(Stroke. 2001;32:1341.)
© 2001 American Heart Association, Inc.
Original Contributions |
Rapid Differential Endogenous Plasminogen Activator Expression After Acute Middle Cerebral Artery Occlusion
From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, Calif.
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Background and Purpose—During focal cerebral ischemia, the microvascular matrix (ECM), which participates in microvascular integrity, is degraded and lost when neurons are injured. Loss of microvascular basal lamina antigens coincides with rapid expression of select matrix metalloproteinases (MMPs). Plasminogen activators (PAs) may also play a role in ECM degradation by the generation of plasmin or by MMP activation.
Methods—The endogenous expressions of tissue-type plasminogen activator (tPA), urokinase (uPA), and PA inhibitor-1 (PAI-1) were quantified in 10-µm frozen sections from ischemic and matched nonischemic basal ganglia and in the plasma of 34 male healthy nonhuman primates before and after middle cerebral artery occlusion (MCA:O).
Results—Within the ischemic basal ganglia, tissue uPA activity and antigen increased significantly within 1 hour after MCA:O (2P<0.005). tPA activity transiently decreased 2 hours after MCA:O (2P=0.01) in concert with an increase in PAI-1 antigen (2P=0.001) but otherwise did not change. The transient decrease in free tPA antigen was marked by an increase in the tPA–PAI-1 complex (2P<0.001). No significant relations to neuronal injury or intracerebral hemorrhage were discerned.
Conclusions—The rapid increase in endogenous PA activity is mainly due to significant increases in uPA, but not tPA, within the ischemic basal ganglia after MCA:O. This increase and an increase in PAI-1 coincided with latent MMP-2 generation and microvascular ECM degeneration but not neuronal injury.
Key Words: basal ganglia • cerebral ischemia, focal • microcirculation • plasminogen activators • tissue plasminogen activator • baboons
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Focal cerebral ischemia is responsible for a loss of microvascular integrity, manifested by increased endothelial cell permeability and loss of basal lamina matrix antigens.1 2 After experimental middle cerebral artery (MCA) occlusion (MCA:O) and during reperfusion (MCA:O/R), the major basal lamina constituents, laminin, collagen IV, and cellular fibronectin, decrease roughly in parallel,1 in association with microvascular hemorrhage.2 Loss of basal laminal integrity coincides with a rapid increase in the expression of latent matrix metalloproteinase (MMP)-2 in the ischemic core (Ic) in a nonhuman primate MCA:O model.3 Plasminogen activators (PAs) may also play several roles in cerebral ischemic injury to facilitate degradation of basal laminal ligands. Collagen, laminin, fibronectin, elastin, and/or myelin basic protein are degraded either directly by plasmin or by select activated MMPs requiring PA expression.4 5 6 7 8 The disruption of normal cell-matrix adhesion has been associated with cell death,9 edema,10 loss of cell viability,11 and hemorrhage3 in several systems. Latent MMP-9 expression is associated with hemorrhagic transformation in the nonhuman primate, but the nature of its activation remains uncertain.3 A contribution of plasmin, via PA generation, to pro-MMP-9 activation in the ischemic parenchyma is a potential mechanism. Some or all of these PA-dependent effects could contribute to central nervous system (CNS) injury. However, during focal cerebral ischemia, the tissue-related contributions of PAs to cerebral vascular and neuronal injury and recovery processes have not been detailed.
PA activities have been described in the normal rodent brain.12 Recent reports have indicated that tissue-type PA (tPA)13 or urokinase (uPA)14 may increase in the rodent brain after experimental MCA:O. Increased PA activity was detected within the caudate putamen after MCA:O in mice.15 But, there has been considerable disagreement about the nature of the PA responses to MCA:O in rodents and their significance. Although uPA activity appears to be increased in several studies,14 16 there is no agreement concerning the responses of tPA-like proteinase activity to MCA:O.13 14 16 17 Wang et al13 have suggested that increases in tPA-like proteinase activity may contribute to excitotoxic neuronal damage in mice. Therefore, the activation of the PA system during focal cerebral ischemia requires further scrutiny. Furthermore, the tissue content of the PAs and their principal inhibitor must be separated from the contributions of plasma.
We hypothesized that the upregulation of tissue-related tPA and/or uPA must occur rapidly to initiate the loss in microvascular integrity1 and accompany the changes in microvascular integrin receptor expression18 and neuronal injury.19 Increased PA expression would suggest a mechanism for pro-MMP-9 activation, whereas a linear association of increased PA expression with neuronal injury would tie the PA to neurodegeneration. We demonstrate rapid significant increases in uPA and PA inhibitor (PAI)-1, but not tPA, in the parenchyma of the ischemic basal ganglia very early after MCA:O. The sources of these PA-PAI axis components appear distinctly separate.
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Materials and Methods |
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The experimental procedures used to obtain the tissues for the present study were approved by the institutional Animal Research Committee and were performed according to standards published by the National Research Council (Guide for the Care and Use of Laboratory Animals) and the US Department of Agriculture Animal Welfare Act. Every effort was made to ensure that the animals were free of pain and discomfort. All experiments and procedures were attended by at least 1 institutional veterinarian, members of the primate handling staff, research associates, and the principal investigator.
Experimental Stroke Model
Cerebral tissues from 34 adolescent male baboons
(Papio anubis/cynocephalus)
were used for the present study. The procedures for development of
the MCA:O/R stroke model have been detailed in previous
studies.1 3 20
All animals were allowed a 7-day procedure-free interval after
transorbital implantation of an eccentric balloon compression device
around the proximal MCA. Twenty baboons underwent MCA:O for 1 hour
(n=4), 2 hours (n=6), or 3 hours or MCA:O with subsequent reperfusion
for 1 hour (n=4), 4 hours (n=3), or 24 hours (n=3). In addition, a
group of baboons that did not undergo device implantation (n=6) and a
group of baboons with lenticulostriatal territory injury harvested 7
days later (n=6) provided additional control materials. Two baboons who
underwent only the implantation procedure served as sham-operated
controls.
Tissue Processing and Sample Extracts
Experiments were terminated under thiopental
Na+ anesthesia by left
ventricular transcardiac perfusion at 180 to
200 mm Hg with chilled perfusate containing heparin (200
IU/L), nitroprusside (1 mg/L), and BSA (50 g/L) (Sigma Chemical Co).
The perfused brains were removed en bloc within 15 minutes of complete
perfusion and were subdivided into 1-cm coronal slices. From each
slice, symmetrically located blocks of both basal ganglia were cut and
embedded in Tissue-Tek OCT (Miles Inc). Three consecutive 10-µm
cryostat sections were minced and dissolved in 100 µL lysis buffer
(50 mmol/L Tris-HCl [pH 7.5], 0.1 mol/L
L-arginine, 150 mmol/L
NaCl, 1.0% Triton X-100, 0.005% Brij 35, and 0.05%
NaN3) for 10 minutes and centrifuged at
4°C for 20 minutes at 9000 rpm. Supernatants were divided into
aliquots and stored at -80°C. Preliminary experiments determined
the optimal conditions for homogenization and
activity extraction.
Blood Sampling
Blood samples were drawn from the
peripheral vein into tubes containing heparin or EDTA at
various times and centrifuged at 3000 rpm for 20 minutes, and
the plasmas were stored at -80°C.
Protein Concentration
The protein contents of tissue and plasma samples
were determined according to the Bradford method with BSA as the
standard. MCA:O produced a significant reduction in protein content per
unit volume in the ischemic basal ganglia
(2P<0.001)
(Figure 1
), consistent with previous
findings.21 All activity and
antigen levels were normalized for protein content to the Ic (see
below). After transcardiac perfusion, plasma protein (IgG)
represented 
0.007% of control basal ganglia.
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Plasminogen-Gelatin
Zymography
Plasminogen-gelatin zymography was
performed by a modification of published
methods.3 22 The
brain tissue and plasma samples (10 µg protein) were resolved under
nonreducing conditions on 8% SDS-polyacrylamide gels
containing 0.01 U/mL plasminogen and 1.5 mg/mL gelatin
(Sigma). For standardization, 10 pg of human melanoma tPA (Sigma) and
10 pg of recombinant human uPA (a kind gift of Drs Andrew P. Mazar,
Attenuon, San Diego, Calif, and Don Eisenhauer, Abbott Laboratories,
Chicago, Ill) were loaded onto each gel. After electrophoresis, the
gels were washed 3 times in 2.5% Triton X-100 solution and then
incubated in 100 mmol/L Tris-HCl (pH 8.2) buffer for 18 hours at
37°C. The gels were stained with 0.1% amido black solution. On
destaining, gelatinolytic activity was demonstrated
as clear zones
(Figure 2).
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To confirm the nature of each band, plasminogen-containing gels were incubated with or without 10 mmol/L CaCl2, 0.1 mmol/L amiloride, 2 mmol/L phenylmethylsulfonyl fluoride (Sigma), or 1 µmol/L trans-epoxysuccinyl-L-leucylamido-butane (E-64, Sigma). In addition, individual samples were incubated with function-blocking anti-human polyclonal antibodies against tPA (American Diagnostica, Inc), uPA (American Diagnostica, Inc), or PAI-1 (a kind gift of Dr David J. Loskutoff, The Scripps Research Institute, La Jolla, Calif) for 60 minutes at 37°C before application to the gel.
Immunoblotting
Samples (100 µg protein per lane) were separated
under nonreducing conditions on 8% SDS-polyacrylamide gels and
electrophoretically transferred to nitrocellulose membranes (Schleicher
& Schuell Inc) by the semidry blotting method. Blocking with 5% nonfat
milk in PBS containing 0.1% Tween 20 (PBST) was followed by incubation
with the primary antibody for 1 hour at 37°C and with horseradish
peroxidase-conjugated anti-rabbit or anti-goat IgG antibody (Santa Cruz
Biotechnology Inc or Vector Laboratories, Inc) at 20°C for 1 hour.
After each incubation, the blots were washed in PBST to remove unbound
antibody. Bound antibody was detected by enhanced chemiluminescence
(NEN Life Science Products Inc).
Goat anti-human melanoma tPA antibody (American Diagnostica, Inc), rabbit anti-human uPA antibody (Dr Andrew P. Mazar, Attenuon, San Diego, Calif), and rabbit anti-human PAI-1 antibody (Dr David J. Loskutoff, The Scripps Research Institute, La Jolla, Calif) were used as the primary antibodies for the respective immunoblot studies. Melanoma tPA, recombinant uPA, and recombinant PAI-1 (Dr David J. Loskutoff) served as controls. To prepare the control for the tPA–PAI-1 and uPA–PAI-1 bound forms, equal amounts of tPA or uPA and PAI-1 were incubated at 37°C for 10 minutes before application for electrophoresis. Goat or rabbit nonimmunized IgG was used as a primary antibody for the control. Absorption studies were performed by using a primary antibody incubated with melanoma tPA or recombinant uPA for 1 hour at 37°C.
Quantification of
Immunoblotting and Zymography
Immunoblot films and zymography gels were
scanned by using a Personal Densitometer SI (Molecular Dynamics Inc)
under the mode of 12 bits per pixel digital resolution and 50-µm
pixel size and quantified by NIH Image 1.61 on a Macintosh platform.
Each measurement was calibrated with a step tablet (Kodak Scanner Step
Tablet, ST-34). The area under the densitometry curve is expressed as
the integrated density (IDA).
Intragel and Intergel Reproducibility
Preliminary plasminogen zymography
experiments with human melanoma tPA and recombinant human uPA (from 1
to 1000 pg) demonstrated that the technique could detect as little as 1
pg of each PA. The log (integrated density) for each sample was linear
with respect to the log (PA content)
(r=0.992 and
2P<0.0001 for tPA,
r=0.988 and
2P<0.0001 for uPA). The PA
activities from both brain and plasma samples were all within that
range. The intragel reproducibility for tPA and uPA were quite
acceptable, with coefficients of variability of 2.3% (n=8) and 5.4%
(n=8), respectively, by use of 10 pg of each PA. The intergel
reproducibility of tPA and uPA was satisfactory, providing coefficients
of variability of 11.7% and 11.0%, respectively (n=6 gels
each).
dUTP Incorporation and Region of Cellular
Injury
Evidence of nuclear DNA scission/repair was taken as
an indication of significant cellular injury, as previously
described.19 Incorporation
of digoxigenin-dUTP on 10-µm cryosections was detected by the DNA
polymerase I–based procedure. The region containing cells with nuclear
dUTP incorporation (dUTP+) was defined as
the Ic. All activities were normalized for the Ic region. The validity
of this approach was confirmed by immunohistochemistry of adjacent
sections (D.-I. Chang, G.J. del Zoppo, unpublished data, 2001).
The absolute numbers and densities of total
dUTP+ cells and
dUTP+ neurons were determined within a
1.5-mm2 region of interest centered within
the Ic by computer-assisted quantitative video-imaging
microscopy.
Enzyme-Linked Immunosorbent Assays
tPA, uPA, and PAI-1 antigen contents in brain tissue
extract and plasma were determined by a commercially available ELISA
kit (American Diagnostica Inc). Assays were performed
according to the manufacturer’s instructions. Antigen concentrations
were calculated in nanograms per milligram
protein.
Analysis of Data
All data are presented as mean±SD.
Differences in the time courses of molecules between ischemic
and matched nonischemic brain samples were assessed by using
2-way ANOVA, with Bonferroni corrections for multiple comparisons.
One-way ANOVA was used for comparison between ischemic and
nonischemic animals or between hemorrhagic and nonhemorrhagic
animals. Associations between PA (activity and antigen) and
ischemic injury were performed by means of linear regression.
Jackknifed standard errors (SEs) of reported correlation coefficients
are provided for assessment of the precision of the estimated
correlations. Significance was set at
2P<0.05.
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Results |
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Zymographic Characterization of uPA, tPA, and PAI-1 Expression
Plasminogen-gelatin zymographic studies displayed clear evidence of tPA and uPA activity within 10-µm frozen sections from both ischemic and nonischemic basal ganglia (Figure 2
). Three bands (105, 61, and 54 kDa) were identified
in all plasma-free brain tissue extracts, and 2 bands (105 and 54 kDa)
were identified in all plasma samples, which were inhibited by
phenylmethylsulfonyl fluoride but not the cysteine protease
inhibitor E-64. No proteolytic bands were seen in the
plasminogen-free gel whether or not the incubation buffer
contained CaCl2. These findings confirmed that
the gelatinolytic activities were serine proteases
that cleave plasminogen to plasmin and were not MMPs.
Plasmin activity was not detectable with plasminogen-free
zymography. The 2 proteolytic bands of 61 and 105 kDa were inhibited by
a function-blocking antibody against human tPA. The 105-kDa band was
also inhibited by the anti–PAI-1 antibody, identifying it with the
tPA–PAI-1 complex. The single proteolytic band of 54 kDa was inhibited
by amiloride and by the function-blocking antibody against human
uPA.
uPA Expression During MCA:O
Parenchymal uPA activity and antigen increased
significantly within 1 to 2 hours after MCA:O in the ischemic
basal ganglia (2P<0.005)
(Figures 2, 3A, and 3B). Both uPA activity and antigen
were significantly greater in the Ic regions than in identical
nonischemic tissues
(2P<0.001)
(Figure 4). No difference in uPA activity or antigen
was observed between the sham-operated and control animals.
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In the plasma, uPA activity, but not antigen, demonstrated a
small but significant increase early after MCA:O
(2P<0.05)
(Figure 3A and 3B). Plasma uPA antigen content did not change
appreciably over the time from MCA:O.
tPA and PAI-1 Expression During MCA:O
A significant increase in PAI-1 antigen
(2P=0.001) and a decrease in
free tPA activity (2P=0.01)
were seen by 2 hours after MCA:O in the ischemic basal ganglia
compared with the nonischemic regions
(Figure 3C and 3E). A transient increase in the tPA–PAI-1
complex and a coordinate decrease in unbound tPA at 2 hours after MCA:O
(2P<0.001) explained these changes in the ischemic tissue
(Figure 5). Total parenchymal tPA antigen content
remained unchanged throughout MCA:O/R
(Figure 3D), and both tPA antigen and activity in the Ic
region were not different in control and sham-operated animals
(2P=0.55)
(Figure 4). But PAI-1 antigen in the Ic region in all
ischemic animals increased significantly
(2P<0.003).
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In plasma, only the gelatinolytic
activity of the tPA–PAI-1 complex was detected. Plasma PAI-1 antigen
increased significantly
(2P<0.05) after MCA:O, in
parallel with the increase in PAI-1 antigen in the ischemic
basal ganglia
(Figure 3E).
PA Expression and Neuronal Injury
The relations of uPA activity/antigen, tPA
activity/antigen, and PAI-I antigen content to the density of
dUTP+ cells and neurons at 2-hour MCA:O were
examined.3 19 No
significant linear relationship between the contents of each PA or PAI
and the density (number per unit area) of
dUTP+ neurons was observed: uPA
activity/antigen=(r=0.739,
SE=0.257,
2P=0.093)/(r=0.803,
SE=0.242, 2P=0.054); tPA
activity/antigen=(r=0.218,
SE=0.539,
2P=0.678)/(r=0.286,
SE=0.257, 2P=0.583); and PAI-1
antigen=(r=0.687, SE=0.232,
2P=0.132). However, a weak
association with uPA content might exist. This is because of the large
SE in the uPA relationships.
Association of PAs With Hemorrhagic
Transformation
At least 1 animal at each time point, 15 in total
(44.1%), demonstrated visible evidence of hemorrhagic transformation
within the Ic region after MCA:O. No significant differences in tPA,
uPA, and PAI-1 contents between the animals that displayed hemorrhagic
transformation and those without hemorrhagic transformation after MCA:O
were detected
(Figure 6).
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Discussion |
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Plasmin, in addition to dissolving fibrin-containing thrombi and fibrinogen, is capable of degrading selected matrix proteins of the basal lamina, elastin, and myelin basic protein either directly or through the activation of select latent MMPs.4 5 6 7 8 The ability of PAs to alter microvascular matrix integrity in response to focal cerebral ischemia has so far not been clearly defined.13 14 23 The initial step is to examine the effect of MCA:O on the PA/PAI axis within the brain parenchyma. From these experiments and those of others,12 there appears to be no zymographic evidence of free plasmin in normal brain tissue. We demonstrate an immediate significant increase in gelatin-proteolytic activity after MCA:O that was mainly due to significant increases in uPA within the ischemic basal ganglia. Parallel increases in PAI-1 were reflected by an increase in tPA–PAI-1 complex formation and a transient decrease in tPA activity in the same territory. uPA antigen and activity and tPA-associated PAI-1 antigen (assigned to the Ic region) increased significantly during focal cerebral ischemia compared with no ischemic injury. However, no relation to neuronal injury was evident. Furthermore, there was no significant difference among the animals that displayed hemorrhagic transformation and those without hemorrhagic transformation with respect to uPA and tPA/PAI-1.
The extracellular matrix provides a substructure for the endothelial cell and astrocyte components of the cerebral microvasculature, contributing a second element of the blood-brain barrier. Intact microvascular basal lamina and integrin-mediated matrix adhesion are required for cell survival.9 11 Focal cerebral ischemia degrades microvascular integrity through major alterations in endothelial cell permeability and loss of the vascular matrix.2 The major basal lamina constituents, including laminin-1, laminin-5, collagen IV, and cellular fibronectin, decrease roughly in parallel during MCA:O/R.1 18 That loss is associated with microvascular hemorrhage.2 From parallel studies, loss of basal laminal integrity and neuronal injury are accompanied by a rapid simultaneous increase in tissue expression of uPA and latent MMP-2, but not tPA, in this model.3 19 PAs may facilitate degradation of basal lamina via several pathways. Plasmin and uPA, but not tPA, can activate latent MMP-1, MMP-3, and MMP-9, or in the case of pro-MMP-2, activation is through the proteolytic activation of membrane type-1–MMP.4 8 24 25 26 Some or all of these PA-dependent effects can contribute to postischemic CNS injury.
With a modification of zymography with the use of gelatin
(collagen) as the plasmin substrate, tPA and uPA activities from
10-µm frozen sections were readily detectable and reproducible.
Within nonischemic basal ganglia, tPA activity (calculated from
the normalized integrated densities) was 
67% of uPA activity in
control animals, although tPA antigen content was 33 times that of uPA
antigen. When casein was used as the substrate for zymography, tPA
activity was 156% of uPA activity in control animals. These
differences may reflect the separate requirements for plasmin formation
by tPA and uPA activity with gelatin as the substrate, the known
contributions of fibrin to optimal in vivo plasminogen
activation by tPA,27 or
other aspects of the activation of plasminogen by tPA.
Nonetheless, the simple extraction procedure used in the present
study released nearly all uPA and tPA antigen/activity into the soluble
phase (96.8%/96.5% and 97.3%/97.4% [n=6], respectively). The
specific activities of uPA or tPA were similar in the soluble and the
nonsoluble phases (data not shown). The low tPA specific activity,
compared with uPA specific activity, is not readily explained by
inhibition with PAI-1, because the tPA–PAI-1 complex levels in
nonischemic tissue were low. Importantly, although the activity
assays attempt to detect matrix-relevant PA activities, the true
condition within cerebral tissue is unknown.
Rosenberg et al14 first described increased uPA-like and decreased tPA-like proteinase activities by 12 to 24 hours after permanent MCA:O in Wistar-Kyoto rats and SHR, whereas Ahn et al16 noted an increase in uPA-like proteinase activity and no change in tPA-like proteinase activity in C57BL/6J mice.16 Wang et al13 suggested that increases in tPA-like proteinase activity after MCA:O contribute to neurodegeneration. tPA, but not uPA, was assigned a role in neuronal injury within the murine hippocampus.13 28 The appearance of both PAs in neuronal cells has been reviewed recently.29 Pfefferkorn et al15 also observed increased PA activity within the caudate putamen by 9 hours after MCA:O in Wistar rats, although the PA was not defined. PAI-1 antigen was apparently increased 4 hours after MCA:O in Wistar rats,30 but no relation of PAI-1 to uPA and tPA activity has been reported. The very rapid (1-hour) increase in uPA, the increase in the tPA–PAI-1 complex, and the transient decrease in free tPA are consistent findings matching the rapid appearance of other gene products in the microvasculature after MCA:O in the nonhuman primate.17 31 32 33 Differences in the species and models used, ancillary effects of the anesthetics in the rodent studies, retained plasma, the use of the more sensitive zymographic assays in the present experiments, and other technical differences may also contribute to the discrepant findings. In recent studies, we have shown that residual plasma significantly decreases the activity of tPA and uPA in rodent brain tissue samples (G.J. del Zoppo, E. Lo, M. Asahi, S.E. Baer, unpublished data, 2000).19
uPA synthesis has been attributed to a number of CNS cell types, including endothelial cells, neurons, astrocytes, and microglia in vivo or in vitro.29 33 34 35 36 Given the rapid and persistent elevation of uPA in the ischemic basal ganglia, it is unlikely that uPA is derived from the plasma compartment. If only a contribution of edema to the tissue content of plasma and stable plasma uPA content are assumed, a mechanism to concentrate uPA 40-fold from plasma would be required to explain the early tissue increase in uPA content.
In contrast, the PAI-1 content of ischemic tissue and plasma increased in parallel, suggesting that plasma PAI-1 entered the edema fluid directly as early as 1 to 2 hours after MCA:O. Although PAI-1 synthesis has been attributed to endothelial cells, neurons, and astrocytes,32 37 38 any in situ production by normal brain tissue might be overwhelmed by plasma PAI-1. Given the parallel increases in tissue and plasma PAI-1, it is likely that a common source of the inhibitor is the endothelium. Increased PAI-1 mRNA and PAI-1 immunoreactive microvessels have been detected 4 hours after MCA:O in Wistar rats.30 Docagne et al17 observed that PAI-1, but not tPA, mRNA expression was increased between 24 hours and 3 days after MCA:O in mice.
In the ischemic basal ganglia, total tPA antigen did
not change, although free tPA activity decreased transiently. The early
decrease in tPA activity is explained by formation of the tPA–PAI-1
complex. tPA, uPA, and PAI-1 secretion have been attributed variously
to stimulated endothelial cells, neurons, astrocytes,
or microglia in vivo or in
vitro,32 33 34 35 36 37 38
but tPA antigen and activity did not accumulate in the ischemic
tissue in the present study. In the nonischemic brain, tPA
antigen is localized to selected noncapillary
microvessels.39 The
differential expression of tPA relative to uPA and PAI-1 might be
explained by early stimulation of uPA and PAI-1 generation by tumor
necrosis factor-
and -ß, transforming growth factor-ß, and
interleukin-1ß (all inflammatory cytokines), which do not
stimulate endothelial cell tPA synthesis and
secretion.17 32 33
Expression of these cytokines increases early within the
ischemic zone in rodents after
MCA:O.40 41 With
human microvascular endothelial cells, epidermal growth
factor increases the synthesis and secretion of tPA, which is inhibited
by tumor necrosis
factor-.42 Therefore,
microvascular endothelium may be responsible for the
increased uPA and PAI-1 antigen, but not tPA antigen.
Recent reports have suggested that tPA might promote neurodegeneration within the rat hippocampus by alteration in laminin integrity.13 43 However, there was no firm relation of uPA, tPA, and PAI-I content to neuronal injury in the ischemic basal ganglia by 2 hours of MCA:O in the present study. This differs from the significant linear relationship of pro-MMP-2 content to neuronal injury in the same setting.3 Recent reports comparing uPA(-/-), tPA(-/-), PAI-1(-/-), plasminogen (-/-), and wild-type mice suggest that the absence of tPA, but not uPA, decreased the apparent volume of ischemic injury.23 Furthermore, plasminogen (-/-) constructs had increased regions of ischemic injury. Those experiments do not address the acute condition and are subject to other concerns noted for selected well-characterized knockout and transgenic mouse preparations subject to MCA:O.23 44 45 46 Despite the lack of change in total tPA antigen, it cannot be excluded that tPA increases in microscopically localized areas and contributes to neuronal injury.
Although no direct association of uPA with hemorrhagic
transformation was observed in the present study
(Figure 6), increased uPA in the ischemic zones of
all animals could promote the activation of pro-MMP-9, which is
associated with hemorrhagic transformation in this model. At the
clinical level, it seems likely that interventional treatment with
exogenous PAs could further increase the risk of hemorrhagic
transformation if endogenous uPA is already increased. This
argument assumes that the extravasation of blood is not itself the
source of the increased pro-MMP-9.
The significant increase in uPA within the ischemic basal ganglia coincides with pro-MMP-2 generation and microvascular matrix degeneration, but not neuronal injury, after MCA:O. Despite the rapid and persistent increase in gelatin-proteolytic activity mainly due to significantly increased uPA and the decreased tPA activity 2 hours after MCA:O, the actual compartmentalization of the endogenous PAs in the CNS and their individual effects on microvascular matrix, neuronal integrity, and clinical outcome remain to be defined.
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Acknowledgments |
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This research was supported by grants RO1 NS-26945 and RO1 NS-38710 of the National Institute of Neurological Disorders and Stroke (Drs del Zoppo and Hosomi) and a fellowship from the Uehara Memorial Foundation (Dr Hosomi).
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Footnotes |
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Reprint requests to Gregory J. del Zoppo, MD, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Rd, MEM-132, La Jolla, CA 92037.
Received August 18, 2000; revision received November 22, 2000; accepted December 22, 2000.
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