(Circulation. 1999;99:2440-2444.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Role of Plasminogen System Components in Focal Cerebral Ischemic Infarction
A Gene Targeting and Gene Transfer Study in Mice
From the Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology (N.N., P.C.), and the Center for Molecular and Vascular Biology (M.D.M., H.R.L., D.C.), KU Leuven, Belgium, and the Department of Physiology, Hamamatsu University School of Medicine, Shizuoka, Japan (N.N.).
Correspondence to Désiré Collen, MD, PhD, Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, KU Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium. E-mail desire.collen{at}med.kuleuven.ac.be
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Abstract |
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Background—The role of plasminogen system components in focal cerebral ischemic infarction (FCI) was studied in mice deficient in plasminogen (Plg-/-), in tissue or urokinase plasminogen activator (tPA-/- or uPA-/-), or in plasminogen activator inhibitor-1 or
2-antiplasmin (PAI-1-/- or
2-AP-/-).
Methods and Results—FCI was produced by ligation of the left
middle cerebral artery and measured after 24 hours by planimetry of
stained brain slices. In control (wild-type) mice, infarct size was
7.6±1.1 mm3 (mean±SEM), uPA-/- mice
had similar infarcts (7.8±1.0 mm3,
P=NS), tPA-/- mice smaller (2.6±0.80
mm3, P<0.0001), PAI-1-/- mice
larger (16±0.52 mm3, P<0.0001), and
Plg-/- mice larger (12±1.2 mm3,
P=0.037) infarcts. 2-AP-/-
mice had smaller infarcts (2.2±1.1 mm3,
P<0.0001 versus wild-type), which increased to
13±2.5 mm3 (P<0.005 versus
2-AP-/-) after intravenous
injection of human 2-AP. Injection into
2-AP-/- mice of Fab fragments of
affinospecific rabbit IgG against human 2-AP, after
injection of 200 µg human 2-AP, reduced FCI from
11±1.5 to 5.1±1.1 mm3 (P=0.004).
Conclusions—Plg system components affect FCI at 2 different
levels: (1) reduction of tPA activity (tPA gene
inactivation) reduces whereas its augmentation (PAI-1
gene inactivation) increases infarct size, and (2) reduction of Plg
activity (Plg gene inactivation or 2-AP
injection) increases whereas its augmentation
(2-AP gene inactivation or
2-AP neutralization) reduces infarct size. Inhibition of
2-AP may constitute a potential avenue to treatment
of FCI.
Key Words: plasminogen • plasminogen activators • cerebral infarction • cerebral ischemia
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Neuronal degeneration in central nervous system diseases such as stroke, epilepsy, and Alzheimer's disease is thought to be stimulated by an excess of the excitatory amino acid glutamate.1 2 Injection of glutamate agonists into the central nervous system indeed induces hippocampal neuronal cell death similar to that observed in neurodegenerative diseases.3 Excitotoxin-induced neuronal degeneration is mediated by tissue plasminogen activator (tPA).4 Consistent with this observation, mice deficient in tPA are resistant to, and infusion of plasminogen activator inhibitor-1 (PAI-1) protects against, excitotoxin-mediated hippocampal neuronal degeneration.4 5 6 Furthermore, deficiency of plasminogen (Plg), the zymogen substrate of tPA, and infusion of
2-antiplasmin
(2-AP), the physiological
plasmin inhibitor, protect mice against excitotoxin-induced
hippocampal neuronal death.5 It has been proposed that
plasmin-mediated degradation of laminin sensitizes hippocampal neurons
to cell death by disrupting neuron–extracellular matrix
interaction.7 Wang et al8 recently demonstrated that neuronal damage after focal cerebral ischemia induced by transient occlusion of the middle cerebral artery (MCA) was also reduced in mice with tPA deficiency and exacerbated by tPA infusion. Thus, the Plg system may be involved both in establishing a cerebral ischemic infarct and in its extension during thrombolytic therapy. We recently demonstrated that the neurotoxic effect of tPA on persistent focal cerebral ischemia also occurred with other thrombolytic agents, including streptokinase and staphylokinase.9 Thus, in patients with persistent cerebral arterial occlusion, thrombolytic therapy for ischemic stroke may cause infarct extension, which would not only partially offset the established overall beneficial effect of arterial recanalization10 11 but would indeed be harmful to a subgroup of patients. Because it is not possible to distinguish between patients who will and those who will not achieve cerebral arterial recanalization with thrombolytic therapy, the development of specific conjunctive strategies to counteract the neurotoxic effects of thrombolytic agents on persisting focal cerebral ischemia appears to be warranted.
Although it is assumed that neuronal injury during focal
ischemia in the brain occurs primarily as a result of
accumulation of excitotoxins, such as glutamates, the role of
plasmin-mediated laminin degradation or alternative mechanisms in the
pathogenesis of cortical neuronal cell death has not been demonstrated.
To delineate the contribution of individual components of the Plg
(fibrinolytic) system on focal cerebral ischemic infarction,
infarct size produced by ligation of the left MCA was quantified
in mice with targeted inactivation of Plg, its activators
tPA or uPA, or the fibrinolytic inhibitors PAI-1 or
2-AP. In addition, the effects of adenoviral
transfer of the tPA and PAI-1 genes and of
infusion of human 2-AP on cerebral infarction
were studied.
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Methods |
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Mice With Targeted Inactivation of Genes Encoding Plg System Components
All mice included in the present study were generated and bred at the Specific Pathogen Free Facility of the Center for Transgene Technology and Gene Therapy, Campus Gasthuisberg, KU Leuven, Belgium. Gene inactivation was obtained by homologous recombination in embryonic stem cells of the genes encoding tPA,12 urokinase plasminogen activator (uPA),12 PAI-1,13 14 Plg,15 or
2-AP,16 as previously described.
Mice with inactivated genes encoding uPA
receptor17 were not included because of the normal results
obtained with uPA-deficient mice.
Adenovirus-Mediated Transfer of tPA or
PAI-1 Genes
The recombinant adenoviruses AdCMVtPA and AdCMVPAI-1 were
generated by homologous recombination in 293 cells essentially as
previously described.18 19 20 After transfection,
recombinant viral plaques were harvested and amplified as
described.21 22 23 Large-scale production of
recombinant adenovirus was performed as described.21 The
kinetics and organ distribution of tPA and PAI-1 expression after
adenoviral transfer by intravenous bolus injection have
been described in detail elsewhere.24 25
Injection of Human 2-AP and Affinospecific Rabbit
Anti-Human 2-AP Fab Fragments
Human 2-AP was prepared from
fresh-frozen plasma as previously described.26 Pooled
rabbit antisera raised against human 2-AP were
chromatographed on protein-A Sepharose, and affinospecific
antibodies were obtained from the IgG pool by
chromatography on a CNBr-activated Sepharose
column substituted with human 2-AP, yielding
0.1 mg specific IgG/mg applied. Fab fragments were obtained from the
affinospecific IgG by digestion with 1% (wt/wt) papain in the presence
of 50 mmol/L cysteine, 1 mmol/L EDTA, 0.1 mol/L phosphate
buffer, pH 7.0, for 5 hours. The reaction was arrested by addition of
iodoacetamide to a final concentration of 75 mmol/L. After
dialysis, the mixture was purified on a protein A Sepharose column
equilibrated with PBS. Fab concentration was determined by ELISA
calibrated against an IgG standard. SDS gel electrophoresis essentially
revealed homogeneous Fab fragments (not shown).
Murine Cerebral Ischemic Infarction Model
Animal experiments were conducted according to the guiding
principles of the American Physiological Society
and the International Committee on Thrombosis and
Hemostasis.27
Focal cerebral ischemia was produced by persistent occlusion of the MCA according to Welsh et al.28 Briefly, mice of either sex, weighing 20 to 30 g, were anesthetized by intraperitoneal injection of ketamine (75 mg/mL, Apharmo) and xylazine (5 mg/mL, Bayer). Atropine (1 mg/kg, Federa) was administered intramuscularly, and body temperature was maintained by keeping the animals on a heating pad. A U-shape incision was made between the left ear and left eye. The top and back segments of the temporal muscle were transected, and the skull was exposed by retraction of the temporal muscle. A small opening (1 to 2 mm in diameter) was made in the region over the MCA with a handheld drill, with saline superfusion to prevent heat injury. The meninges were removed with a forceps, and the MCA was occluded by ligation with 10-0 nylon thread (Ethylon) and transected distally to the ligation point. Finally, the temporal muscle and skin were sutured back in place.
AdCMVtPA, AdCMVPAI-1, or AdRR5 was given as an intravenous
bolus injection of 1.3x109 cfu 4 days before
ligation of the MCA. Human 2-AP
(h2-AP) was given intravenously,
divided into 2 injections, given 1 minute before and 30 minutes after
ligation of the MCA, respectively. Fab fragments were injected
intravenously as a bolus 10 minutes after the second
h2-AP injection.
The animals were allowed to recover and were then returned to their
cages. After 24 hours, the animals were killed with an overdose of
Nembutal (500 mg/kg, Abbott Laboratories) and decapitated. The brain
was removed and placed in a matrix for sectioning into 1-mm segments.
The sections were immersed in 2%
2,3,5-triphenyltetrazolium chloride (TTC)
in saline,29 incubated for 30 minutes at 37°C, and
placed in 4% formalin in PBS. With this procedure, the necrotic
infarct area remains unstained (white) and is clearly distinguishable
from stained (brick red) viable tissue (Figure 1
). The sections were photographed and
subjected to planimetry. The infarct volume was defined as the sum of
the unstained areas of the sections multiplied by their thickness. A
highly significant correlation has previously been observed between
infarct size determined by TTC staining and clinical score
(r=0.67, P<0.0001) in rabbits 5 hours after
thrombotic focal cerebral infarction.30
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Immunohistochemical Analyses
For immunostaining of laminin on paraffin
sections, sections were incubated with a primary rabbit anti-laminin
antibody (Sigma Chemical Co) diluted 1:50, followed by a
peroxidase-labeled swine anti-rabbit IgG (Dako) diluted 1:50.
Fibrin(ogen) was stained via a 3-step procedure with a goat anti-mouse
fibrinogen (Nordic Immunologies) diluted 1:200, followed by rabbit
anti-goat IgG (Dakopatts) diluted 1:100 and goat
peroxidase–anti-peroxidase complex (Dakopatts) diluted 1:50.
Peroxidase activity was developed by incubating sections in 0.05 mol/L
Tris-HCl buffer (pH 7.0) containing 0.06% 3,3'-diaminobenzidine and
0.01% H2O2, followed by
counterstaining with hematoxylin.
Statistical Analysis
The data are represented as mean±SEM of n
determinations. The significance of differences was determined by ANOVA
followed by Fisher's protected least significant difference test with
the StatView software package.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Cerebral Ischemic Infarct Size in Mice With Targeted Inactivation of Genes Encoding Plg System Components
Ligation of the left MCA induced a cerebral infarct with a volume of 7.6±1.1 mm3 (n=11) in wild-type mice with a mixed (50% S129 and 50% C57BL/6) genetic background, a volume of 9.3±2.7 mm3 (n=6) in inbred C57BL/6 mice, and a volume of 6.4±1.3 mm3 (n=6) in inbred S129 mice (P=NS versus mixed background, results not shown).
Inactivation of the tPA gene was associated with a
significant reduction of infarct size to 2.6±0.80
mm3 (n=11) (P<0.0001 versus wild-type
mice), whereas inactivation of the uPA gene had no effect on
infarct size (7.8±1.0 mm3, n=8,
P=NS versus wild-type). Inactivation of the PAI-1
gene was associated with a significant increase in infarct size
(16±0.52 mm3, n=6, P<0.0001
versus wild-type) (Figure 2). In mice
with inactivated Plg genes, cerebral infarct
size was significantly larger than in wild-type mice (12±1.2
mm3, n=9, P=0.037 versus wild-type),
whereas, conversely, in 2-AP
gene–deficient mice, infarct size was markedly reduced (2.2±1.1
mm3, n=7, P=0.0001 versus wild-type)
(Figure 2).
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Effect of tPA and PAI-1 Gene Transfer
on Cerebral Infarct Size
Injection of 1.3x109 pfu of AdCMVtPA in 6
tPA-/- mice 4 days before MCA ligation was
associated with a cerebral infarct size of 6.0±1.3
mm3, significantly larger than the infarcts in 5
tPA-/- mice injected with the control virus
AdRR5 (1.8±0.63 mm3, P=0.028) (Figure 3A). Conversely, injection of
1.3x109 pfu of AdCMVPAI-1 in 5
PAI-1-/- mice was associated with a cerebral
infarct size of 10±1.4 mm3, significantly
smaller than the infarcts in 5 PAI-1-/- mice
injected with the control virus AdRR5 (13±1.0
mm3, P=0.019) (Figure 3B).
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Effect of 2-AP on Cerebral Infarct Size
Cerebral infarct size correlated with
2-AP gene dose, corresponding to
11±2.0, 4.9±2.0, and 2.2±1.1 mm3 in
wild-type and heterozygously and homozygously deficient mice,
respectively (Figure 4A). Injection of
h2-AP in
2-AP-/- mice increased
the infarct size to 13±2.5 mm3 (n=4) with a
1-mg total dose and to 11±1.5 mm3 (n=6)
with a 0.2-mg total dose. Injection of 1.7 mg affinospecific Fab
against h2-AP in mice given 0.2 mg
h2-AP reduced the cerebral infarct size to
5.1±1.1 mm3 (n=7, P=0.004 versus
0.2 mg h2-AP) (Figure 4B).
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Immunochemical Staining
Light-microscopic analysis of brain sections stained
for laminin revealed clear immunoreactivity in the hippocampus but not
outside the vessels in the ischemic or the viable cortex region
(Figure 5A).
Immunostaining of fibrin(ogen) revealed increased
extravascular reactivity in PAI-1-/- mice in
the infarcted zone and its penumbra and some intravascular fibrin
deposition in the infarcted region of the other genotypes,
without a clear difference in density of immunoreactivity between
Plg-/- and
2-AP-/- mice (apart
from the differences in infarct size).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the present study, the effect of specific deficiencies of Plg system components on focal cerebral ischemic infarct size in mice was studied in an effort to gain further insight into the mechanism of infarct expansion by endogenous as well as exogenous tPA.8 9 A better understanding of this phenomenon might indeed lead to development of conjunctive treatments to reduce potential neurotoxic side effects of ischemic stroke and of thrombolytic therapy in patients with persisting focal cerebral ischemia.
The synergistic action of Plg system activation and excitotoxins
on neuronal cell death in the hippocampus has been studied in
significant detail.4 5 6 7 Using a mouse model with
stereotactic injection of the glutamate analogue kainic
acid in the hippocampus, they found that local tPA administration
exacerbated whereas tPA deficiency or PAI-1 injection reduced
hippocampal neuronal cell death. Furthermore, both Plg deficiency and
local 2-AP injection protected against
excitotoxin-induced neuronal degeneration. All findings were compatible
with a mechanism in which tPA-mediated plasmin generation induced
laminin degradation, resulting in disturbed neuron–extracellular
matrix interaction. The extrapolation from these observations was that
inhibitors of the hippocampal extracellular tPA/plasmin
proteolytic cascade might protect neurons against excitotoxin-mediated
brain disorders.
Focal cerebral ischemia induced by MCA occlusion is also
associated with excitotoxin-mediated enhanced neuronal cell death and
administration of tPA exacerbates persistent ischemic infarct
size,8 9 suggesting that cortical ischemic
neuronal cell death may be sensitive to a similar tPA/plasmin
proteolytic cascade activation. This working hypothesis could not be
confirmed in the present study, however. Whereas the
findings4 5 6 7 that tPA deficiency protects against focal
cerebral ischemic infarction were fully confirmed and extended
by the observation that PAI-1 deficiency resulted in significantly
larger infarcts, the observation that Plg deficiency protects against
excitotoxin-induced neuronal cell death could not be confirmed.
Instead, we found that focal cerebral infarct size was significantly
larger in mice with Plg deficiency and, conversely, significantly
smaller in mice with 2-AP deficiency. This
internal consistency makes it unlikely that the discrepancy
with the earlier observations on hippocampal neuronal degeneration
might be coincidental or relate to differences in genetic background
between the strains used in the two studies. Furthermore, although it
has been shown that procedural and strain-related variables may
significantly affect outcome in a murine model of focal cerebral
ischemia,31 we have not observed significant
differences in infarct size in mice with a C57BL/6, S129, or mixed
background.
Immunohistochemical staining revealed the presence of laminin in the
hippocampus but not outside the vessels in the ischemic or
contralateral cortex. Fibrin(ogen) staining revealed increased
extravascular reactivity in PAI-1-/- mice,
suggestive of altered vascular permeability. Intravascular fibrin
deposition was observed to a comparable extent in
Plg-/- and in
2-AP-/- mice, although
the area in which this deposition occurred was much larger in the
Plg-/- mice.
In aggregate, our findings are incompatible with the unique linked
cause-and-effect pathway demonstrated in the hippocampus (tPA-mediated
plasmin generation, laminin degradation, and neuronal cell death).
Instead, the data are suggestive of 2 independent mechanisms operating
in opposite directions. At present, one can only speculate about
possible mechanisms. tPA (and other exogenous
thrombolytic agents) might affect the permeability of
the blood-brain barrier, either directly or via activation of a
substrate different from Plg, allowing excitotoxins to diffuse more
readily and cause infarct extension. Altered permeability is suggested
by the increased extravascular fibrin(ogen) deposition in the infarct
zone of PAI-1-/- mice, but this will need
further confirmation. Conversely, reduction of Plg activity might shift
the hemostatic balance in the penumbra of the infarct toward transient
intravascular deposition of fibrin. Although the density of fibrin
deposition was not different between Plg-/- and
2-AP-/- mice, the
total fibrin burden was much larger in the former because of the
severalfold larger infarct size.
The internally consistent observations with
2-AP were unexpected but are potentially
relevant for the treatment of ischemic stroke. First, a
correlation was found between infarct size and genotype, with
heterozygotes displaying infarct sizes between those of the wild-type
and homozygous phenotypes. Second, bolus injection of
h2-AP in
2-AP-/- mice caused a
dose-related infarct expansion. Finally, and importantly, Fab fragments
from affinospecific polyclonal rabbit
anti–h2-AP antibodies significantly reduced
the cerebral ischemic infarct size. This observation suggests
that 2-AP inhibitors (eg,
neutralizing monoclonal antibodies) might counteract focal
ischemic infarction.
To be applicable to humans, the present observations obtained in a
"heterologous and discontinuous" system, ie, using bolus
h2-AP in
2-AP-/- mice, need to
be extrapolated to the "autologous continuous" system in wild-type
mice, eg, with a neutralizing monoclonal antibody. Efforts to produce
monoclonal antibodies neutralizing murine 2-AP
have been initiated by immunization of
2-AP-/- mice with
purified murine 2-AP. On the basis of previous
experience with other Plg system components,
2-AP-/- mice are
anticipated to be able to produce neutralizing monoclonal antibodies
against murine 2-AP.32 The
concentration of 2-AP in human plasma is
1 µmol/L,33 corresponding to a total body pool of
500 mg. An equivalent dose of a monoclonal Fab fragment would be
400 mg, which would be high but not excessive for a single
administration. Furthermore, the observation that infarct size is
proportional to the 2-AP level (derived from
the gene dose effect and the dose-response effect of
h2-AP transfusion) suggests that even a
partial reduction of the plasma level might have a beneficial effect.
In view of the excessive morbidity associated with ischemic
stroke, further exploration of this potential avenue to reduction of
focal cerebral ischemic infarct size would seem to be
warranted.
Received October 14, 1998; revision received January 21, 1999; accepted January 25, 1999.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Coyle JT, Puttfarcken P. Oxidative stress, glutamate and neurodegenerative disorders. Science. 1993;262:689–695.
2.
Lipton SA, Rosenberg PA. Excitatory amino acids as a
final common pathway for neurological disorders. N Engl
J Med. 1994;330:613–622.
3. Coyle JT, Molliver ME, Kuhar MJ. In situ injection of kainic acid: a new method for selectively lesioning neuronal cell bodies while sparing axons of passage. J Comp Neurol. 1978;180:301–323.[Medline] [Order article via Infotrieve]
4. Tsirka S, Gualandris A, Amaral DG, Strickland S. Excitotoxin induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature. 1995;377:340–344.[Medline] [Order article via Infotrieve]
5.
Tsirka S, Rogove AD, Bugge TH, Degen JL, Strickland S.
An extracellular proteolytic cascade promotes neuronal degeneration in
the mouse hippocampus. J Neurosci. 1997;17:543–552.
6. Tsirka S, Rogove AD, Strickland S. Neuronal cell death and t-PA. Nature. 1996;384:123–124.[Medline] [Order article via Infotrieve]
7. Chen ZL, Strickland S. Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell. 1997;91:917–925.[Medline] [Order article via Infotrieve]
8. Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nat Medi. 1998;4:228–231.[Medline] [Order article via Infotrieve]
10.
The National Institute of Neurological Disorders, and
Stroke rt-PA Stroke Study Group. Tissue plasminogen
activator for acute ischemic stroke. N
Engl J Med. 1995;333:1581–1587.
11.
Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von
Kummer R, Boysen G, Bluhmki E, Haxter G, Mahagne MH, Hennerici M, for
the ECASS Study Group. Intravenous
thrombolysis with recombinant tissue
plasminogen activator for acute hemispheric
stroke: the European Cooperative Acute Stroke Study (ECASS).
JAMA. 1995;274:1017–1025.
12. Carmeliet P, Schoonjans L, Kieckens L, Ream B, Degen J, Bronson R, De Vos R, van den Oord J, Collen D, Mulligan R. Physiological consequences of loss of plasminogen activator gene function in mice. Nature. 1994;368:419–424.[Medline] [Order article via Infotrieve]
13. Carmeliet P, Kieckens L, Schoonjans L, Ream B, Van Nuffelen A, Prendergast G, Cole M, Bronson R, Collen D, Mulligan RC. Plasminogen activator inhibitor-1 gene-deficient mice, I: generation by homologous recombination and characterization. J Clin Invest. 1993;92:2746–2755.
14. Carmeliet P, Stassen JM, Schoonjans L, Ream B, van den Oord JJ, De Mol M, Mulligan RC, Collen D. Plasminogen activator inhibitor-1 deficient mice, II: effects on hemostasis, thrombosis and thrombolysis. J Clin Invest. 1993;92:2756–2760.
15.
Poplis VA, Carmeliet P, Vazirzadeh S, Van Vlaenderen I,
Moons L, Plow EF, Collen D. Effects of disruption of the
plasminogen gene on thrombosis, growth, and health in mice.
Circulation. 1995;92:2585–2593.
16.
Lijnen HR, Okada K, Matsuo O, Collen D, Dewerchin
M. 2-Antiplasmin gene-deficiency in mice
is associated with enhanced fibrinolytic potential without overt
bleeding. Blood. 1999. In press.
17. Dewerchin M, Van Nuffelen A, Wallays G, Bouché A, Moons L, Carmeliet P, Mulligan RC, Collen D. Generation and characterization of urokinase receptor-deficient mice. J Clin Invest. 1996;97:870–878.[Medline] [Order article via Infotrieve]
18. McGrory WJ, Bautista DS, Graham FL. A simple technique for the rescue of early region 1 mutations into infectious human adenovirus type 5. Virology. 1988;163:614–617.[Medline] [Order article via Infotrieve]
19.
Gomez-Foix AM, Coats WS, Baque S, Alam T, Gerard RD,
Newgard CB. Adenovirus-mediated transfer of the muscle glycogen
phosphorylase gene into hepatocytes confers
altered regulation of glycogen metabolism. J
Biol Chem. 1992;267:25129–25134.
20.
Graham FL, Smiley J, Russel WC, Nairn R.
Characteristics of a human cell line transformed by DNA from human
adenovirus type 5. J Gen Virol. 1977;36:59–74.
21. Gerard RD, Meidell RS. Adenovirus vectors. In: Hames BD, Glover D, eds. DNA Cloning: A Practical Approach: Mammalian Systems. Oxford, UK: Oxford University Press; 1995:285–307.
22.
Alcorn JL, Gao E, Chen Q, Smith ME, Gerard RD,
Mendelson CR. Genomic elements involved in transcriptional regulation
of the rabbit surfactant protein-A gene. Mol Endocrinol. 1993;7:1072–1085.
23.
Kopfler WP, Willard M, Betz T, Willard JE, Gerard RD,
Meidell RS. Adenovirus-mediated transfer of a gene encoding human
apolipoprotein A-I into normal mice increases circulating high-density
lipoprotein cholesterol. Circulation. 1994;90:1319–1327.
24.
Carmeliet P, Stassen JM, Van Vlaenderen I, Meidell RS,
Collen D, Gerard RD. Adenovirus-mediated transfer of tissue-type
plasminogen activator augments
thrombolysis in tissue-type plasminogen
activator-deficient and plasminogen
activator inhibitor-1-overexpressing mice.
Blood. 1997;90:1527–1534.
25.
Carmeliet P, Moons L, Lijnen R, Janssens S, Lupu F,
Collen D, Gerard RD. Inhibitory role of
plasminogen activator inhibitor-1
in arterial wound healing and neointima
formation. Circulation. 1997;96:3180–3191.
26.
Lijnen HR, Holmes WE, Van Hoef B, Wiman B, Rodriguez H,
Collen D. Amino-acid sequence of human
2-antiplasmin. Eur J Biochem. 1987;166:565–574.[Medline]
[Order article via Infotrieve]
27. Giles AR. Guidelines for the use of animals in biomedical research. Thromb Haemost. 1987;58:1078–1084.[Medline] [Order article via Infotrieve]
28. Welsh FA, Sakamoto T, McKee AE, Sims RE. Effect of lactacidosis on pyridine nucleotide stability during ischemia in mouse brain. J Neurochem. 1987;49:846–851.[Medline] [Order article via Infotrieve]
29.
Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis
RL, Bartkowski HM. Evaluation of
2,3,5-triphenyltetrazolium chloride as a
stain for detection and quantification of experimental cerebral
infarction in rats. Stroke. 1986;17:1304–1308.
30.
Vanderschueren S, Van Vlaenderen I, Collen D.
Intravenous thrombolysis with recombinant
staphylokinase versus tissue-type plasminogen
activator in a rabbit embolic stroke model.
Stroke. 1997;28:1783–1788.
31. Connolly ES Jr, Winfree CJ, Stern DM, Solomon RA, Pinsky DJ. Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia. Neurosurgery. 1996;18:523–532.
32.
Declerck P, Carmeliet P, Verstreken M, De Cock F,
Collen D. Generation of monoclonal antibodies against autologous
proteins in gene-inactivated mice. J Biol
Chem. 1995;270:8397–8400.
33.
Collen D, Wiman B. Turnover of antiplasmin, the
fast-acting plasmin inhibitor of plasma. Blood. 1979;53:313–324.
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 H. Yuan, K. M. Vance, C. E. Junge, M. T. Geballe, J. P. Snyder, J. R. Hepler, M. Yepes, C.-M. Low, and S. F. Traynelis The Serine Protease Plasmin Cleaves the Amino-terminal Domain of the NR2A Subunit to Relieve Zinc Inhibition of the N-Methyl-D-aspartate Receptors J. Biol. Chem., May 8, 2009; 284(19): 12862 - 12873. [Abstract] [Full Text] [PDF]
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 A. K. M. T. Zaman, C. J. French, D. J. Schneider, and B. E. Sobel A Profibrotic Effect of Plasminogen Activator Inhibitor Type-1 (PAI-1) in the Heart Experimental Biology and Medicine, March 1, 2009; 234(3): 246 - 254. [Abstract] [Full Text] [PDF]
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 C. Zhang, J. An, D. K. Strickland, and M. Yepes The Low-Density Lipoprotein Receptor-Related Protein 1 Mediates Tissue-Type Plasminogen Activator-Induced Microglial Activation in the Ischemic Brain Am. J. Pathol., February 1, 2009; 174(2): 586 - 594. [Abstract] [Full Text] [PDF]
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M. Thiyagarajan, J. A. Fernandez, S. M. Lane, J. H. Griffin, and B. V. Zlokovic Activated Protein C Promotes Neovascularization and Neurogenesis in Postischemic Brain via Protease-Activated Receptor 1 J. Neurosci., November 26, 2008; 28(48): 12788 - 12797. [Abstract] [Full Text] [PDF]
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 N. Lebeurrier, S. Launay, R. Macrez, E. Maubert, H. Legros, A. Leclerc, S. P. Jamin, J.-Y. Picard, S. Marret, V. Laudenbach, et al. Anti-Mullerian-hormone-dependent regulation of the brain serine-protease inhibitor neuroserpin J. Cell Sci., October 15, 2008; 121(20): 3357 - 3365. [Abstract] [Full Text] [PDF]
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 J. An, C. Zhang, R. Polavarapu, X. Zhang, X. Zhang, and M. Yepes Tissue-type plasminogen activator and the low-density lipoprotein receptor-related protein induce Akt phosphorylation in the ischemic brain Blood, October 1, 2008; 112(7): 2787 - 2794. [Abstract] [Full Text] [PDF]
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F. Adhami, D. Yu, W. Yin, A. Schloemer, K. A. Burns, G. Liao, J. L. Degen, J. Chen, and C.-Y. Kuan Deleterious Effects of Plasminogen Activators in Neonatal Cerebral Hypoxia-Ischemia Am. J. Pathol., June 1, 2008; 172(6): 1704 - 1716. [Abstract] [Full Text] [PDF]
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R. Polavarapu, J. An, C. Zhang, and M. Yepes Regulated Intramembrane Proteolysis of the Low-Density Lipoprotein Receptor-Related Protein Mediates Ischemic Cell Death Am. J. Pathol., May 1, 2008; 172(5): 1355 - 1362. [Abstract] [Full Text] [PDF]
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M. A. Ozog, G. Modha, J. Church, R. Reilly, and C. C. Naus Co-administration of Ciliary Neurotrophic Factor with Its Soluble Receptor Protects against Neuronal Death and Enhances Neurite Outgrowth J. Biol. Chem., March 7, 2008; 283(10): 6546 - 6560. [Abstract] [Full Text] [PDF]
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 C. Orset, R. Macrez, A. R. Young, D. Panthou, E. Angles-Cano, E. Maubert, V. Agin, and D. Vivien Mouse Model of In Situ Thromboembolic Stroke and Reperfusion Stroke, October 1, 2007; 38(10): 2771 - 2778. [Abstract] [Full Text] [PDF]
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X. Zhang, R. Polavarapu, H. She, Z. Mao, and M. Yepes Tissue-Type Plasminogen Activator and the Low-Density Lipoprotein Receptor-Related Protein Mediate Cerebral Ischemia-Induced Nuclear Factor-{kappa}B Pathway Activation Am. J. Pathol., October 1, 2007; 171(4): 1281 - 1290. [Abstract] [Full Text] [PDF]
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R. Polavarapu, M. C. Gongora, H. Yi, S. Ranganthan, D. A. Lawrence, D. Strickland, and M. Yepes Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit Blood, April 15, 2007; 109(8): 3270 - 3278. [Abstract] [Full Text] [PDF]
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K. Benchenane, H. Castel, M. Boulouard, R. Bluthe, M. Fernandez-Monreal, B. D. Roussel, J. P. Lopez-Atalaya, S. Butt-Gueulle, V. Agin, E. Maubert, et al. Anti-NR1 N-terminal-domain vaccination unmasks the crucial action of tPA on NMDA-receptor-mediated toxicity and spatial memory J. Cell Sci., February 15, 2007; 120(4): 578 - 585. [Abstract] [Full Text] [PDF]
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 S. Akkawi, T. Nassar, M. Tarshis, D. B. Cines, and A. A.-R. Higazi LRP and {alpha}vbeta3 mediate tPA activation of smooth muscle cells Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1351 - H1359. [Abstract] [Full Text] [PDF]
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R. Polavarapu, M. C. Gongora, J. A. Winkles, and M. Yepes Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Increases the Permeability of the Neurovascular Unit through Nuclear Factor-{kappa}B Pathway Activation J. Neurosci., November 2, 2005; 25(44): 10094 - 10100. [Abstract] [Full Text] [PDF]
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C. Reddrop, R. X. Moldrich, P. M. Beart, M. Farso, G. T. Liberatore, D. W. Howells, K.-U. Petersen, W.-D. Schleuning, and R. L. Medcalf Vampire Bat Salivary Plasminogen Activator (Desmoteplase) Inhibits Tissue-Type Plasminogen Activator-Induced Potentiation of Excitotoxic Injury Stroke, June 1, 2005; 36(6): 1241 - 1246. [Abstract] [Full Text] [PDF]
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M. Yepes, S. A.N. Brown, E. G. Moore, E. P. Smith, D. A. Lawrence, and J. A. Winkles A Soluble Fn14-Fc Decoy Receptor Reduces Infarct Volume in a Murine Model of Cerebral Ischemia Am. J. Pathol., February 1, 2005; 166(2): 511 - 520. [Abstract] [Full Text] [PDF]
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M. Fernandez-Monreal, J. P. Lopez-Atalaya, K. Benchenane, M. Cacquevel, F. Dulin, J.-P. Le Caer, J. Rossier, A.-C. Jarrige, E. T. MacKenzie, N. Colloc'h, et al. Arginine 260 of the Amino-terminal Domain of NR1 Subunit Is Critical for Tissue-type Plasminogen Activator-mediated Enhancement of N-Methyl-D-aspartate Receptor Signaling J. Biol. Chem., December 3, 2004; 279(49): 50850 - 50856. [Abstract] [Full Text] [PDF]
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M. Yepes and D. A. Lawrence New Functions for an Old Enzyme: Nonhemostatic Roles for Tissue-Type Plasminogen Activator in the Central Nervous System Experimental Biology and Medicine, December 1, 2004; 229(11): 1097 - 1104. [Abstract] [Full Text] [PDF]
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Y. Suzuki, F. Chen, Y. Ni, G. Marchal, D. Collen, and N. Nagai Microplasmin Reduces Ischemic Brain Damage and Improves Neurological Function in a Rat Stroke Model Monitored With MRI Stroke, October 1, 2004; 35(10): 2402 - 2406. [Abstract] [Full Text] [PDF]
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D.N. Atochin, J.C. Murciano, Y. Gursoy-Ozdemir, T. Krasik, F. Noda, C. Ayata, A.K. Dunn, M.A. Moskowitz, P.L. Huang, and V.R. Muzykantov Mouse Model of Microembolic Stroke and Reperfusion Stroke, September 1, 2004; 35(9): 2177 - 2182. [Abstract] [Full Text] [PDF]
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B.-Q. Zhao, Y. Ikeda, H. Ihara, T. Urano, W. Fan, S. Mikawa, Y. Suzuki, K. Kondo, K. Sato, N. Nagai, et al. Essential role of endogenous tissue plasminogen activator through matrix metalloproteinase 9 induction and expression on heparin-produced cerebral hemorrhage after cerebral ischemia in mice Blood, April 1, 2004; 103(7): 2610 - 2616. [Abstract] [Full Text] [PDF]
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T. Nassar, S.'e. Akkawi, A. Shina, A. Haj-Yehia, K. Bdeir, M. Tarshis, S. N. Heyman, and A. A.-R. Higazi In vitro and in vivo effects of tPA and PAI-1 on blood vessel tone Blood, February 1, 2004; 103(3): 897 - 902. [Abstract] [Full Text] [PDF]
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T. Hoekstra, J. M. Geleijnse, C. Kluft, E. J. Giltay, F. J. Kok, and E. G. Schouten 4G/4G Genotype of PAI-1 Gene Is Associated With Reduced Risk of Stroke in Elderly Stroke, December 1, 2003; 34(12): 2822 - 2828. [Abstract] [Full Text] [PDF]
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J. P. Melchor, R. Pawlak, and S. Strickland The Tissue Plasminogen Activator-Plasminogen Proteolytic Cascade Accelerates Amyloid-{beta} (A{beta}) Degradation and Inhibits A{beta}-Induced Neurodegeneration J. Neurosci., October 1, 2003; 23(26): 8867 - 8871. [Abstract] [Full Text] [PDF]
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E. M. Conway, F. Zwerts, V. Van Eygen, A. DeVriese, N. Nagai, W. Luo, and D. Collen Survivin-Dependent Angiogenesis in Ischemic Brain: Molecular Mechanisms of Hypoxia-Induced Up-Regulation Am. J. Pathol., September 1, 2003; 163(3): 935 - 946. [Abstract] [Full Text] [PDF]
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E. Dai, H. Guan, L. Liu, S. Little, G. McFadden, S. Vaziri, H. Cao, I. A. Ivanova, L. Bocksch, and A. Lucas Serp-1, a Viral Anti-inflammatory Serpin, Regulates Cellular Serine Proteinase and Serpin Responses to Vascular Injury J. Biol. Chem., May 9, 2003; 278(20): 18563 - 18572. [Abstract] [Full Text] [PDF]
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G. T. Liberatore, A. Samson, C. Bladin, W.-D. Schleuning, and R. L. Medcalf Vampire Bat Salivary Plasminogen Activator (Desmoteplase): A Unique Fibrinolytic Enzyme That Does Not Promote Neurodegeneration Stroke, February 1, 2003; 34(2): 537 - 543. [Abstract] [Full Text] [PDF]
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P. A. Lapchak, D. M. Araujo, S. Pakola, D. Song, J. Wei, and J. A. Zivin Microplasmin: A Novel Thrombolytic That Improves Behavioral Outcome After Embolic Strokes in Rabbits Stroke, September 1, 2002; 33(9): 2279 - 2284. [Abstract] [Full Text] [PDF]
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 Z. Zhang, L. Zhang, M. Yepes, Q. Jiang, Q. Li, P. Arniego, T. A. Coleman, D. A. Lawrence, and M. Chopp Adjuvant Treatment With Neuroserpin Increases the Therapeutic Window for Tissue-Type Plasminogen Activator Administration in a Rat Model of Embolic Stroke Circulation, August 6, 2002; 106(6): 740 - 745. [Abstract] [Full Text] [PDF]
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 N. Mataga, N. Nagai, and T. K. Hensch Permissive proteolytic activity for visual cortical plasticity PNAS, May 28, 2002; 99(11): 7717 - 7721. [Abstract] [Full Text] [PDF]
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M. Xue and M. R. Del Bigio Acute Tissue Damage After Injections of Thrombin and Plasmin into Rat Striatum Stroke, September 1, 2001; 32(9): 2164 - 2169. [Abstract] [Full Text] [PDF]
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N. Hosomi, J. Lucero, J. H. Heo, J. A. Koziol, B. R. Copeland, and G. J. del Zoppo Rapid Differential Endogenous Plasminogen Activator Expression After Acute Middle Cerebral Artery Occlusion Stroke, June 1, 2001; 32(6): 1341 - 1348. [Abstract] [Full Text] [PDF]
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N. Nagai, M. De Mol, B. Van Hoef, M. Verstreken, and D. Collen Depletion of circulating {alpha}2-antiplasmin by intravenous plasmin or immunoneutralization reduces focal cerebral ischemic injury in the absence of arterial recanalization Blood, May 15, 2001; 97(10): 3086 - 3092. [Abstract] [Full Text] [PDF]
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 B. D. Car and V. M. Eng Special Considerations in the Evaluation of the Hematology and Hemostasis of Mutant Mice Vet. Pathol., January 1, 2001; 38(1): 20 - 30. [Abstract] [Full Text] [PDF]
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M. Yepes, M. Sandkvist, M. K. K. Wong, T. A. Coleman, E. Smith, S. L. Cohan, and D. A. Lawrence Neuroserpin reduces cerebral infarct volume and protects neurons from ischemia-induced apoptosis Blood, July 15, 2000; 96(2): 569 - 576. [Abstract] [Full Text] [PDF]
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 K. Akassoglou, K. W. Kombrinck, J. L. Degen, and S. Strickland Tissue Plasminogen Activator-Mediated Fibrinolysis Protects against Axonal Degeneration and Demyelination after Sciatic Nerve Injury J. Cell Biol., May 29, 2000; 149(5): 1157 - 1166. [Abstract] [Full Text] [PDF]
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N. Nagai, I. Vanlinthout, and D. Collen Comparative Effects of Tissue Plasminogen Activator, Streptokinase, and Staphylokinase on Cerebral Ischemic Infarction and Pulmonary Clot Lysis in Hamster Models Circulation, December 21, 1999; 100(25): 2541 - 2546. [Abstract] [Full Text] [PDF]
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 M. D. Ginsberg On Ischemic Brain Injury in Genetically Altered Mice Arterioscler Thromb Vasc Biol, November 1, 1999; 19(11): 2581 - 2583. [Full Text] [PDF]
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