNA sequencing was performed to verify the orientation and integrity of the retroviral construct.
Rat SMC Transduction
Viral packaging was performed according to Miller and Rosman.13 Two packaging cell lines were used. PE501 cells were transfected with the retroviral constructs and used to produce replication-defective retrovirus to infect the amphotropic packaging cell line PA317. Viral titers were ≈1×106 plaque-forming units per milliliter for LPAISN and LXSN. Rat SMCs were enzymatically isolated from male Fischer 344 rat aortas and then cultured in DMEM with 10% FBS. Supernatant harvested at 16 hours from the packaging cell lines was used to infect rat SMCs. Colonies of G418 (1 mg/mL, GIBCO/BRL)–resistant cells were isolated.
RNA Isolation and Northern Analysis
Total cellular RNA was isolated from SMCs grown in DMEM containing 10% FBS by using guanidinium isothiocyanate–phenol–chloroform extraction as previously described.14 Isolated RNA was resuspended in 0.5% SDS and quantified spectrophotometrically. RNA samples were separated in a 1% agarose/formaldehyde gel.15 RNA was transferred to a Zeta-probe nylon membrane (Bio-Rad) as described by the manufacturer and cross-linked to the membrane by using UV light (Stratagene). Filter hybridizations were carried out as described by Church and Gilbert.16 rRNA bands (18S) were visualized with UV light to document equal loading.
PAI-1 Activity Assay In Vitro and Carotid Extracts
PAI-1 activity was determined by reverse zymography as previously described.17 Equal amounts of protein from cells grown in culture or from homogenized carotid arteries were separated on an SDS-polyacrylamide gel, and the gel was overlaid onto an agar gel containing plasminogen, uPA, and casein. PAI-1 inhibits the caseinolytic activity of plasminogen activated by urokinase. The reverse zymogram was developed at 4°C overnight and then at 37°C for 3 to 6 hours. Dark-field illumination makes regions of undigested casein appear white when viewed on a black background; quantification of bands was performed by densitometry.
Zymography was also performed on carotid extracts by using the methods described above, except that uPA was omitted in the agar gel. Amiloride (1 mmol/L final concentration, Sigma Chemical Co) was added to some agar underlays to inhibit uPA activity. Rat urine was collected from euthanized rats by use of a hypodermic needle and syringe.
Migration, Attachment, and Proliferation In Vitro
The ability of rat SMCs to invade the matrix and migrate toward a chemoattractant was assayed by using 2 experimental approaches. The first approach made use of a modified Boyden chamber method that involved a 48-well microchemotaxis chamber (Neuro Probe) and polycarbonate filters (Nucleopore Corp) with 10-μm pores. The filters were precoated with 2.7 μg per well of basement membrane matrix (Matrigel, Collaborative Research) in 0.5× PBS and dried overnight. Thirty minutes before use, the matrix was reconstituted in 0.5× PBS, and the filter was assembled on top of the lower chamber containing 0.67 nmol/L platelet-derived growth factor (PDGF)-BB (Zymogenetics). Cultured SMCs were trypsinized, washed 3 times in serum-free medium, and resuspended at a concentration of 5×105 cells per milliliter in serum-free DMEM, and then 40 μL of the cell suspension was added to the upper chamber. The chemotaxis chamber was incubated for 5 hours at 37°C under 5% CO2. At the end of the assay, the upper side of the filter was scraped clean, and the cells that had migrated to the bottom of the filter were stained with Diff-Quick (Baxter) and quantified.
Attachment assays were conducted as described for the Boyden chamber above, but no chemoattractant was added to the lower wells. After 60 minutes of incubation, the filters were gently washed 3 times in 1× PBS, and the cells were counted on the upper side of the filter.
For longer term migration studies, a thick layer of collagen over pluronic gel containing PDGF-BB was used. Two milliliters of Pluronic gel (F-127, Sigma) per well in a 24-well plate containing 0.67 nmol/L PDGF-BB was allowed to gel at 37°C for 2 hours before adding 1 mL of chilled Vitrogen (Cohesion Technologies), which was allowed to polymerize for 2 hours at 37°C. SMCs were trypsinized (0.05% trypsin), counted, and suspended in DMEM containing 10% FBS (GIBCO/BRL) with 5 mmol/L hydroxyurea (Sigma) to inhibit proliferation. Rabbit anti-rat PAI-1 IgG (5 μg/mL, No. 1062, American Diagnostica) or control rabbit IgG (5 μg/mL, Santa Cruz Biotechnology) was added to the cells before they were plated on Vitrogen. Cells were plated on the surface of the polymerized Vitrogen at a density of 1.0×103 cells per square millimeter. After 48 hours, the medium was removed, and each well was fixed with 100% methanol and stained with Diff-Quick stain. The number of cells that had migrated into the collagen was determined by phase microscopy in 4 fields per well. The experiments were performed in duplicate or triplicate 4 times.
To measure growth rates in vitro, 103 cells per square centimeter were cultured in DMEM containing 10% FBS (GIBCO) in triplicate and then at various time points were trypsinized and counted in a hemocytometer.
SMC Seeding In Vivo
Male Fischer 344 rats (250 to 300 g) were anesthetized,18 and the left common artery was surgically exposed and stripped of endothelium by the passage of a balloon catheter. Transduced SMCs were seeded onto the luminal surface as previously described.10 SMCs (105) were infused into the carotid artery and allowed to attach for 10 minutes.
At various times, rats were killed, and the carotid arteries were either removed or surgically exposed for further analysis. The rats were cared for according to the Principles of Laboratory Animal Care (formulated by the National Society of Medical Research) and the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985). One day before the rats were killed, one 5-bromo-2′-deoxyuridine (BrdU) tablet (50 mg, Boehringer-Mannheim) was implanted subcutaneously to measure proliferation.
PAI Activity in Carotid Extracts
Plasmin generation was measured as an indicator of net PAI/PA activity in seeded carotid arteries essentially as described previously.19 20 Carotid arteries seeded with PAI-1 (LPAISN) or control SMCs (LXSN) were excised and homogenized in cold buffer (50 mmol/L Tris, pH 9.0). Protein (10 μg) was added to human Glu-type plasminogen (0.5 μmol/L final, American Diagnostica), fibrinogen (100 μg/mL final, American Diagnostica), and a chromogenic substrate of plasmin S-2251 (400 nmol/L final, Chromogenix), and the optical density at 405 nm was measured after 30 minutes.
Tissue Preparation, Immunohistochemistry, Morphometry, and Endothelial Staining
At various time points, PAI-1–overexpressing and control cell–seeded carotid arteries were flushed clear of blood with lactated Ringer’s solution (Baxter) and perfusion-fixed with 4% formalin in PBS (Fisher Scientific) at 120 mm Hg pressure. Endothelial regeneration was visualized by Evans blue staining. Evans blue dye (60 mg/kg, E-2129, Sigma) was injected in the tail vein 60 minutes before the animals were killed.21 The blue-white boundary defines the limit of endothelial ingrowth. Endothelial ingrowth was measured from the proximal and distal ends of the injured carotid by using the carotid bifurcation and proximal tie as reference markers. When excised for measurement, the injured region of the carotid artery is ≈10 mm long. Vessels were embedded in paraffin and cross-sectioned for histology and immunohistochemistry.
Proliferating SMCs were quantified on histological cross sections after immunostaining for BrdU. BrdU was detected immunohistochemically and reported as percent BrdU-positive cells in the intima.22
Morphometric analysis of the lumen, intima, and media was performed by using a camera lucida linked to a computer-driven digitizing pad and software (Opelco)
Carotid arteries were flushed with Ringer’s solution and then perfusion-fixed at 120 mm Hg with 4% paraformaldehyde for 3 minutes. Tissue intended for transmission electron microscopy was embedded in Epon (Polysciences) for sectioning and subsequent transmission electron microscopy analysis on a JEOL 100B (Japan Optics Electron Laboratory) at 60 kV. Tissue intended for scanning electron microscopy was pinned out to expose the luminal surface and fixed in 2% osmium tetroxide before sputter coating (n=3 in each group at 1 and 2 weeks, n=2 in each group at 4 weeks.) The entire seeded area and several millimeters of carotid proximal to the seeded area were examined. Representative areas of minimal and maximal platelet accumulation were photographed.
Quantification of cell volume–to–matrix ratios were performed on cross sections of PAI-1 and control cell–seeded carotid arteries as described previously.23
All values are expressed as mean±SD. Comparisons between PAI-1 and control groups were made by Mann-Whitney nonparametric tests (SPSS, version 8.0.0). Statistical significance was set at P≤0.05 by 2-tailed tests.
Total RNA from PAI-1–overexpressing and control cells was analyzed by Northern blotting to verify expression of the PAI-1 cDNA (Figure⇑ I, published online at http://atvb. ahajournals.org/cgi/content/full/20/3/853/DC1). A 4.2-kb PAI-1 transcript was easily identified in PAI-1–overexpressing cells. This transcript was of the expected size and is composed of 3.1 kb of the retroviral vector sequence and 1.1 kb of rat PAI-1 open reading frame. The control vector alone and PAI-1–overexpressing SMCs show a band at 3.0 kb corresponding to the endogenous message. Endogenous PAI-1 mRNA is diminished when serum is withdrawn (data not shown). Clonal isolates of PAI-1–overexpressing SMCs had a similar expression of PAI-1 compared with pooled groups of transduced SMCs. Pooled groups of PAI-1–overexpressing SMCs were used for all experiments in the present study.
PAI-1–overexpressing SMCs have the same morphological and growth properties as control vector alone–transduced SMCs and passage-matched primary arterial SMC isolates. There were no significant differences in the growth rate of PAI-1–overexpressing SMCs compared with SMCs transduced with the control vector (Figure⇑ II, published online at http://atvb.ahajournals.org/cgi/content/full/20/3/853/DC1).
PAI-1 activity in cultured cells was measured by their ability to reduce plasmin generation in a reverse zymogram. There was an ≈4-fold increase in PA inhibitory activity in the PAI-1–overexpressing SMCs compared with control SMCs (Figure⇑ III, published online at http://atvb.ahajournals.org/cgi/content/full/20/3/853/DC1).
SMC migration through matrix is dependent on proteolytic activity.24 25 26 Therefore, we assayed the ability of PAI-1–overexpressing SMCs to migrate through Matrigel in a modified Boyden chamber. No significant differences were observed in migration over a 5-hour time period or in attachment to Matrigel (Figure⇑ IV, published online at http://atvb.ahajournals.org/cgi/content/full/20/3/853/DC1). In longer migration assays in which SMCs migrate into a collagen gel for 48 hours, PAI-1–overexpressing SMCs had reduced levels of migration compared with control SMCs. This reduced migration was reversed by the addition of a PAI-1–blocking antibody (Figure 2⇓). PAI-1–blocking antibodies did not significantly increase control SMC migration.
Effects of PAI-1 Overexpression on Neointima
PAI-1–overexpressing SMCs were seeded into the lumen of balloon-injured carotid arteries. Increased PAI-1 activity was independently confirmed by measuring net plasmin generation ex vivo. We have measured the residual active PA and amount of plasmin generated in the seeded carotid arteries and found that PAI-1 overexpression reduces local PA activity and subsequent plasmin generation in the carotid by ≈50%, as measured by chromogenic substrate S-2251 (Figure 3⇓). The extraction buffer used does not activate latent PAI-1, and the direct plasmin generation assay without SDS present gives us confidence that PAI-1 produced by the seeded SMCs is biologically active.
Carotids seeded with PAI-1–overexpressing SMCs have increased PAI-1 immunoreactivity compared with uninjured or control cell–seeded carotid arteries (Figure 4⇓). The media of all vessels shows little PAI-1 immunoreactivity. The neointima is composed of seeded and endogenous SMCs. The even distribution of PAI-1 immunoreactivity in the neointima suggests that PAI-1–overexpressing SMCs have migrated throughout the neointima and that not all PAI-1 immunoreactivity is cell-associated.
There was increased PA inhibitory activity in the PAI-1–seeded vessels at 1 week as measured by reverse zymography (Figure 5⇓, lanes 1 and 2). At earlier development times, the reverse zymograph showed faint PAI-1 activity in the control lane (data not shown). There was an ≈8-fold increase in PAI-1 activity in the PAI-1–overexpressing carotid extracts compared with control cell–seeded carotid arteries. Carotid arteries overexpressing PAI-1 did show an increase in a low molecular weight caseinolytic activity compared with control cell–seeded vessels (Figure 5⇓, lanes 3 and 4). This 28-kDa caseinolytic activity might be a fragment of uPA in view of the fact that it was inhibited with 1 mmol/L amiloride.
PAI-1 overexpression resulted in increased mural thrombus compared with either control cell–seeded carotid arteries (Figure 6⇓) or regions beyond the zone of PAI-1 seeding (data not shown). Areas of mural thrombus formation with clearly identifiable fibrin accumulation were seen only in PAI-1–overexpressing carotid arteries (Figure 6D⇓). This increase in platelet adhesion and mural thrombus persisted for 1 week. At 2 and 4 weeks, platelets and fibrin were not visible on the luminal surface.
PAI-1 overexpression decreased neointimal areas by 30% and 25% at 1 week and 2 weeks, respectively (Figure 7⇓). By 1 month, the intimal areas of the PAI-1–overexpressing and control carotid arteries were not significantly different. Medial areas and internal elastic lamina lengths were not significantly different at any time tested. There were also no significant differences in proliferation rates in vivo at 1, 7, and 14 days after seeding (Figure⇑ V, published online at http://atvb.ahajournals.org/cgi/content/full/20/3/853/DC1). To determine whether the loss of reduced intimal thickening at 1 month was due to a loss of gene expression, PAI-1 activity was measured by reverse zymography at 1 month. There was no apparent loss of elevated PAI-1 expression compared with control cell–seeded vessels or vessels receiving injury alone (data not shown).
Changes in the proteolytic balance could result in changes in extracellular matrix composition or volume. At early time points after cell seeding, we found differences in platelet and fibrin accumulation. There were no significant differences in the ratio of matrix to cell volume in the neointima between control cell– and PAI-1–seeded carotid arteries at 1 week (Figure⇑ VI, published online at http://atvb.ahajournals.org/cgi/content/full/20/3/853/DC1). Ultrastructural morphology within the neointima did not show any clear differences between control cell– and PAI-1–seeded carotid arteries, although the luminal surface of the PAI-1–overexpressing carotid arteries at 4 days had more platelets (data not shown).
Reendothelialization was accelerated in PAI-1–overexpressing neointimas. At early time points, the entire area of the PAI-10–overexpressing neointima was covered by patches of platelet-rich mural thrombus (Figure 6⇑). At later time points (2 weeks and 1 month), the surface was free of platelets, and the PAI-1–overexpressing neointima was rapidly reendothelialized (Figure 8⇓). In the injured rat carotid artery, endothelial cells migrate and proliferate from the proximal and distal uninjured regions adjoining the denuded areas. PAI-1–overexpressing carotid arteries had a 2-fold increase in endothelial coverage of the seeded area at 2 weeks and 4 weeks compared with control cell–seeded vessels. The control cell–seeded vessel was reendothelialized at a rate similar to that of the unseeded balloon-injured vessel (data not shown).
Localized and stable overexpression of PAI-1 was achieved by using a novel cell-based gene transfer technique involving SMCs seeded onto denuded carotid arteries. Increased PAI-1 expression altered the development of the neointima after arterial injury. In the present study, we report that PAI-1 overexpression in the neointima enhanced early thrombosis, reduced intimal thickening, and accelerated endothelial regrowth. These results support the conclusion that PAI-1 plays a direct role in regulating intimal formation in injured arteries and may play a role in atherosclerosis and restenosis.
Effects of PAI-1 Overexpression on Thrombosis
At early times after arterial injury in the rat, there is some platelet adherence along with limited fibrin deposition on the luminal surface that clears after 1 day. PAI-1 is transiently induced during the first 24 hours after injury and may serve to limit fibrinolysis.17 PAI-1 overexpression during this early repair period extended the duration of platelet adhesion and fibrin accumulation through 1 week.
Increased platelet and fibrin accumulation does not persist beyond 1 week, even though plasmin generation remains suppressed. This result suggests either that increased levels of PAI-1 are able only to slow the removal of fibrin or that compensatory increases in PA activity counteract increased PAI-1. We detected no changes in uPA or tissue PA in PAI-1–overexpressing carotid arteries; however, we did see the appearance of a 28-kDa caseinolytic activity. This 28-kDa caseinase is likely to be a low molecular weight fragment of uPA, because it is inactivated by amiloride. We conclude that a sustained increase in PAI-1 is sufficient to delay but not prevent the removal of fibrin on the luminal surface.
PAI-1 overexpression in the injured rat carotid artery accelerated reendothelialization. The mechanism for accelerated regrowth is not known, but several previous publications have addressed this question. Lindner et al27 compared gentle filament denudation with balloon catheter injury and found that gentle denudation increased platelet adherence and accelerated endothelial regrowth. Cell-based tissue factor overexpression increases platelet and fibrin accumulation and also accelerates endothelial regrowth, suggesting that platelets or fibrin could stimulate endothelial regrowth.27a However, Carmeliet et al8 compared endothelial regrowth rates in PAI-1–deficient mice and mice with normal PAI-1 levels and did not find any differences at 1 week after injury. This result suggests that PAI-1 does not have a direct role in endothelial regrowth, at least in the murine electric injury model. Increased PAI-1 expression in vitro inhibits endothelial migration.28 Taking these potentially conflicting results together, we believe that increased PAI-1 could accelerate endothelial regrowth by increasing the duration or amount of platelet adherence to the vessel wall or by increasing the amount of fibrin on the luminal surface after injury. The lack of visible platelets or fibrin at any time after 1 week in either tissue factor–overexpressing, PAI-1–overexpressing, or gently denuded carotid arteries combined with prolonged increases in endothelial migration suggests that fibrin breakdown products or platelet factors released early after injury could continue to drive endothelial migration at later time points.
Intimal areas were significantly reduced at 1 and 2 weeks after injury in PAI-1–overexpressing carotid arteries. At these times, SMCs are migrating from the media to the intima. It is interesting to note that treatments that reduce migration through a wide variety of methods tend to block intimal growth for ≈2 weeks after injury. After this time, the intimas seem to catch up with controls. Carotid arteries overexpressing PAI-1 also follow this same pattern, which might be attributed to a decrease in migration followed by compensatory low level increases in proliferation or matrix accumulation over a several-week period. Proliferation indices (BrdU labeling) at 1, 7, and 14 days were the same in PAI-1–overexpressing and LXSN control cell–seeded arteries (Figure⇑ V). This finding is consistent with other experiments in which inhibitors of PAs or matrix metalloproteinases (tranexamic acid, tissue inhibitor of metalloproteinases type 1, and BB-94) significantly inhibited SMC migration and reduced neointimal formation up to 2 weeks but did not sustain this effect at later times.29 30 In our short-term in vitro migration assays, PAI-1–overexpressing SMCs migrated at the same rate as did LXSN control SMCs. In longer term experiments, PAI-1–overexpressing SMCs exhibited decreased rates of migration, and this reduced migration was reversed when PAI-1 was inhibited with a blocking antibody.
Increased PAI-1 expression could alter the proteolytic balance in the arterial wall, resulting in changes in the ratio of cells to the surrounding matrix. PAI-1–overexpressing carotid arteries were examined at 1 week by transmission electron microscopy and histochemistry for relative changes in cell and matrix components. At this time point, we did not detect any changes in cell-to-matrix ratios or extracellular matrix composition between PAI-1–overexpressing SMCs and LXSN control cell–seeded carotid arteries.
Our results from overexpressing PAI-1 in the rat carotid artery confirm previous reports by Carmeliet and colleagues8 9 showing that PAI-1 can inhibit intimal formation after electrical arterial injury in mice. However, our experiments also demonstrate significant differences, including the degree of inhibition and effects on endothelial regrowth. These differences may be due to the systemic release of PAI-1, lack of sustained expression in adenovirus-mediated PAI-1 expression in mice, and the increased severity of the electrical injury.
Relevance of the Rat Model to Advanced Atherosclerosis
In the present study, we demonstrate that localized overexpression of PAI-1 can modulate intimal biology. Our studies might also help resolve the dilemma posed in the introduction.6 Overexpression of PAI-1 in the intima models several aspects of the human lesion, including prolonged elevation of PAI-1 localized to the intima and increased mural thrombus. PAI-1 might perform the same functions in the atherosclerotic plaque as in the intima generated by cell seeding. It might also contribute to plaque fragility by preventing SMC migration into the fibrous cap.6 After the fibrous cap ruptures, it would be expected to prevent dissolution of the thrombus formed at the site of plaque rupture. In addition, it might encourage endothelial regeneration over the disrupted intima but suppress SMC migration and intimal hyperplasia. In summary, PAI-1 expression might encourage rapid repair and limit scarring at the risk of increasing thrombotic complications at sites of vascular injury.
This study was supported by National Institutes of Health (NIH) grant HL-52459. D.H. was supported by NIH training grant T32 HL-07312. We thank Dr Thomas D. Gelehrter (University of Michigan, Ann Arbor) for the rat PAI-1 cDNA.
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