© 1995 American Heart Association, Inc.
Articles |
Quantification of Plasminogen Activators and Their Inhibitors in the Aortic Vessel Wall in Relation to the Presence and Severity of Atherosclerotic Disease
From the Division of Hematology/Oncology, Department of Internal Medicine (T.P., M.S., J.K.), and the Institute of Pathology (K.W.S.), University of Münster (Germany), and the Gaubius Laboratory IVVO-TNO (J.J.E.), Leiden, the Netherlands.
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Abstract Increased expression of plasminogen activator inhibitor–1 (PAI-1) has been demonstrated in the human atherosclerotic vessel wall and may contribute to the impaired plasma fibrinolytic capacity in patients at high risk of atherothrombotic events. In addition, the mural PA/plasmin system may have important pathobiologic functions during atherogenesis. We quantitatively analyzed PAs of the tissue type (TPA) and urokinase type (UPA), PAIs, and plasminogen in protein extracts from different layers of human aorta in relation to the presence and severity of atherosclerotic lesions. In comparison with normal control vessels, intimal and neointimal TPA concentrations were reduced in atherosclerotic aortas except in the necrotic core areas of advanced plaques, where TPA was mainly complexed to PAI-1 in extracellular matrix deposits. In the media, TPA antigen was higher in lesional segments and closely associated with smooth muscle cells. UPA antigen was increased in the intima of atherosclerotic lesions and colocalized with tissue-infiltrating macrophages and neointimal smooth muscle cells. By spectrophotometric assay, neither TPA nor UPA activity could be detected in intimal or medial extracts. PAI-1 concentrations increased significantly in the intima of atherosclerotic segments compared with adjacent uninvolved areas or control aortas. The immunohistochemical distribution of PAI-1 was similar to that observed for TPA. A large excess of PAI-1 over PA concentrations, particularly in the intimal layer, characterizes atherosclerotic lesions of the human aorta and suggests that PA action is locally confined and counterbalanced by enhanced PAI expression and accumulation.
Key Words: fibrinolysis • plasminogen activators • vessel wall • atherosclerosis • plasminogen activator inhibitors
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Introduction |
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Local proliferation and migration of smooth muscle cells (SMCs) are considered key events in the development of atherosclerotic lesions1 and are subject to stimulation by a variety of growth factors.2 The plasminogen activator/plasmin (PA/plm) system, on the other hand, is involved in the regulation of growth factor activities3 and contributes to proteolytic degradation of the extracellular matrix,4 5 which is considered a prerequisite for migration of SMCs.6
Fibrinogen, fibrin, and their plm-induced degradation products are found in the intimal and subintimal layers of the arterial vessel wall. Their local concentrations vary considerably, depending on the presence and severity of atherosclerotic lesions,7 thus suggesting a possible pathogenetic role for intramural fibrin accumulation and degradation. Fibrin(ogen) degradation products have, indeed, a variety of biological effects that may be relevant in atherogenesis. These include inhibition of SMC proliferation,8 disorganization of the vascular endothelium,9 increase in vascular permeability,10 and monocyte chemotaxis and secretory stimulation.11 12
Moreover, clinical studies have demonstrated elevated plasma levels of plasminogen activator inhibitor–1 (PAI-1) and tissue-type plasminogen activator (TPA) antigen, but not TPA activity, in patients with coronary artery disease13 14 15 16 and in individuals at high risk of future myocardial infarction and stroke.17 18 19 20 21 These findings gave rise to the hypothesis that plasma concentrations of these proteins increase as a consequence of progressing atherosclerosis or, in turn, that the reduced plasma fibrinolytic capacity favors local fibrin deposition and extracellular matrix accumulation.
Recently, several authors have reported increased PAI-1 expression in the human atherosclerotic vessel wall.22 23 24 However, until now, the quantitative distribution of both PAs and PAIs in the human arterial wall has not been studied in a comparative manner in either normal or atherosclerotic vessels. Therefore, in this study, we quantitatively analyzed the antigen and activity levels of individual components of the PA/plm system in the different layers of normal and diseased human aortic tissue. Our aim was to investigate whether quantitative alterations in the PA/plm system within the vascular wall are related to the presence and severity of atherosclerotic lesions.
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Methods |
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Materials
Upper abdominal aortic tissue from patients with (n=8) and without (n=8) atherosclerotic disease was obtained at autopsy within 24 hours of death due to any cause. However, samples were excluded when the autopsy showed any evidence of infectious disease.
The samples were processed immediately. After removal of connective tissue and adherent blood, the specimens were divided into grossly homogeneous parts. Aortic wall segments were classified by their macroscopic appearance according to the presence and severity of atherosclerotic lesions. The following groups were established: (1) controls (C's): specimens from aortas without any macroscopic evidence of atherosclerotic disease; (2) macroscopically normal areas (MNAs): macroscopically normal-appearing segments from otherwise atherosclerotic vessels; (3) early lesions (ELs): segments with a macroscopically detectable white or yellowish thickening limited to the intimal layer (gelatinous lesions); macroscopically, the tunica media in these segments had a normal appearance; and (4) advanced lesions (ALs): samples with deep lesions (fibrous plaque type) extending into the media. Samples containing macroscopic calcification, thrombosis, or ulceration were excluded.
To confirm the validity of the macroscopic classification, representative samples of each type of segment were examined histologically. The intima, media, and adventitia of each specimen were separated while being viewed under a magnifier. The media was further dissected into a luminal and an abluminal part (inner and outer media). The intima of advanced lesions was divided into a luminal (cap-AL) and a central (core-AL) part. After dissection, all tissue samples were immediately snap-frozen in liquid N2 and stored at -80°C.
Extraction Method
Tissue extraction was performed as previously
described.25 In brief, individual tissue specimens were
pulverized in liquid N2, weighed, and
homogenized in extraction buffer (40 mg/mL) for 1 minute at
4°C by using an Ultra-Turrax T25 homogenizer. The
average absolute amount of pulverized tissue used for each extraction
was 97±46 mg (mean±SD). As described previously,25 26
the components of the PA/plm system were extracted in an acid acetate
buffer (75 mmol/L acetic acid, 225 mmol/L NaCl, 75 mmol/L KCl, 10
mmol/L EDTA, 100 mmol/L arginine, and 0.25% [vol/vol] Triton X-100,
pH 4.2). The tissue homogenate was centrifuged for
10 minutes at 3000g at 4°C. Thereafter, the supernatant
was filtered (Sartorius filter; pore size, 1.2 µm) and stored at
-80°C until assayed.
To control for the efficiency of the extraction procedure, the protein content of the extracts was measured after repetitive precipitation of the proteins with 10% cold trichloroacetic acid.27 Because no significant differences in the protein content of the extracts were observed between groups, the results are presented per 100 mg of wet tissue weight.
Assays of PAs
TPA antigen levels were determined by a commercial enzyme-linked
immunosorbent assay (ELISA, Imulyse). According to the manufacturer,
the capture antibody is goat anti-human TPA IgG and the detecting
antibody is a horseradish peroxidase–labeled goat anti-human TPA IgG.
This assay quantitatively detects single-chain and two-chain
forms of TPA with an immunoreactivity towards TPA:PAI complexes of
>90%. Urokinase-type PA (UPA) antigen levels were evaluated with an
ELISA (EU-5 Monozyme). This assay, which employs two different
monoclonal antibodies (mouse IgG1), recognizes pro-UPA;
free, two-chain, high-molecular-weight UPA; and the UPA:PAI-1 complex
but does not detect low-molecular-weight UPA or the amino-terminal
fragment of UPA.
Total PA activity in tissue extracts was analyzed spectrophotometrically28 and corrected for the plm inhibitory activity found in the respective sample.29 To evaluate TPA and UPA activities, samples were assayed in the presence or absence of excess quenching rabbit anti-TPA Ig (Organon Teknika) or goat anti-UPA Ig (kindly provided by G. Dooijewaard, IVVO-TNO, Leiden, the Netherlands). In addition, UPA activity was determined by a biologic immunoassay (BIA) purchased from Biopool. In this test, pro-UPA and UPA (53-kD form only) in the samples were allowed to bind to an immobilized mouse monoclonal anti–pro-UPA antibody. After activation of pro-UPA to the two-chain active molecule, total UPA activity was measured by using glu-plasminogen and a plm-sensitive chromogenic substrate (D-butyl-CHT-Lys-p-nitroaniline).
PA activities were visualized by fibrin zymography after subjecting the tissue extracts (without prior reduction) to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) on a 9% separating gel and a 4% stacking gel.30 After extensive washing in 2.5% Triton X-100 aqueous solution, zymograms were allowed to develop for 24 to 48 hours at 37°C on a plasminogen-rich, fibrin-agarose underlay.31 PA bands in the zymograms were identified by using human TPA, UPA, and the TPA:PAI-1 complex as standards. Samples were also incubated with anti-human TPA Ig or anti-human UPA Ig for 10 minutes before electrophoresis to confirm the identity of the PA bands. Areas of lysis detected in the PA:PAI-1 position were always abolished by incubation with anti-TPA Ig but were not affected by anti-UPA Ig.
Assays of PAIs
PAI-1 antigen was measured by a commercial ELISA (Monozyme) that
detects active and latent PAI-1 as well as PAI-1 complexed to TPA and
UPA. PAI-2 antigen was determined by an enzyme immunoassay kindly
provided by Behringwerke AG. PAI activity was evaluated
spectrophotometrically as described by Verheijen et
al.32
Assay of Plasminogen Activity
Plasminogen activity was determined
spectrophotometrically after activation of the samples with
streptokinase (Kabikinase, Kabi) as described by Friberger et
al,33 with minor modifications. In brief, 45 µL buffer
(0.05 mol/L Tris-HCl, 0.012 mol/L NaCl, and 0.1% [vol/vol] Tween 80,
pH 7.4), 15 µL sample, and 20 µL streptokinase (10 000 IU/mL) were
placed into microtiter wells. After incubation for 15 minutes at
37°C, 150 µL S-2251 (0.9 mmol/L in Tris-HCl buffer) was added.
Absorbance was measured at 405 nm after a 30-minute incubation at
37°C. Human plasminogen (Chromogenix) was used as a
standard. Plasminogen activity was corrected for anti-plm
activity as described previously29 and is expressed as
casein units (CUs) per 100 mg of wet tissue.
Immunohistochemistry
The aortic specimens were fixed in 4%
paraformaldehyde, embedded in paraffin (Paraplast Plus,
Sigma Chemical Co), sectioned into 8-µm-thick slices, and mounted on
STAR-FROST adhesive slides (Engelbrecht).
Serial sections were processed for immunohistochemistry by using the
following murine monoclonal antibodies: anti-human TPA (3 VPA,
Technoclone; 20 µg/mL), anti-human UPA (MAb 3688, American
Diagnostica; 20 µg/mL), anti-human PAI-1 (MAb 3785,
American Diagnostica; 10 µg/mL), anti-human
macrophage (DAKO-CD68, DAKO; working dilution, 1:50),
anti-human 
–smooth muscle actin (clone 1A4, DAKO; working dilution,
1:25), and anti-human von Willebrand factor (DAKO-vWF, DAKO;
working dilution, 1:25). For negative controls, mouse IgG (Sigma; 20
µg/mL) was used as a substitute for the first antibody.
Immunohistolocalization was performed by the peroxidase-conjugated
biotin-avidin technique. Before being stained, tissue sections were
deparaffinized in xylene, incubated with 1% (vol/vol)
H2O2 in methanol (10 minutes) to block
endogenous peroxidase activity, and rehydrated in a graded
ethanol series. Samples were permeabilized by treatment
with 0.23% (wt/vol) pepsin (Sigma) for either 3 (PAI-1, TPA, and UPA
detection) or 6 (CD68, -actin, and vWF detection) minutes. The
primary antibodies were applied for 2.5 hours at 37°C, followed by a
biotinylated goat anti-mouse IgG antibody (Amersham; working dilution,
1:50) and a streptavidin–horseradish peroxidase conjugate (Amersham;
working dilution, 1:100) for 30 minutes each. Peroxidase activity was
revealed by using an aminoethylcarbazole substrate kit (Zymed). After
immunoreaction, the sections were counterstained with hematoxylin and
mounted in a glycerol/vinyl alcohol solution (Zymed).
Statistics
Data are presented as medians and interquartile ranges.
Statistical significance of differences between MNA and either EL or AL
groups in atherosclerotic vessels was analyzed by the Wilcoxon
matched-pairs signed rank test. Differences between the MNA group in
atherosclerotic vessels and the C group were estimated by the
Mann-Whitney rank sum test for independent groups.34 A
value of P
.05 was considered significant, with corrections
made according to the Bonferroni procedure in case of multiple
comparisons. Graphic presentation of the data is made by
box-and-whisker plots.35
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Results |
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Antigen and activity levels of the components of the PA/plm system were determined in extracts from defined areas of upper abdominal aortic tissue specimens from eight patients with overt atherosclerotic disease. Representative segments of areas without macroscopic lesions (ie, MNAs), with early lesions (ELs), and with advanced lesions (ALs) were isolated from each aortic vessel wall specimen. In addition, tissue samples from aortas without any evidence of atherosclerosis from eight different patients were used as controls (C's). Gender distribution and the time from death to autopsy (range, 12 to 24 hours) were not different between C and atherosclerotic patients. Inevitably, individuals in the C group were significantly younger than patients with atherosclerosis (median age and range, 44 and 13 to 65 years, respectively, versus 69 and 55 to 80 years; P<.01). Causes of death were as follows: in the C group, pulmonary embolism (n=2), cancer (n=2), cardiomyopathy (n=1), cerebral hemorrhage (n=1), renal failure (n=1), and unknown (n=1); in the atherosclerosis group, coronary heart disease (n=5), pulmonary embolism (n=1), stroke (n=1), and pancreatitis (n=1).
PAs in the Intima and Media
Control Vessels
Both TPA and UPA antigens were consistently detected in
extracts of normal aortic intima and media (Figs 1
and 2). In the intima, the median TPA antigen content was
5.7 times higher than the median UPA antigen (13.0 ng/100 mg tissue
versus 2.3 ng/100 mg tissue, P<.01) and 2.5 times higher
than median TPA antigen levels in the inner (P<.01; cf Figs 1 and 2) and outer (P=.03) media. No significant differences
in TPA or UPA content were found between the inner and outer media.
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). The 
in A indicates a
significant difference between C and MNA groups (Mann-Whitney test,
significance for two-tailed P<.05). EL and AL (cap-AL and
core-AL) groups were compared with the MNA group by the Wilcoxon
test; significant differences (*) were accepted for
P<.016 according to Bonferroni's procedure. TPA indicates
tissue-type plasminogen activator; UPA,
urokinase-type plasminogen activator.
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Atherosclerotic Intima
Compared with the intima of C aortas, TPA antigen was
significantly decreased in MNAs of atherosclerotic vessels (Fig 1A
).
This trend was observed regardless of whether TPA antigen was related
to the amount of wet tissue extracted or to the amount of extractable
protein (median TPA concentrations in C versus MNA: 13.0 versus 5.6
ng/100 mg wet tissue, P=.035; 8.0 versus 3.3 ng/mg extracted
protein, P=.031).
In the intima of ELs and the intimal cap of AL segments, TPA antigen
levels were not significantly different from those in MNAs. However, a
significant increase in TPA content was observed in the core-AL
(P=.014), with median concentrations comparable with those
in the C intima. A different pattern emerged for UPA antigen in
intimal extracts. Whereas UPA antigen levels in MNAs were low and
not different from those in C vessels, there was a significant increase
in ELs and ALs (P<.01, Fig 1B).
By spectrophotometric assay, neither TPA nor UPA activity could be
detected in the intimal layer. However, when extracts were
analyzed by fibrin-agarose zymography after SDS-PAGE (Fig 3), two bands at the position of free TPA and another
two at the position of the TPA:PAI-1 complex were evident in all
samples. None of these bands was detected in samples previously
incubated with anti-TPA, but the bands were not affected by prior
incubation with anti-UPA. Zymograms of extracts from C aortas and MNAs
of atherosclerotic vessels showed a thin band at a higher position than
the TPA:PAI complex. This band, which was not present in samples
treated with anti-TPA, may correspond to a complexed form of TPA with a
high-molecular-weight inhibitor. Zymograms of extracts from
ALs showed weaker bands in the free-TPA position, whereas the TPA:PAI-1
bands became more intense. No UPA activity was detected by zymography
in either C or atherosclerotic intimas. With use of a BIA, which
detects both the single-chain zymogen and the active two-chain forms, a
60% increase in functional UPA was found in pools of intimal
segments with ELs or ALs (C=2.5, MNA=2.2, EL=3.9, cap-AL=4.0, and
core-AL=3.6 ng/100 mg of wet tissue).
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Atherosclerotic Media
Values for TPA and UPA antigen in extracts of C segments did not
differ significantly from those of MNAs in the inner (Fig 2) and outer
(not shown) medial layers. In the inner media of atherosclerotic
vessels, TPA antigen was slightly but significantly increased in areas
with ELs and ALs compared with MNAs (P=.014, Fig 2A). No
significant differences in UPA antigen levels were found in the media
between segments with and without lesions (Fig 2B; outer media not
shown). This may in part be due to considerable interindividual
variability.
PA activity could not be detected spectrophotometrically in any of the media extracts. However, as in the intimal extracts, functional UPA could be demonstrated by BIA in pooled extracts of the inner and outer media (inner media: C=6.8, MNA=6.1, EL=4.8, and AL=7.9 ng/100 mg wet tissue; outer media: C=5.1, MNA=4.2, EL=4.7, and AL=4.4 ng/100 mg wet tissue).
PAIs in the Intima and Media
In the intima as well as in the inner and outer media, levels of
extractable PAI-1 antigen did not differ significantly between C
segments and MNAs (Fig 4; outer media not shown).
However, PAI-1 content was significantly increased in intimal extracts
of ELs and ALs (P<.01, Fig 4A). An increase in PAI-1 was
also measured in extracts from the inner media of ELs
(P=.01, Fig 4B), whereas no significant changes were
detected in the outer medial layer. PAI-2 antigen could not be detected
in any of the tissue extracts; the detection limit of this assay was 2
ng/mL in the extraction buffer.
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Using a spectrophotometric assay, we found median PAI activities of 2
U/100 mg tissue in the intima and inner media (Table 1).
There were no significant differences between C and MNA segments. In
the atherosclerotic vessels, PAI activity did not show a significant
difference between MNA and lesional segments. No PAI activity was
detected in the outer medial layer. On the basis of the assumption that
the specific PAI-1 activity is 650 000 U/mg protein,36
one can calculate that <15% of the PAI-1 antigen in intimal and
medial extracts was active. To avoid underestimating PAI activity in
the tissue extracts because of the low pH of the extraction
buffer,37 the samples were neutralized to pH 7.7 prior to
PAI activity measurements.
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PAs and PAIs in the Adventitia
TPA was the prevailing PA in the adventitia of both C and
atherosclerotic vessels, with a median 10-fold excess of antigen over
UPA (median TPA antigen >50 ng/100 mg tissue; median UPA antigen <5
ng/100 mg tissue). In the adventitial extracts of all segments studied,
TPA antigen levels also exceeded PAI-1 content (median, <7 ng/100 mg
tissue). Thus, net PA activity, largely attributable to TPA, was
detected in all segments. There was considerable variability in PA
activity between individuals. However, PA activity in the adventitia
was unrelated to the presence and severity of atherosclerotic lesions.
PAI-1 antigen levels in the adventitia were low, and no free PAI
activity was detected.
Plasminogen in the Intima, Media, and
Adventitia
Low but detectable levels of plasminogen activity were
extracted from the intima and media of C aortas (Table 2). Plasminogen activity did not show any
significant difference in extracts of the intimal layer in relation to
the presence and severity of atherosclerotic lesions. However, a
significant increase in plasminogen activity was observed
in inner medial extracts of EL and AL segments compared with the MNAs
(P=.014). Stable though consistently low activity
levels were observed in the outer media (Table 2) and adventitia (not
shown).
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Immunohistolocalization of PAs and PAI-1
Serial sections of ELs and ALs were analyzed for
immunoreactivity with anti-TPA, anti-UPA, and anti–PAI-1 antibodies
(Fig 5). The specimens were also reacted with anti-vWF,
anti–-actin, and anti-CD68 to ascertain the identity of
endothelium, SMCs and macrophages,
respectively. The luminal endothelium was
morphologically intact in ELs and stained positive for TPA, PAI-1 (Fig 5A and 5C), and vWF (not shown). In contrast, luminal
endothelial cells were frequently absent in ALs. Strong
TPA-specific staining was observed in the medial and deeper intimal
layers of ELs (Fig 5A) in close association with -actin–positive
SMCs (not shown). In ALs the TPA signal colocalized mainly with
amorphous material in and around the core region of the plaques (Fig 5B). A similar staining pattern was demonstrated for PAI-1, with the
exception of a more "spotty" labeling in the inner part of the
media (Fig 5C and 5D). Strongly positive UPA staining was detected in
the thickened intimas of ELs (Fig 5E), matching the scattered
infiltration by CD68-positive macrophages (Fig 5F). However, a
UPA signal was also associated with SMCs in the abluminal part of the
intima and adjacent media. No staining was detected by
immunohistochemistry when nonimmune murine Ig was substituted for
either of the first antibodies.
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Discussion |
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Controversial findings have been reported on the fibrinolytic activity in the atherosclerotic vessel wall.38 39 40 41 However, most of the earlier reports were based on overall assessment of tissue-associated fibrinolytic activity, eg, by fibrin plate or fibrin slide methods. The question of whether local concentrations of PAs and their principal inhibitor PAI-1 are related to the development and progression of human atherosclerotic lesions has only recently been addressed.22 23 24 42 43 These studies have mainly focused on the expression of individual components of the PA/plm system, in particular PAI-1, in different arterial territories, with the use of in situ hybridization and immunohistochemical analyses.
In contrast to previous investigations on vascular tissue obtained from organ donors and patients undergoing atherectomy or vascular reconstructive surgery, we analyzed serial sections of aortic tissue obtained during autopsy. This allowed comparative quantification and immunohistolocalization of PAs and PAI-1 in representative samples from different individuals. However, in our experience as well as others,44 autopsy specimens have been found to be unsuitable for analyses at the mRNA level. Thus, we can only speculate on the origin of these components in the aortic vessel wall.
Excess active TPA over UPA and PAI-1 concentrations was demonstrated in the adventitia and appeared to be unrelated to atherosclerotic involvement of the aorta. TPA antigen was also detected in the luminal endothelium as well as in association with myointimal cells in ELs. Local concentrations of TPA were increased in the necrotic core of ALs and in the media of lesional sites, where it colocalized with SMCs. In this latter respect, our observations accord with the findings of Clowes et al,45 who demonstrated enhanced TPA expression by intimal and medial SMCs after mechanical injury in rat carotid arteries. These authors concluded that preferential expression of TPA is a feature of migrating SMCs. Reilly and coworkers44 also detected TPA expression in the intima of normal and diseased aortas but found its expression in the media to be restricted to diseased areas. Thus, TPA expression by arterial SMCs appears to be a local phenomenon related to injury and remodeling of the vascular wall. Most interesting in this context is the recent work by Herbert et al,46 showing that TPA is a direct and selective mitogen for human aortic SMCs and may act as an autocrine growth factor.
The accumulation of TPA in and surrounding the necrotic centers of advanced plaques is a finding that has not previously been reported. This finding would be consistent with the increase in extractable TPA activity documented for endarterectomy cores from atheromatous coronary arteries.43 However, we were unable to detect active TPA in intimal extracts of aortic lesions, and zymographic analysis revealed that most of the TPA in the core-ALs is complexed to PAI-1. These divergent results suggest quantitative differences in the modulation of intramural TPA by PAI-1 in aortic and coronary vessel walls. Our own preliminary results, indeed, demonstrate excess extractable TPA activity in atherosclerotic coronary arteries47 as opposed to the excess of inhibitor found in the atherosclerotic intima of human aortas. Complexation of TPA to matrix-bound PAI-1 may account for local accumulation of the former in the core-ALs, as both proteins show a similar intimal distribution on immunohistochemical analyses. From our studies we cannot exclude, however, that intimal fibrin deposits also bind and accumulate TPA from adjacent cells or even from the circulation.
It is important to note that the TPA antigen extractable from the intima was lower in atherosclerotic aortas than in C vessels, with the exception of core-ALs. This observation has been independently confirmed by a recent preliminary report on the localization and quantification of fibrinolytic proteins in the human aorta.48 The decrease in TPA content cannot be attributed to endothelial denudation, because the luminal endothelium was morphologically intact in MNA and EL areas of the atherosclerotic aortas. It can be argued that we observed a relative decrease in TPA content owing to the presence of cellular infiltrates, extracellular matrix deposits, or lipid accumulations, any or all of which would increase tissue wet weight or soluble protein content without producing TPA. This cannot be ruled out from our studies, nor can we prove decreased synthesis or increased release of TPA by the endothelium. However, our data clearly demonstrate an increase in PAI-1 over TPA concentrations in the intima of lesional areas.
UPA antigen levels were found to be increased in the intima of ELs and ALs. The lack of UPA activity on zymographic analyses, together with the increase in UPA detected by BIA, suggests that most intimal UPA represents its zymogen form, pro-UPA. A similar distribution pattern of UPA and CD68 antigen in the luminal part of the lesional intima further suggests that the increase in UPA is largely attributable to tissue infiltration by macrophages, which are known to express pro-UPA.49 In the abluminal intima and adjacent media, an unambiguous UPA signal was evident in association with SMCs, which have been shown to preferentially express UPA during proliferation.45 By analogy to the role of UPA during tumor cell invasion and metastasis,50 local UPA expression may favor the pericellular matrix proteolysis that is required for subendothelial migration of macrophages, as well as for the proliferation and migration of SMCs during atherogenesis.
Recent experimental work in rabbits has demonstrated increased PAI-1
expression in aortic neoendothelial cells, intimal
macrophages, and SMCs beneath the internal elastic lamina in
response to sustained mechanical injury, particularly when accompanied
by hypercholesterolemia.51 The increase in
PAI-1 transcription paralleled the severity of the vascular lesions
induced. These findings are in line with the reported increase in PAI-1
expression in the intima and media of human atherosclerotic lesions
from various vascular territories.22 23 24 Our results
confirm these observations in a quantitative manner, on the basis of
protein measurements in tissue extracts. They further demonstrate that
the increase in intimal PAI-1 concentrations is a local phenomenon
restricted to areas of atherosclerotic involvement and suggest that
enhanced PAI-1 expression in the media is a feature of early rather
than advanced atherosclerosis. The immunohistochemical
analyses have revealed that most PAI-1 protein in the thickened
intima of ELs is located in and around neointimal SMCs and
possibly macrophages, whereas matrix deposits of PAI-1 prevail
in the core-ALs. As indicated by the zymographic results, PAI-1 in
these areas is, to a large extent, complexed with TPA, suggesting that
PAI-1 has an important function in modulating mural TPA activity. Its
pathobiologic role in atherogenesis, however, remains speculative and
may reside in the prevention of extracellular matrix breakdown and
tissue repair as well as in the inhibition of TPA-stimulated SMC
proliferation.46 In addition, it is tempting to
hypothesize that increased intimal PAI-1 could favor local fibrin
deposition during plaque rupture. Indeed, we observed a trend towards
increasing PAI-1 activities in the neointima of ELs and ALs
(Table 1). However, in contrast to the results on PAI-1 antigen, this
trend failed to reach the level of statistical significance.
It is presently unclear whether intimal TPA and PAI-1 expression is related to their respective plasma concentrations. Increased plasma PAI-1 and TPA antigen, but not TPA activity, have been reported in patients with advanced coronary artery disease.13 14 16 52 However, there is apparently no gradational relationship with respect to the extent of coronary atherosclerosis.52 Similarly, elevated levels of TPA antigen, but not of TPA activity, predict an increased risk of future myocardial infarction or stroke, as demonstrated in healthy, middle-aged men17 18 and patients with angina pectoris.19 53 These findings are likely to reflect an impaired plasma fibrinolytic capacity due to inhibition of active TPA by PAI-1 and resulting in increased levels of circulating TPA:PAI-1 complex. Furthermore, plasma PAI-1 is elevated in patients with the insulin resistance syndrome, which is known to be associated with coronary heart disease, and atherogenic lipoproteins, such as VLDL, oxidized LDL, and Lp(a), have been shown to induce endothelial PAI-1 production (reviewed in Reference 1515 ). These atherogenic risk factors seem likely to be involved in the upregulation of PAI-1 synthesis in the atherosclerotic vascular wall. However, it remains to be demonstrated whether the observed increase in intimal concentrations of PAI-1 and TPA:PAI-1 complex in turn has an effect on circulating plasma levels, which could then serve as markers of progressing atherosclerosis.
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Acknowledgments |
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This work was supported by the Dr-Karl-Wilder-Stiftung, Verband der Deutschen Lebensversicherer. Dr Padró was the recipient of a grant from the Alexander von Humboldt-Stiftung. We thank Dr E. Schüler, Behringwerke AG, for evaluating PAI-2 antigen in tissue extracts.
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Footnotes |
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Reprint requests to J. Kienast, MD, Division of Hematology/Oncology, Department of Internal Medicine, University of Münster, Albert-Schweitzer Str 33, 48129 Münster, Germany.
Received December 1, 1994; accepted April 11, 1995.
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References |
|---|
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Ross R. The pathogenesis of atherosclerosis—an update. N Engl J Med. 1986;314:488-500. [Medline] [Order article via Infotrieve]
2. Ross R. Polypeptide growth factors and atherosclerosis. Trends Cardiovasc Med. 1991;1:277-282.
3.
Flaumenhaft R, Abe M, Sato Y, Harpel J, Hedin CH,
Rifkin D. Role of the latent TGF-ß binding protein in the
activation of latent TGF-ß by co-cultures of
endothelial and smooth muscle cells.
J Cell Biol. 1993;120:995-1002.
4. Machovich R, Owen WG. An elastase dependent pathway of plasminogen activation. Biochemistry. 1989;28:4517-4522. [Medline] [Order article via Infotrieve]
5. Murphy G, Reynolds JJ, Hembry RM. Metalloproteinases and cancer invasion and metastasis. Int J Cancer. 1989;44:757-760. [Medline] [Order article via Infotrieve]
6.
Sperti G, van Leeuwen RTJ, Quax PHA, Maseri A, Kluft
C. Cultured rat aortic vascular smooth muscle cells digest
naturally produced extracellular matrix. Circ
Res. 1992;71:385-392.
7.
Smith EB, Keen A, Grant A, Stirk C. Fate of
fibrinogen in human arterial intima.
Arteriosclerosis. 1990;10:263-275.
8. Ishida T, Takada K. Effect of fibrin and fibrinogen degradation products on the growth of aortic smooth muscle cells in culture. Atherosclerosis. 1982;44:161-174. [Medline] [Order article via Infotrieve]
9.
Dang CV, Bell WR, Kaiser D, Wong A.
Disorganization of cultured vascular endothelial
cell monolayers by fibrinogen fragment D. Science. 1985;227:1487-1490.
10. Gerdin B, Saldeen T. Effect of fibrin degradation products on microvascular permeability. Thromb Res. 1978;13:995-1006. [Medline] [Order article via Infotrieve]
11. Richardson DL, Pepper DS, Kay AB. Chemotaxis for human monocytes by fibrinogen-derived peptides. Br J Haematol. 1976;32:507-513. [Medline] [Order article via Infotrieve]
12.
Hamaguchi M, Morishita Y, Takabashi I, Ogura M,
Takanotsu J, Saito H. FDP D-dimer induces the
secretion of interleukin-1, urokinase-type plasminogen
activator and plasminogen
activator-2 in a human promonocytic leukemia cell
line. Blood. 1991;77:94-100.
13. Paramo JA, Colucci M, Collen D. Plasminogen activator inhibitor in the blood of patients with coronary artery disease. Br Med J. 1985;291:573-574.
14. Aznar J, Estelles A, Tormo G, Sapena P, Tormo V, Blanch S, España F. Plasminogen activator inhibitor activity and other fibrinolytic variables in patients with coronary artery disease. Br Heart J. 1988;58:535-541.
15. Juhan-Vague I, Alessi MC. Plasminogen activator inhibitor 1 and atherothrombosis. Thromb Haemost. 1993;70:138-143. [Medline] [Order article via Infotrieve]
16. Olofsson BO, Dahlen G, Nilsson TK. Evidence for increased levels of plasminogen activator inhibitor and tissue plasminogen activator in plasma of patients with angiographically verified coronary artery disease. Eur Heart J. 1990;10:77-82.
17. Ridker PM, Vaughan DE, Stampfer MJ, Manson JE, Hennekens CH. Endogenous tissue-type plasminogen activator and risk of myocardial infarction. Lancet. 1993;341:1165-1168. [Medline] [Order article via Infotrieve]
18. Ridker PM, Hennekens CH, Stampfer MJ, Manson JE, Vaughan DE. Prospective study of endogenous tissue plasminogen activator and risk of stroke. Lancet. 1994;343:940-943. [Medline] [Order article via Infotrieve]
19.
Jansson JH, Nilsson TK, Olofsson BO. Tissue
plasminogen activator and other risk factors as
predictors of cardiovascular events in patients with
severe angina pectoris. Eur Heart J. 1991;12:157-161.
20. Hamsten A, de Faire U, Walldius G, Dahlen G, Szamosi A, Landou C, Blombäck M, Wiman B. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet. 1987;2:3-9.[Medline] [Order article via Infotrieve]
21.
Munkvad S, Gram J, Jespersen J. A depression of
active tissue plasminogen activator in plasma
characterizes patients with unstable angina pectoris. Eur
Heart J. 1990;11:525-528.
22.
Schneiderman J, Sawdey SM, Keeton MR, Bordin GM,
Bernstein EF, Dilley RB, Loskutoff DJ. Increased type 1
plasminogen activator inhibitor
gene expression in atherosclerotic human arteries. Proc
Natl Acad Sci U S A. 1992;89:6998-7002.
23. Chomiki N, Henry M, Alessi MC, Anfosso F, Juhan-Vague I. Plasminogen activator inhibitor-1 expression in human liver and healthy or atherosclerotic vessel walls. Thromb Haemost. 1994;72:44-53. [Medline] [Order article via Infotrieve]
24.
Lupu F, Bergonzelli GE, Heim DA, Cousin E, Genton CY,
Bachmann F, Kruithof EKO. Localization and production of
plasminogen activator inhibitor-1
in human healthy and atherosclerotic arteries.
Arterioscler Thromb. 1993;13:1090-1100.
25. Padró T, van den Hoogen CM, Emeis JJ. Distribution of tissue-type plasminogen activator (activity and antigen) in rat tissues. Blood Coagulation Fibrinol. 1990;1:601-608. [Medline] [Order article via Infotrieve]
26. Camiolo SM, Siuta MR, Madeja JM. Improved medium for extraction of plasminogen activator from tissue. Prep Biochem. 1982;12:297-305. [Medline] [Order article via Infotrieve]
27.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.
Protein measurement with the Folin phenol reagent.
J Biol Chem. 1951;193:265-275.
28. Verheijen JH, Mullaart E, Chang GTG, Kluft C, Wijngaards G. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemost. 1982;48:255-269.
29. Padró T, van den Hoogen CM, Emeis JJ. Plasmin inhibitory effect of rat tissues: comparison with antiplasmin activity of plasma. Scand J Clin Lab Invest. 1991;51:599-603. [Medline] [Order article via Infotrieve]
30. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-682. [Medline] [Order article via Infotrieve]
31.
Granelli-Piperno A, Reich E. A study of
proteases and protease-inhibitor complexes in biological
fluids. J Exp Med. 1978;148:223-234.
32. Verheijen JH, Chang GTG, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemost. 1984;51:392-395. [Medline] [Order article via Infotrieve]
33. Friberger P, Knös M, Gustavsson S, Aurell L, Claeson G. Methods for determination of plasmin, antiplasmin and plasminogen by means of substrate S-2551. Haemostasis. 1978;7:138-145. [Medline] [Order article via Infotrieve]
34. Siegel S. Nonparametric Statistics for the Behavioral Sciences. New York, NY: McGraw-Hill Book Co; 1956:116-127.
35. Frigge M, Hoagland DC, Iglewicz B. Some implementations of the boxplot. Am Stat. 1989;43:50-54.
36. Urden G, Hamsten A, Wiman B. Comparison of plasminogen activator inhibitor activity and antigen in plasma samples. Clin Chim Acta. 1987;169:189-196. [Medline] [Order article via Infotrieve]
37. Rånby M, Sundell IB, Nilsson TK. Blood collection in strong acidic citrate anticoagulant used in a study of dietary influence on basal t-PA activity. Thromb Haemost. 1989;62:917-922. [Medline] [Order article via Infotrieve]
38. Astrup T, Coccheri S. Thromboplastic and fibrinolytic activity of the arteriosclerotic human aorta. Nature. 1962;193:182-183. [Medline] [Order article via Infotrieve]
39. Noordhoek HV. Distribution and variation of fibrinolytic activity in the walls of human artery and veins. Haemostasis. 1976;5:355-372. [Medline] [Order article via Infotrieve]
40. Donner L, Klener P, Roth Z. The plasminogen activator of the arterial wall. Thromb Haemost. 1977;37:436-443. [Medline] [Order article via Infotrieve]
41. Ljungnér H, Bergquist D. Decreased fibrinolytic activity in human atherosclerotic vessels. Atherosclerosis. 1984;50:113-116. [Medline] [Order article via Infotrieve]
42. Smokovitis A, Kokolis N, Alexaki-Tzivanidou E. Fatty streaks and fibrous plaques in human aorta show increased plasminogen activator activity. Haemostasis. 1988;18:146-153. [Medline] [Order article via Infotrieve]
43.
Underwood MJ, Debono DP. Increased fibrinolytic
activity in the intima of atheromatous coronary
arteries—protection at a price. Cardiovasc Res. 1993;27:882-885.
44. Reilly JM, Sicard GA, Lucore CL. Abnormal expression of plasminogen activators in aortic aneurysmal and occlusive disease. J Cardiovasc Surg. 1994;19:865-872.
45.
Clowes AW, Clowes MM, Au YPT. Smooth
muscle cells express urokinase during mitogenesis and tissue-type
plasminogen activator during migration in
injured rat carotid artery. Circ Res. 1990;67:61-67.
46.
Herbert JM, Lamarche I, Prabonnaud V, Dol F, Gauthier
T. Tissue-type plasminogen activator is
a potent mitogen for human aortic smooth muscle cells.
J Biol Chem. 1994;269:3076-3080.
47. Padró T, Steins M, Hammel D, Kienast J. Atherosclerosis-related alterations of the plasminogen activator/plasmin (PA/plm) system in vascular wall: studies in human coronary arteries. Thromb Haemost. 1993;69:971. Abstract.
48. Robbie LA, Booth NA, Brown PAJ, Croll AM, Bennett B. Localisation and quantification of proteins of the fibrinolytic system in normal and atherosclerotic vessels. Fibrinolysis. 1994;8(suppl 1):86. Abstract.
49.
Vassalli JD, Dayer JM, Wohlwend A, Belin D.
Concomitant secretion of prourokinase and a
plasminogen activator-specific
inhibitor by cultured human
monocytes-macrophages. J Exp Med. 1984;159:1653-1668.
50. Vassalli JD, Sappino AP, Belin D. The plasminogen activator/plasmin system. J Clin Invest. 1991;88:1067-1072.
51.
Sawa H, Sobel BE, Fujii S. Potentiation by
hypercholesterolemia of the induction of aortic
intramural synthesis of plasminogen activator
inhibitor type 1 by endothelial
injury. Circ Res. 1993;73:671-680.
52.
Thompson SG, van de Loo JCW. ECAT angina
pectoris study: baseline associations of haemostatic factors with
extent of coronary arteriosclerosis and
other coronary risk factors in 3000 patients with angina
pectoris undergoing coronary angiography. Eur
Heart J. 1993;14:8-17.
53. Haverkate F, van de Loo JCW, Thompson SG. Haemostasis risk markers, inflammatory and endothelial reactions in angina pectoris: results of the ECAT angina pectoris study. Thromb Haemost. 1993;69:969. Abstract.
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