© 1997 American Heart Association, Inc.
Articles |
Changes in Arterial Expression of Fibrinolytic System Proteins in Atherogenesis
From the Departments of Medicine, Pathology (D.J.T.) and Surgery (J.C.R., B.J.L.), University of Vermont, Burlington, Vt.
Correspondence to David J. Schneider, MD, Department of Medicine, Cardiovascular Division, E217 Given Building, University of Vermont, Burlington, Vt. E-mail djschnei{at}zoo.uvm.edu
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Abstract |
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Abstract Plasminogen activators (PAs) and their inhibitor, plasminogen activator inhibitor type-1 (PAI-1), have been implicated in modulation of luminal fibrinolysis and mural proteolysis contributing to atherogenesis.
Expression of PAs/PAI-1 (normalized to extracted tissue protein)
was delineated by assays of conditioned media and of extracts from
walls of human arterial segments in culture.
Arterial specimens (n=39 from 26 subjects) were divided
into four groups: normal (n=14), fatty streak (n=6), moderate
atherosclerosis (mural thickening with <70% lumen
obstruction, n=5), and severe atherosclerosis (mural
thickening with >70% lumen obstruction, n=14). Paired samples from
the same individual comprising a normal arterial segment
and an atherosclerotic segment were evaluated also. A fourfold molar
excess in PAI-1:t-PA was seen in conditioned media from samples with
any evidence of atherosclerosis compared with normal
specimens (normal 21±4, diseased 82±21, P
.05).
Compared with normal pairs, the tissue content of PAI-1 (ng) was
increased in fatty streak lesions (n=3, normal 35±12, fatty streak
50±8, P.05); stable to decreased in moderate
atherosclerosis (n=3, normal 34±3, moderate 22±7,
P=.16); and increased in severe
atherosclerosis (n=6, normal 48±9, severe 85±19,
P.05). The tissue content of PAs (ng), though not
increased in fatty streak lesions, was elevated in moderately and
severely atherosclerotic segments (normal 0.7±0.2, moderate 1.6±0.1;
normal 0.8±0.3, severe 2.1±0.3, P.05 for each
comparison).
Atherogenesis is associated with decreased luminal fibrinolytic capacity that may exacerbate thrombosis. Decreased mural proteolysis in early atherogenesis may exacerbate matrix accumulation. Increased mural proteolysis later is associated with, and may potentiate, smooth muscle cell migration and proliferation.
Key Words: atherosclerosis • plasminogen activators • plasminogen activator inhibitor type I • arterial wall
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Introduction |
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Atherogenesis involves deposition of lipoproteins, chemotaxis of inflammatory cells (particularly monocytes), migration and proliferation of smooth muscle cells, and accumulation of extracellular matrix and is often associated with thrombosis.1,2 Accumulation of lipoproteins and extracellular matrix within the arterial wall predominates in early lesions.3–5 Subsequently, proliferation and migration of smooth muscle cells, chemotaxis of macrophages, and ultimately fibrosis and calcification result in mural thickening and reduced luminal cross-sectional area.1–5 Altered expression of fibrinolytic system proteins may potentiate atherogenesis by modifying the extent and persistence of thrombi in the arterial lumen and vascular smooth muscle cell proliferation and migration and the accumulation or degradation of extracellular matrix in the walls.6–8
Thrombosis and deposition of fibrin(ogen) may accelerate atherogenesis by exposing vascular luminal surfaces to clot associated mitogens such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-ß), and thrombin.9 Sequelae of mural exposure to thrombi and fibrin include disorganization of endothelial cells,10 increased vascular permeability,11 smooth muscle cell migration and proliferation,12–16 and chemotaxis of monocytes/macrophages.17 The local extent and persistence of thrombi are influenced by vascular wall plasminogen activators, particularly tissue type plasminogen activator (t-PA). Urokinase type plasminogen activator (u-PA) is involved primarily in extracellular proteolysis occurring in tissues. An important physiological inhibitor of both t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1).18 Arterial constituents, particularly endothelial cells, are thought to be a primary source of endogenous t-PA and PAI-1.19,20 Accordingly, intraluminal fibrinolysis in response to thrombosis depends on a dynamic equilibrium between local concentrations of t-PA and PAI-1.
Previous studies describing arterial expression of fibrinolytic system proteins have evaluated tissue expression in arterial segments obtained primarily at autopsy.21–24 Two seemingly contradictory views of the presence and significance of mural fibrinolytic system proteins have emerged. According to the first, increased mural expression of PAI-1 potentiates atherogenesis by impairing degradation of extracellular matrix.22,23 According to the second, increased mural expression of plasminogen activators potentiates macrophage migration and smooth muscle cell proliferation and migration.6,8,21 The present study was designed to delineate potential roles and interrelationships of both.
Freshly obtained arterial segments from surgical specimens were maintained in organ culture for up to 4 days to delineate expression of fibrinolytic system proteins in media and within the arterial tissue. The dynamic expression of fibrinolytic system proteins was characterized, and the relative amount of the proteins was quantified in two compartments: 24-hour conditioned media and the arterial wall. The vessels that were studied ranged from morphologically normal to severely atherosclerotic. In addition, morphologically normal tissue and atherosclerotic tissue from the same individuals were evaluated for 12 subjects.
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Methods |
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Arterial Culture
Arteries, isolated from pathologic specimens obtained at the time of clinically mandated vascular surgical procedures and organ harvests, were used in protocols approved by the University of Vermont Institutional Review Board. A segment of artery was removed from the pathologic specimen in the operating room and placed immediately in a buffered solution containing glucose (sterile Hanks' buffer with 300 U/mL of penicillin and 300 µg/mL of streptomycin [Gibco BRL]) at 4°C. For instance, a segment of tibial artery was isolated from a lower extremity amputation, and segments of aorta and renal artery were trimmed from the vascular pedicle that was removed during an organ harvest. Arterial specimens used include the aorta, renal, internal mammary, inferior epigastric, femoral, popliteal, and tibial arteries. Subsequent processing of the specimens was performed in a laminar flow hood. After removal of adherent connective tissue, arterial segments were cut into rings that were 1 to 3 mm in width. Segments of aorta were cut into 3- to 5-mm pieces. The arterial rings or segments were then placed in Dulbecco's modified Eagle medium with Ham's nutrient mixture F12 ([DME/F12], Gibco BRL) with 1% bovine serum albumin (Sigma Chemical Co). The rings were cultured in a water-jacketed incubator at 37°C in an atmosphere enriched with 5% CO2. Media were changed daily. Media and reagents were screened regularly for detection of endotoxin contamination with the use of the Limulus amoebocyte lysate assay (Associates of Cape Cod) and did not exhibit contamination exceeding 0.01 ng/mL (100-fold less than concentrations required to stimulate expression of PAI-1).
35S-Methionine Incorporation
Arterial rings in culture were exposed to DME/F12
devoid of methionine for 30 minutes followed by a 1-hour exposure to
DME/F12 containing 0.1 mCi/mL of Transmet (ICN). Protein was extracted
from tissues as described below and precipitated with trichloroacetic
acid (10% w/v). Protein-associated radioactivity was quantified by
liquid scintillation spectrometry with Atomlight (NEN).
Extraction of Tissue Protein
Arterial rings were washed three times in PBS and
processed at 4°C. After wet weight had been obtained, the tissues
were pulverized in liquid nitrogen and homogenized in RIPA
buffer (10 mmol/L Tris pH 7.4, 150 mmol/L NaCl,
1% nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium
dodecyl sulfate, 1 mmol/L iodoacetamide, and 1
mmol/L phenylmethylsulfonyl fluoride [Sigma]).
Cellular debris were removed by centrifugation
(12 000g for 15 minutes). The supernatant fraction was
stored at -20°C until assays were performed. Total protein was
quantified conventionally with the method of
Bradford.25 Albumin was quantified by a
colorimetric assay with the use of bromcresol green
(Sigma). The albumin content in tissue lysates accounted for
less than 0.02% of the total protein.
Quantification of DNA
The DNA content in tissue lysates was quantified with the use of
a DNA-binding fluorochrome H33258.26
Concentrations of DNA were determined by comparing emission at a
wavelength of 450 nm with that of a known amount of calf thymus DNA
(excitation wavelength 365 nm).
Quantification of Fibrinolytic System Proteins
PAI-1 was measured by ELISA as previously
described27 with antibodies kindly provided by
Professor Desiré Collen. The interassay coefficient of variation
was 3.6%, and the intraassay coefficient of variation was 2.5%. t-PA
and u-PA were measured by ELISA (Imubind, [American
Diagnostica]). The coefficients of variation for t-PA
determinations were 9.5% (interassay) and 4.3% (intraassay). The
coefficients of variation for u-PA determination were 10% (interassay)
and 4.6% (intraassay). Functional activity of PAI-1 protein is labile,
particularly in nonserum-containing media. Accordingly, all
determinations of fibrinolytic system protein concentrations were
determined by ELISA. In each case, the ELISA for PAI-1, t-PA, and u-PA
recognized both free and complexed protein.
The recoveries of PAI-1, u-PA, and t-PA were determined under three conditions: (1) the addition of each protein to RIPA buffer containing 1% BSA and subsequent homogenization and processing in a manner similar to that used for arterial rings; (2) the addition of each protein to RIPA buffer containing a "normal" arterial segment with the "spiked" segment and then processing in the standard fashion; and (3) the addition of each protein to RIPA buffer containing an atherosclerotic arterial segment and the spiked segment and then processing in the standard fashion. In each case, there was no difference in the recovery of PAI-1, t-PA, or u-PA with processing in association with a normal segment or an atherosclerotic segment. The recoveries were 35±6% for PAI-1, 56±6% for t-PA, and 30±4% for u-PA. The lack of difference in recovery between normal and atherosclerotic arteries suggests that differences we observed in the experiments performed were not a reflection of differential recovery.
Statistics
Determination of significance of differences between paired
samples was performed with paired t tests. Differences
between groups were evaluated with Student's t tests.
Values are mean±SEM.
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Results |
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Tissue Characterization
On the basis of macroscopic appearance, arterial segments were categorized as (1) morphologically normal (normal), (2) fatty streak lesions present, (3) moderate atherosclerosis (evidence of mural thickening with less than 70% obstruction of the lumen), and (4) severe atherosclerosis (evidence of mural thickening with greater than 70% obstruction of the lumen). Arterial segments were obtained from 26 subjects, and 39 segments of arteries were cultured (normal=14, fatty streak=6, moderate=5, severe=14). The age, gender, and underlying medical conditions of patients from whom the arterial segments were taken are shown in Table 1
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Tissue viability was assessed on the basis of structural integrity and continued protein synthesis (35S-methionine incorporation). All arterial segments cultured were viewed macroscopically and microscopically with the use of an inverted microscope and did not show evidence of degradation. Severely atherosclerotic arteries contained substantial amounts of noncellular, calcified material.
No significant change in the rate of protein synthesis was seen through 4 days with either normal or atherosclerotic arteries in culture. The incorporation of 35S-methionine over 1 hour (CPM/µg wet weight) was 13.7±0.9 immediately after procurement, 10±1.9 after 1 day, 9.9±1.4 after 2 days, 9.9±1.5 after 3 days, and 11.2±2 after 4 days in culture (n=3 subjects from whom normal arteries were obtained and n=2 subjects from whom atherosclerotic arteries were obtained, P=NS).
The wet weight of arterial segments before processing and the DNA and total protein content of arterial tissue lysates were measured. Morphologically normal tissue had higher ratios of DNA to weight and protein to weight than those in severely atherosclerotic tissue (µg DNA/mg weight, normal: 0.27±0.04, atherosclerotic: 0.14±.03, P=.016; µg protein/mg weight, normal: 79±14, atherosclerotic: 22±3, P<.001). The decreased protein-to-weight and DNA-to-weight ratios in severely atherosclerotic tissue were associated with the macroscopic appearance of accumulation of fibrocalcific material. A decreased ratio of protein to DNA was determined also in lysates from severely atherosclerotic arteries compared with those from normal arteries (µg protein/µg DNA, normal: 0.29±0.05, atherosclerotic: 0.16±0.03, P=.027). All results were normalized with respect to the wet weight and tissue protein concentrations that were determined after culture.
Expression of Fibrinolytic System Proteins
Accumulation of PAI-1 and t-PA was determined daily in 24-hour
conditioned media (Tables 2 and 3). The
concentrations of PAI-1, t-PA, and u-PA were determined in the protein
extracts from the arterial segments after 3 or 4 days in
culture (Tables 2 and 3). Measurement of u-PA in the conditioned media
was not performed because this protein is generally not released into
media from vessel wall either in vivo or in vitro. In each case, the
total amount of each fibrinolytic system protein was quantified in
conditioned media and protein extracts, and the results were normalized
to the wet weight as well as to the amount of protein extracted from
the tissue.
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The accumulation of PAI-1 in 24-hour conditioned media from all
arterial segments with atherosclerotic changes was
increased regardless of severity and regardless of whether results were
normalized to wet weight or tissue protein (Table 2
). Decreased tissue
content of PAI-1 was observed in arterial segments with
moderate atherosclerosis compared with the tissue
content of PAI-1 in morphologically normal arterial
segments when normalized to wet weight and to tissue protein (Table 2).
By contrast, tissue content of PAI-1 in severely atherosclerotic
specimens was decreased when normalized to wet weight but tended to be
increased when normalized to tissue protein content (Table 2). This
difference is likely to be explained by the substantial amount of
calcified material and noncellular debris seen in the severely
atherosclerotic specimens.
The total amount of PAI-1 (average of media + tissue content) was
increased in the arterial segments with severe
atherosclerosis compared with normal segments (PAI-1
ng/mg protein, normal: 51.5±7.2, severe: 131.4±20.3,
P.001). A trend toward increased PAI-1 was observed in
fatty streak segments (fatty streak: 84.2±21.4, P=.079
compared with normal segments). By contrast, no increase in PAI-1 was
observed in the segments with moderate atherosclerosis
(moderate: 54.8±13.5, P=NS compared with that in normal
segments and P.05 compared with that in severely
atherosclerotic segments).
When plasminogen activator accumulation in
conditioned media and content in tissue were normalized to wet weight,
the only difference that was detected was a decrease in the mean
accumulation in media conditioned by segments with fatty streak lesions
compared with normal segments (Table 3). By contrast, both the
accumulation in conditioned media and content in tissue were increased
in arterial segments with moderate and severe
atherosclerosis when results were normalized to tissue
protein (Table 3). The total amount of plasminogen
activators (PA, accumulation of t-PA in conditioned media +
tissue content of t-PA and u-PA) was increased from
arterial segments with moderate and severe
atherosclerosis (ng PA/mg tissue protein, normal:
1.8±0.3, fatty streak: 2±0.6, moderate: 5.2±2.7,* severe:
6.1±1.1**; *P.05 and **P.001 compared with
normal).
We characterized expression of fibrinolytic proteins in two
compartments, in the conditioned media and in the vessel wall, to
assess both luminal fibrinolytic capacity (potentially influencing
activity in intraluminal blood in vivo) and mural proteolytic capacity.
This assessment is limited in that the accumulation of proteins in
conditioned media may reflect elaboration not only from the luminal
surface but also from the cut surface of the rings. Despite the
increase in both PAI-1 accumulation and t-PA accumulation in media
conditioned by moderately and severely atherosclerotic
arterial segments, the molar ratio of PAI-1:t-PA was
increased by fourfold when this ratio, in all diseased segments, was
compared with that in normal segments (molar ratio PAI-1:t-PA, normal:
21.1±3.5, all atherosclerotic segments: 82.3±20.7,
P.05).
Expression of Fibrinolytic System Proteins in Paired Samples From
the Same Patient
Because differences observed from arterial segments
may reflect systemic differences present in the patient from which
the sample was obtained, the expression of fibrinolytic system proteins
was quantified in conditioned media and in tissue lysates from normal
and diseased arterial segments obtained from the same
patient (Tables 4 and 5).
Three groups of paired specimens were
obtained from 12 subjects: (1) Specimens from three subjects with fatty
streak lesions were cultured such that sections from the same vessel
that appeared normal were cultured separately from sections of the
vessel that had fatty streak lesions. In each case, the specimen was
obtained from an organ donor without a medical history of important
illness. (2) Specimens from three subjects with moderate
atherosclerosis were compared in each case with
normal-appearing arterial samples from the same subjects.
The segments consisted of two pairs of vessels comprising a
morphologically normal inferior epigastric artery paired
with a segment of atherosclerotic aorta removed during repair of an
abdominal aortic aneurysm and a segment of a popliteal artery
with atherosclerosis involving approximately 50% of
the mural surface paired with a branch of the same vessel without
morphologic evidence of atherosclerosis. (3) Specimens
from six subjects with severe atherosclerosis were
compared with normal-appearing arterial segments from the
same subjects. In each case, the paired samples were removed during
peripheral vascular repair or a lower extremity amputation
necessitated by hypoperfusion. The normal arterial segments
were either branch vessels or collateral vessels in the vicinity of the
segment of diseased artery.
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Accumulation of PAI-1 was increased in conditioned media from the
diseased arterial segments (fatty streak, moderate, and
severe atherosclerosis) when compared with the
morphologically normal sample from the same subject. This increment was
present regardless of whether the results were normalized to wet
weight or tissue protein (Table 4). Similar to results observed with
the unpaired samples, the accumulation of plasminogen
activators in conditioned media was not increased when
normalized to wet weight of tissue; however, the arterial
segments with moderate and severe atherosclerosis
displayed increased accumulation of t-PA when compared with normal
segments taken from the same individual when results were normalized to
tissue protein (Table 5).
The content of PAI-1 in the tissue lysate from the arterial
segments with fatty streak lesions was increased in comparison with
normal tissue obtained from the same individual (Table 4 and Fig 1). No difference was detected in the
fibrinolytic (proteolytic) protein content in the fatty streak segments
compared with the content in normal segments (Table 5 and Fig 1). Thus,
the fibrinolytic (proteolytic) balance in the arterial wall
was decreased in arterial tissue with fatty streaks
compared with normal arterial tissue.
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The tissue content of PAI-1 in the moderately and severely
atherosclerotic specimens was not increased (it tended to be decreased)
when normalized to wet weight (Table 4). When normalized to tissue
protein, the tissue content of PAI-1 tended to be decreased in
moderately diseased segments and was increased in the severely
atherosclerotic arterial segments compared with that in its
normal counterpart (Table 4 and Fig 1). Thus, the tissue content of
PAI-1 associated with atherosclerosis appeared to
depend on severity, with increased PAI-1 present in fatty streaks
and severe atherosclerosis and stable to decreased
PAI-1 present in moderately diseased segments in which
proliferation appears to be present on the basis of macroscopic
appearance.
In comparison with the normal pair, the tissue content of t-PA and u-PA
was increased in the arterial specimens with moderate and
severe disease only when normalized to tissue protein (Table 5 and Fig 1). Thus, as a ratio of plasminogen activators
to all proteins present in moderately and severely atherosclerotic
arterial segments, the fibrinolytic (proteolytic) capacity
was increased in comparison with that in morphologically normal
arterial segments. These results are consistent
with a significant increase in tissue content of
plasminogen activators in segments from
moderately and severely atherosclerotic arteries compared with segments
from unpaired normal arteries.
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Discussion |
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Increased concentrations of PAI-1 in blood have been seen in diverse groups of patients with atherosclerosis.28–37 Activity of PAI-1 is increased in blood in patients early after acute myocardial infarction,30,31 late after infarction in young long-term survivors,32 and in patients with angiographically defined coronary artery disease.33 The ARIC study found that concentrations of PAI-1 in blood were increased in patients with early evidence of carotid atherosclerosis detected by ultrasonic interrogation.38
In the present study, results parallel to those in blood from subjects with atherosclerosis were observed. We found that the molar excess of the ratio of PAI-1:t-PA in conditioned media from morphologically normal vessels was increased fourfold in conditioned media from arterial segments with atherosclerotic changes that ranged from fatty streak lesions to severe atherosclerosis. Accordingly, the overall balance of the amount of fibrinolytic system proteins favored limitation of fibrinolysis. The fourfold excess of PAI-1 compared with t-PA could be anticipated to impair the fibrinolytic response to thrombi. A resulting exuberant formation and persistence of thrombi may occur and predispose atherosclerotic arteries to acute thrombotic events underlying acute coronary syndromes and potentially to increased exposure to mitogens found in thrombi. The increased PAI-1 in blood from patients with atherosclerosis may be secondary to increased expression of PAI-1 by arteries harboring atheroma, and the increased systemic concentrations of PAI-1 may be but the "tip of an iceberg" of increased and potentially widespread local expression of PAI-1 by atherosclerotic vessels. Because the increased elaboration of PAI-1 is present in arteries with the earliest manifestations of disease, it may be a marker or potentiator of atherogenesis, or both.
Plasminogen activation by t-PA and u-PA influences extracellular proteolysis by activation of proteolytic enzymes including collagenase and elastase.6–8,38,39 Thus, the mural balance of fibrinolytic system proteins could be expected to influence accumulation of extracellular matrix. The importance of mural expression of fibrinolytic system proteins is underscored by the production of these proteins by smooth muscle cells. In this study, we observed changes in the balance of fibrinolytic (proteolytic) capacity in relation to the degree of atherosclerosis present. Increased mural content of PAI-1 combined with no change in the content of plasminogen activators was seen when tissue with a fatty streak lesion was compared with adjacent morphologically normal tissue. By contrast, lesions with moderate macroscopic transmural thickening were associated with either a decrease or no change in the content of PAI-1 and an increase in the content of plasminogen activators when characterized as a fraction of the total protein present in the arterial segment. An analogous increase in tissue content of plasminogen activators has been seen in tissue taken from abdominal aortic aneurysms.40 Severely atherosclerotic segments were associated with increased tissue content of both plasminogen activators and PAI-1 as a fraction of total protein content.
Decreased tissue fibrinolysis (proteolysis) may accentuate accumulation of extracellular matrix by impairing matrix degradation.41 The predisposition to accumulation of extracellular matrix potentiated by PAI-1 appears to be predominant in early atherogenesis. This accumulation of extracellular matrix may provide a stimulus for subsequent migration and proliferation of smooth muscle cells and infiltration by macrophages.
By contrast, increased local concentrations of plasminogen activators may favor smooth muscle cell proliferation and migration.6,39 Local and cell surface expression of u-PA correlates with invasiveness and metastatic activity of human tumors.42 Arteries with moderate atherosclerosis characterized by cellular proliferation exhibited an increase in u-PA and t-PA without a comparable increase in PAI-1 content compared with morphologically normal arteries. Increased expression of plasminogen activators by smooth muscle cells appears to account for the bulk of the increment in tissue content of plasminogen activators.38,39 Severely atherosclerotic arterial segments display both an increase in plasminogen activators and PAI-1. The increments in both PAI-1 and plasminogen activators could potentiate both further proliferation of cellular elements and accumulation of noncellular debris in distinct zones within the arterial wall. In addition, the increased tissue content of plasminogen activators may predispose to plaque rupture, particularly in vulnerable shoulder regions with limited cellularity.43
Our results suggest that arterial segments remain viable in culture and that the expression of both plasminogen activators and PAI-1 in culture is parallel to changes in concentrations in the blood of subjects with atherosclerosis28–38 and to results observed with immunohistochemistry and in situ hybridization performed on intact arteries.19–24,40 The increased content of PAI-1 in conditioned media and in the arterial wall of specimens with fatty streak lesions and severe atherosclerosis are consistent with results observed in animal preparations after arterial injury.44,45
Arterial organ culture allows culture of an intact vessel wall. An advantage is that phenotypic changes that are observed when isolated mural constituents are cultured should be limited. In addition, the local milieu within the arterial wall is maintained. Limitations associated with the culture of arterial segments include the loss of exposure of the arterial lumen to circulating blood and the loss of pulsatile flow associated with a specific distending pressure.
Our preparation of specimens for culture included sectioning of the arterial segment. Injury associated with the preparation of specimens for culture may alter expression of plasminogen activators and PAI-1 as has been seen in animal preparations in vivo.44,45 Because all specimens were handled in a similar manner, however, differences between groups attributable to this potential artifact should be minimal.
In summary, arterial mural expression of fibrinolytic system proteins is altered in relation to the severity of atherosclerosis present. Decreased fibrinolytic capacity is likely to occur in fatty streak lesions and within distinct zones of end-stage lesions. Relative to the total protein content present in arterial segments, the content of plasminogen activators was increased in moderately and severely diseased segments with mural thickening associated with cellular proliferation. These mural changes occur under conditions in which a consistent excess of PAI-1 is elaborated from arteries with fatty streak or complex lesions. These results are consistent with the hypothesis that excess luminal PAI-1 accelerates atherogenesis by predisposing the arterial wall to exposure to excessive amounts of clot associated mitogens (eg, PDGF, TGF-ß, and thrombin) secondary to persistence of thrombi. Decreased mural proteolysis associated with fatty streaks may potentiate accumulation of extracellular matrix. An increase in complex lesions may facilitate vascular smooth muscle cell migration and proliferation, hallmarks of neointimalization in rapidly evolving lesions, and facilitate plaque rupture.43
Received August 12, 1996; accepted May 29, 1997.
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