Role of Plasminogen System Components in Focal Cerebral Ischemic Infarction : A Gene
Targeting and Gene Transfer Study in Mice
N. Nagai, M. De Mol, H. R. Lijnen, P. Carmeliet and D. Collen
Circulation. 1999;99:2440-2444
doi: 10.1161/01.CIR.99.18.2440
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Basic Science Reports
Role of Plasminogen System Components in Focal Cerebral
Ischemic Infarction
A Gene Targeting and Gene Transfer Study in Mice
N. Nagai, PhD; M. De Mol; H.R. Lijnen, PhD; P. Carmeliet, MD, PhD; D. Collen, MD, PhD
Background—The role of plasminogen system components in focal cerebral ischemic infarction (FCI) was studied in mice
deficient in plasminogen (Plg2/2), in tissue or urokinase plasminogen activator (tPA2/2 or uPA2/2), or in plasminogen
activator inhibitor-1 or a2-antiplasmin (PAI-12/2 or a2-AP2/2).
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 7.661.1 mm3 (mean6SEM), uPA2/2
mice had similar infarcts (7.861.0 mm3, P5NS), tPA2/2 mice smaller (2.660.80 mm3, P,0.0001), PAI-12/2 mice
larger (1660.52 mm3, P,0.0001), and Plg2/2 mice larger (1261.2 mm3, P50.037) infarcts. a2-AP2/2 mice had smaller
infarcts (2.261.1 mm3, P,0.0001 versus wild-type), which increased to 1362.5 mm3 (P,0.005 versus a2-AP2/2) after
intravenous injection of human a2-AP. Injection into a2-AP2/2 mice of Fab fragments of affinospecific rabbit IgG
against human a2-AP, after injection of 200 mg human a2-AP, reduced FCI from 1161.5 to 5.161.1 mm3 (P50.004).
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 a2-AP injection) increases whereas its augmentation (a2-AP gene inactivation or a2-AP
neutralization) reduces infarct size. Inhibition of a2-AP may constitute a potential avenue to treatment of FCI.
(Circulation. 1999;99:2440-2444.)
Key Words: plasminogen n plasminogen activators n cerebral infarction n cerebral ischemia
N
euronal 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 – 6 Furthermore, deficiency of plasminogen
(Plg), the zymogen substrate of tPA, and infusion of a2antiplasmin (a2-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 plasminmediated laminin degradation or alternative mechanisms in
Received October 14, 1998; revision received January 21, 1999; accepted January 25, 1999.
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 [email protected]
© 1999 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
2440
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Nagai et al
May 11, 1999
2441
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 a2-AP. In addition, the effects of adenoviral transfer
of the tPA and PAI-1 genes and of infusion of human a2-AP
on cerebral infarction were studied.
Methods
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 a2AP,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 –20 After transfection, recombinant viral
plaques were harvested and amplified as described.21–23 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 a2-AP and Affinospecific
Rabbit Anti-Human a2-AP Fab Fragments
Human a2-AP was prepared from fresh-frozen plasma as previously
described.26 Pooled rabbit antisera raised against human a2-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 a2-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
Figure 1. TTC-stained brain from wild-type mouse. Infarct is
identified as unstained (white) necrotic area surrounded by
stained (brick red) viable tissue.
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 1.33109 cfu 4 days before ligation of the
MCA. Human a2-AP (ha2-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 ha2-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 (r50.67, P,0.0001) in rabbits 5 hours after thrombotic focal cerebral infarction.30
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%
3,39-diaminobenzidine and 0.01% H2O2, followed by counterstaining
with hematoxylin.
Statistical Analysis
The data are represented as mean6SEM 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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2442
Plasminogen System and Cerebral Infarction
Figure 2. Effect of deficiency of Plg system components on focal
ischemic cerebral infarct size. WT indicates wild-type (pooled values of 50% C57BL/6 50% S129, 100% C57BL6, and 100% S129
genetic background) and wild-type littermates of different gene-inactivated groups). Data represent mean values and SEM (vertical
bars), with number of experiments given in columns.
Results
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 7.661.1 mm3 (n511) in wild-type mice with a mixed
(50% S129 and 50% C57BL/6) genetic background, a volume of
9.362.7 mm3 (n56) in inbred C57BL/6 mice, and a volume of
6.461.3 mm3 (n56) in inbred S129 mice (P5NS versus mixed
background, results not shown).
Inactivation of the tPA gene was associated with a significant
reduction of infarct size to 2.660.80 mm3 (n511) (P,0.0001
versus wild-type mice), whereas inactivation of the uPA gene
had no effect on infarct size (7.861.0 mm3, n58, P5NS versus
wild-type). Inactivation of the PAI-1 gene was associated with a
significant increase in infarct size (1660.52 mm3, n56,
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 (1261.2 mm3, n59, P50.037 versus wild-type),
whereas, conversely, in a2-AP gene–deficient mice, infarct size
was markedly reduced (2.261.1 mm3, n57, P50.0001 versus
wild-type) (Figure 2).
Figure 3. Effect of adenoviral transfer of tPA or PAI-1 genes on
focal ischemic cerebral infarct size in tPA- or PAI-1– deficient
mice, respectively.
heterozygously and homozygously deficient mice, respectively
(Figure 4A). Injection of ha2-AP in a2-AP2/2 mice increased the
infarct size to 1362.5 mm3 (n54) with a 1-mg total dose and to
1161.5 mm3 (n56) with a 0.2-mg total dose. Injection of 1.7 mg
affinospecific Fab against ha2-AP in mice given 0.2 mg ha2-AP
reduced the cerebral infarct size to 5.161.1 mm3 (n57, P50.004
versus 0.2 mg ha2-AP) (Figure 4B).
Effect of tPA and PAI-1 Gene Transfer on
Cerebral Infarct Size
Injection of 1.33109 pfu of AdCMVtPA in 6 tPA2/2 mice 4
days before MCA ligation was associated with a cerebral
infarct size of 6.061.3 mm3, significantly larger than the
infarcts in 5 tPA2/2 mice injected with the control virus
AdRR5 (1.860.63 mm3, P50.028) (Figure 3A). Conversely,
injection of 1.33109 pfu of AdCMVPAI-1 in 5 PAI-12/2 mice
was associated with a cerebral infarct size of 1061.4 mm3,
significantly smaller than the infarcts in 5 PAI-12/2 mice
injected with the control virus AdRR5 (1361.0 mm3,
P50.019) (Figure 3B).
Effect of a2-AP on Cerebral Infarct Size
Cerebral infarct size correlated with a2-AP gene dose, corresponding to 1162.0, 4.962.0, and 2.261.1 mm3 in wild-type and
Figure 4. Effect of a2-AP on focal ischemic cerebral infarct size.
A, Effect of a2-AP genotype on cerebral infarct size. B, Effect of
injection of ha2-AP or of ha2-AP followed by anti– ha2-AP Fab
fragments on cerebral infarct size.
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Nagai et al
Figure 5. Immunohistochemical analysis of laminin and fibrin(ogen). A, Brain slice stained for laminin, which is abundant in hippocampus (A.1) but undetectable outside blood vessels in cortex
(in both necrotic and viable areas, A.2). B, Brain slices stained for
fibrin(ogen), revealing increased extravascular reactivity in PAI-12/2
mice (B.2) and comparable intravascular reactivity in other genotypes (tPA2/2, B.1; Plg2/2, B.3; and a2-AP2/2, B.4).
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-12/2 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 Plg2/2 and
a2-AP2/2 mice (apart from the differences in infarct size).
Discussion
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.4–7 Using a mouse model with stereotactic injection of the glutamate analogue kainic acid in the hippocampus, they
May 11, 1999
2443
found that local tPA administration exacerbated whereas tPA
deficiency or PAI-1 injection reduced hippocampal neuronal cell
death. Furthermore, both Plg deficiency and local a2-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 findings4–7 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 a2-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-12/2 mice,
suggestive of altered vascular permeability. Intravascular
fibrin deposition was observed to a comparable extent in
Plg2/2 and in a2-AP2/2 mice, although the area in which this
deposition occurred was much larger in the Plg2/2 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-12/2 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 Plg2/2
and a2-AP2/2 mice, the total fibrin burden was much larger in
the former because of the severalfold larger infarct size.
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2444
Plasminogen System and Cerebral Infarction
The internally consistent observations with a2-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 ha2-AP in a2-AP2/2
mice caused a dose-related infarct expansion. Finally, and
importantly, Fab fragments from affinospecific polyclonal
rabbit anti– ha2-AP antibodies significantly reduced the cerebral ischemic infarct size. This observation suggests that
a2-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 ha2-AP in a2-AP2/2 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 a2-AP have been
initiated by immunization of a2-AP2/2 mice with purified
murine a2-AP. On the basis of previous experience with other
Plg system components, a2-AP2/2 mice are anticipated to be
able to produce neutralizing monoclonal antibodies against
murine a2-AP.32 The concentration of a2-AP in human
plasma is 1 mmol/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 a2-AP level (derived from
the gene dose effect and the dose-response effect of ha2-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.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
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