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Regular Articles |
From the Department of Molecular Cell Biology,*
the Max Planck Institute for Physiological and Clinical Research, Bad
Nauheim, Germany; and the University of
Caen,
UMR 6551-CNRS, Caen, France
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Angiogenesis is defined as the formation of new blood vessels by sprouting of endothelial cells from pre-existing vessels.3 During the process of sprouting, endothelial cells degrade the underlying basement membrane, migrate into neighboring tissue, proliferate, and assemble into tubes. Finally, tube-to-tube connections are made and blood flow is established. The ability of the mature vasculature to adapt to changing demands requires both soluble factors and cell-cell as well as cell-matrix interactions. Among the factors capable of modulating angiogenesis characterized to date, vascular endothelial growth factor (VEGF) is the best candidate for a specific regulator of endothelial cell growth and differentiation.4 VEGF, also known as vascular permeability factor, is a dimeric glycoprotein that is mitogenic for endothelial cells and enhances vascular permeability.5 VEGF is expressed in the normal adult brain, mainly in the epithelial cells of the choroid plexus, but also in astrocytes and neurons, such as granule cells of the cerebellum.6,7 It binds to two endothelial tyrosine kinase receptors, VEGF receptor-1 (VEGFR-1) (Flt-1) and VEGFR-2 (Flk-1/KDR).5
It has been shown that hypoxia is a strong inducer of VEGF mRNA
expression in many cells in vitro.8,9
In
addition, in vivo experiments have revealed that
systemic hypoxia is capable of inducing the expression of both VEGF and
VEGFR-1 in various organs, including the brain.6
Furthermore, VEGF expression is clearly induced in hypoxic regions in
the vicinity of tumor necroses10-12
and in various models
of ischemia.13-18
However, there are conflicting results
concerning the temporal kinetics and localization of induction of VEGF
and its receptors after cerebral ischemia. Moreover, there is no
evidence to connect VEGF to angiogenesis after cerebral ischemia.
Finally, very little is known about the mechanisms by which VEGF gene
expression is regulated during cerebral ischemia. In vitro
studies have identified three mechanisms that are responsible for the
increase in biologically active VEGF secreted by cells exposed to
hypoxia. One is an increased transcription rate mediated by binding of
HIF-1 to a hypoxia-responsive element in the 5'-flanking region of the
VEGF gene,19-21
and the second is increased VEGF mRNA
stability,8,22
probably due to binding of the RNA-binding
protein HuR.23
And finally, an internal ribosome entry
site ensures efficient translation of VEGF mRNA, even under
hypoxia.24
HIF-1 is a basic helix-loop-helix heterodimeric
transcription factor activated by reduced oxygen tension. HIF-1 is
composed of a hypoxia-regulated 
-subunit and a
ß-subunit.25
A homolog of HIF-1, named
HIF-2,25
has recently been cloned26,27
and
shown to be involved in the regulation of VEGF gene expression as
well.28
HIF-2 has an additional role in the regulation of
VEGFR-2.29
However, little is known about the involvement
and the activation of these two factors during cerebral ischemia.
In the present study we investigated the hypothesis that the hypoxic environment at the border of an infarcted area can lead to new vessel growth, thereby minimizing the detrimental effects of cerebral ischemia. We demonstrate that the tissue bordering the infarcted area, the penumbra, is indeed hypoxic. Our data further indicate that both VEGF and the VEGF receptors (VEGFR-1 and VEGFR-2) are up-regulated by hypoxia in the brain after cerebral ischemia. These could mediate the angiogenic response, observed in the ischemic border zone and extending toward the core region of the infarcted area.
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Materials and Methods |
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Surgical protocols were approved by the local ethics committee and governed by the pertinent national legislation. Focal ischemia was induced in OF1 mice (Iffa Credo, France) by the permanent occlusion of the left middle cerebral artery (MCAO) under chloral hydrate anesthesia as reported previously.30-32 In this model, ischemia is restricted to the neocortex. At different times after occlusion, mice were anesthetized and the brains were removed.
Semiquantitative Reverse Transcription-Polymerase Chain Reaction
Total RNA was prepared from cerebral cortices by phenol-chloroform
extraction as described.32
Reverse transcription (RT) and
polymerase chain reaction (PCR) amplification were performed as
described.6
For VEGF, the primers and PCR
profile were as described6
: for mouse HIF-1, the
primers 5'-TGAGGCTCACCATCAGTTAT-3' (sense) and
5'-TAACCCCATGTATTTGTTC-3' (antisense) resulted in a 187-bp product
(94°C for 1 minute, 50°C for 1 minute, 72°C for 2 minutes for 35
cycles), and for ß-actin the primers 5'-TGTGATGGTGGGAATGGGTCAG-3'
(sense) and 5'-TTTGATGTCACGCACGATTTCC-3' (antisense) gave rise to a
514-bp product (94°C for 0.75 minute, 60°C for 1 minute, 72°C for
1 minute for 35 cycles).
In Situ Hybridization
The techniques and 35S-UTP-labeled
single-stranded RNA probes for VEGF, VEGFR-1, and VEGFR-2 used for
in situ hybridization were essentially as
described.6,33
The HIF-1 and the HIF-2 probe have
been described before.12,34
Hybridization was performed on
cryostat-cut coronal brain sections (10 µm) with 2.5–5 x
104
cpm/µl 35S-labeled
RNA probe overnight at 48°C. Sections were washed, dehydrated, coated
with Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY), developed
after 12–28 days of exposure, and counterstained with 0.02% toluidine
blue.
Immunohistochemistry
The immunohistochemical staining was performed as described previously.35 The following primary antibodies were used: MEC 13.3 rat monoclonal anti-mouse PECAM-1/CD31 antibody (a gift from E. Dejana, Milan, Italy), polyclonal rabbit anti-mouse Ki67 antibody (Dianova), and rat monoclonal anti-mouse flk-1 (VEGFR-2) antibody (a gift from H. Kataoka, Kyoto, Japan). PECAM-1-positive endothelial cells and Ki67-positive proliferating cells were counted under the microscope in four to eight randomly chosen high-magnification fields (x100) in the ischemic border zone and a mean value was calculated. To map hypoxic regions in vivo, 250 µl of 10 mmol/L EF536 was administered intravenously to control animals or to mice subjected to cerebral ischemia, 20 hours after MCAO. Four hours later, all animals were killed and the brains were removed. Immunofluorescence for EF5 with Cy3-conjugated ELK3–51 monoclonal antibody (a gift from C. Koch, Philadelphia, PA) was performed as described.36,37
Mouse VEGF Immunoassay
Cortical tissue lysates from ipsilateral and contralateral brain hemispheres were prepared at different times after occlusion by homogenization in lysis buffer (100 mmol/L NaCl, 20 mmol/L Tris (pH 7.5), 1 mmol/L EDTA) supplemented with a cocktail of proteinase inhibitors (1 µg/ml pepstatin, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride). Mouse VEGF was quantitated using a commercially available immunoassay kit (Quantikine M; R&D Systems).
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Results |
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To determine the distribution pattern of endothelial cells, we
performed immunohistochemistry on frontal sections of adult mouse
brain, using an antibody against PECAM-1 as a marker for
endothelial cells. Positively stained endothelial cells were detected
in the whole brain, mainly in the choroid plexus, in capillaries in the
neocortex and hippocampus, and in pial vessels at the surface of the
normal brain (Figure 1a)
. Using an
antibody against Ki67, a cell proliferation marker, we confirmed that
cell proliferation was absent in normal adult brain parenchyma (Figure 1b)
. The staining pattern was similar after 0.5, 3, and 12 hours of
MCAO, although staining for PECAM-1 was reduced in the ischemic core
region (data not shown). After 24 hours, there was a slight increase in
the number of PECAM-1-positive cells, but still hardly any Ki67-labeled
cells were detected (Figure 2)
. After 48
and 72 hours of occlusion, however, a major change was observed, for
both PECAM-1 and Ki67 staining. PECAM-1 staining was strongly increased
at the border zone around the infarcted area and in the ischemic core
(Figure 1c)
, and the number of endothelial cells and proliferating
cells increased dramatically (Figure 2)
. Strong expression of Ki67 in
the same area of PECAM-1 staining suggested that endothelial cells had
resumed cell proliferation (Figure 1d)
. Immunohistochemistry staining
for both antigens was therefore performed on serial sections, where
individual vessels could be followed up. Figure 1, e and f
, shows the
same pial vessel with proliferating endothelial cells. Figure 1, g and h
, shows proliferating endothelial cells within a vessel that invades
from the ischemic border toward the core region of the infarcted area.
At 7 days after occlusion, the number of PECAM-1-positive endothelial
cells in vessel-like structures in the ischemic hemisphere was still
enhanced compared to the contralateral side (Figure 2)
. In the
contralateral hemisphere, Ki67 staining was absent at all time points,
and no change in PECAM-1 staining could be observed (data not shown).
These results demonstrate that an angiogenic reaction starts between 24
and 48 hours after cerebral infarction. This angiogenic reaction
appears to originate from the borders of the infarcted area and from
the pial vessels at the brain surface.
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After occlusion of a cerebral artery, oxygenation of the brain
tissue supplied by this vessel is impaired, resulting in subsequent
tissue hypoxia and even cell death. We hypothesized that tissue hypoxia
might be the stimulus responsible for the observed new vessel growth.
To map hypoxic tissue in vivo, we injected the
nitroimidazole compound EF5 intravenously 20 hours after MCAO and
analyzed the distribution of this hypoxia marker 4 hours after
injection, using immunofluorescence techniques on cryosections of the
infarcted brain. We found hypoxic regions around the infarcted area and
observed the strongest hypoxia signal in the cortex directly adjacent
to the infarction (Figure 3a)
. This
region, known as the penumbra, although perfused, suffers from hypoxia
and corresponds completely to the area where the angiogenic response
was detected 24 hours later (Figure 1c)
. This suggests that hypoxia, in
fact, could be the inducer of blood vessel formation. In contrast, no
EF5 staining was detectable in the contralateral hemisphere (Figure 3b)
or in normal brain (Figure 3c)
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To further delineate the mechanisms by which hypoxia could lead to
angiogenesis, we analyzed the expression pattern of VEGF and its
receptors by RT-PCR and in situ hybridization. VEGF is the
main regulator of angiogenesis during development.3
We
have recently stated that VEGF gene expression is inducible by hypoxia
in vitro and in vivo.6,8
VEGF mRNA
was not detectable by RT-PCR in control brain or in the contralateral
hemisphere at any time after occlusion, or in the ipsilateral side
after 3 hours of occlusion. After 6 and 12 hours, however, mRNA for
both VEGF120 and VEGF 164
was strongly induced in the ischemic hemisphere, but not in the
contralateral side (Figure 4)
. At later
time points VEGF mRNA again became undetectable. To identify regional
localization of VEGF mRNA expression, in situ hybridization
was performed. VEGF was expressed throughout the normal brain at a low
level and in the choroid plexus at higher levels (Figure 5, a and b)
. Changes in the expression
pattern of the VEGF gene in situ were detected 12 hours
after occlusion. VEGF gene expression was induced at the border of the
infarcted area (Figure 5d)
, in the same location where we had
identified hypoxic tissue by using EF5 staining (Figure 3a)
. VEGF mRNA
levels were strongly increased at a very restricted penumbral area
after 24 hours of MCAO (Figure 5e)
. They remained slightly elevated 72
hours after occlusion (Figure 5f)
but returned to normal levels after
168 hours of MCAO (Figure 5g)
. VEGF gene expression was also induced in
the pia above the infarcted area (Figures 5, d and e)
but was absent in
the ischemic core. To further quantify this reaction, VEGF protein was
measured by using a specific enzyme-linked immunosorbent assay. The
mean VEGF protein concentration in brain cortex was 4.15 ± 0.69
ng VEGF/g total protein (n = 4). Induction of
VEGF was not detected in the contralateral hemisphere at any time after
occlusion. In the ischemic hemisphere, however, VEGF protein levels
increased up to twofold between 12 and 48 hours of occlusion. By 168
hours, levels had returned to basal values (Figure 6)
. Thus VEGF protein induction followed
the increase seen in RNA levels.
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, in the choroid plexus, as
well as in single glial cells throughout the neocortex and the
hippocampus (Figure 7e)
. Twenty-four hours after MCAO, VEGFR-1
expression was lost in the ischemic core but was otherwise unchanged
(Figure 7b)
. After 48 hours of occlusion, however, VEGFR-1 mRNA levels
were induced at the border of the infarct and in the pia, in addition
to vessel-like structures invading the core region of the infarcted
area (Figure 7c)
. This suggests that the newly forming vessels express
VEGFR-1. By 7 days after occlusion, expression levels returned to
normal (not shown).
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. Like that
of VEGFR-1, VEGFR-2 expression was lost in the ischemic core but
remained unchanged in the other brain regions 24 hours after MCAO
(Figure 8b)
. At about 48 hours after occlusion VEGFR-2 was induced at
the border of the infarction and in the pia (Figure 8c)
, with strong
expression at 72 hours (Figure 8d)
. Expression was found in vessel-like
structures at the border of the infarction (Figure 8c)
and at later
times in vessels invading the core region of the infarcted area (Figure 8d)
. Increased VEGFR-2 mRNA levels persisted for 7 days after MCAO
(Figure 8e)
. These mRNA data were confirmed at the protein level, using
a monoclonal antibody against VEGFR-2. In the ipsilateral hemisphere we
found vessel-like structures invading from pial vessels and in the core
region of the infarct itself that expressed VEGFR-2 (Figure 9a)
, whereas no VEGFR-2 protein was
detectable in the contralateral hemisphere (Figure 9b)
, where no new
vessel growth was observed.
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. Expression in the hippocampus was
visible 24 hours after infarction and was sustained for at least 7 days
(Figure 8e)
. This was observed in the hippocampus of the ipsilateral
side as well as the contralateral hemisphere (Figure 8f)
but was absent
in control, sham-operated animals (not shown). A subset of hippocampal
cells strongly expressed mRNA for VEGFR-2, as detected by a specific
cRNA antisense probe (Figure 8, g, h, and k)
, whereas signal was absent
when the corresponding sense probe was used (Figure 8i)
. This finding
was confirmed by detection of VEGFR-2 protein in cells within the same
region by using the anti-VEGFR-2 antibody (Figure 9c)
, whereas the
PECAM-1 antigen, the endothelial cell marker, was clearly present in
different cells (Figure 9d)
. Taken together, these results demonstrate that VEGF gene expression is induced only in the most hypoxic region adjacent to the infarction. This is followed by up-regulation of VEGFR-1 and VEGFR-2, initially at the penumbra, with subsequent expression along vessel-like structures that invade the core region of the infarcted tissue. We also show that nonendothelial cells in the hippocampus, probably neuronal cells, can express VEGFR-2.
Expression of HIF-1 and HIF-2
To further analyze the mechanisms by which angiogenesis occurs
during cerebral ischemia, we studied the expression of the
transcription factors HIF-1 and HIF-2. Both factors are involved in
hypoxia-induced transcriptional regulation of both VEGF and its
receptors. HIF-1 and HIF-2 consist of two subunits, and ß; the
-subunit is hypoxia responsive.25
Expression analysis, however, is hampered by the fact that
hypoxic regulation of HIF-1 and HIF-2 activity is regulated at the
protein level and not at the mRNA level.25,38
Accordingly,
no significant changes at the mRNA level of HIF-1 were detected at
any time after occlusion by RT-PCR analysis (Figure 4)
.
Nevertheless, in situ hybridization analysis showed a
remarkable change in the expression pattern. Whereas HIF-1 was
expressed ubiquitously at low levels with enhanced expression in the
hippocampus and the dentate gyrus, expression of HIF-2 was
restricted to vessels (data not shown) as has been reported
before.27,28
Up to 24 hours after occlusion, ie, at a time
at which we could clearly identify hypoxic tissue and up-regulated VEGF
gene expression, the expression of both HIF-1 and HIF-2 did not
change in the penumbra, but again was completely lost in the ischemic
core (data not shown), supporting the finding that hypoxia does not
activate transcription of these factors. After 48 hours, and more
pronouncedly 72 hours after MCAO, though, there was a marked localized
increase in mRNA levels for both factors (Figure 10, a and b)
. Whereas HIF-2
expression was restricted to vessel-like structures at the border of
the infarction (Figure 10, d and f)
, HIF-1 was expressed in cells
directly adjacent to the infarction (Figure 10, c and e)
.
Unfortunately, analysis of HIF-1 and HIF-2 protein expression in
tissue sections is hampered by the lack of commercially available
antibodies against these factors for immunohistochemistry. However, the
late, strictly localized induction of both HIF-1 and HIF-2 mRNA
expression at 72 hours implies that stimuli, maybe other than hypoxia,
are able to induce transcription of these genes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With the discovery of HIF-142
and HIF-2,25
the first transcription factors have been identified that mediate
hypoxia-induced gene expression at physiologically relevant oxygen
tensions.43
The fundamental role of HIF-1 for angiogenesis
was demonstrated by targeted disruption of both HIF-1 and HIF-1ß.
Mice that are deficient for either subunit die around midgestation and
demonstrate defects in angiogenesis as well as reduced levels of VEGF
gene expression.44-46
However, the hypoxic response is
not totally abrogated but only partially eliminated.47
The
remaining hypoxic response might be due to hypoxically induced
stabilization of the VEGF mRNA, which is probably mediated
independently of HIF-1 through an element in the 3'-untranslated region
of the VEGF transcript.12
On the other hand, VEGF mRNA
levels were increased in HIF-1-deficient ES cells by deprivation of
glucose,44
and recently elevated VEGF mRNA levels were
also found in HIF-1-null embryos.48
As glucose
deprivation mainly stabilizes VEGF mRNA,49
reduced glucose
levels may function as a hypoxia- and therefore HIF-1-independent
mechanism for VEGF induction. This could occur in situations where
tissue perfusion is inadequate and results in combined oxygen and
glucose deficiency as it occurs in stroke or even during embryogenesis,
when tissues are rapidly expanding.48
HIF-1 and HIF-2
activities are not regulated at the mRNA level, but at the level of
protein stability.25,38,50
This would explain the lack of
HIF-1 and HIF-2 mRNA induction 24 hours after occlusion, although
we found hypoxic cells and strong VEGF gene induction at this time.
Unfortunately, detection of HIF-1 and HIF-2 protein by
immunohistochemical methods was hampered by the lack of suitable
commercially available antibodies. In addition, the very short
half-life of these proteins under normoxic conditions50,51
makes it extremely difficult to prepare appropriate brain lysates for
the detection of this protein. We were unable to detect any HIF-1 or
HIF-2 protein by immunoblot analysis in nuclear extracts freshly
prepared from ischemic brain hemispheres. However, we found an
induction of both HIF-1 and HIF-2 mRNA expression
after 3 days of occlusion. Thus there might be factors other than
hypoxia involved that are able to induce the expression of HIF-1 and
HIF-2 at the transcriptional level. These could be
associated with other cellular events ongoing at the time,
such as endothelial cell proliferation, astrocyte activation, or
microglia invasion.
We found the induction of VEGF gene expression after tissue hypoxia.
However, there might be other stimuli involved in this gene activation
as well. As mentioned above, glucose deficiency is likely to occur
during ischemic disease and may contribute to increased VEGF
expression. In addition, a whole range of growth factors and cytokines
such as platelet-derived growth factor, transforming growth factor ß,
tumor necrosis factor , or interleukin 1ß52
are known
inducers of VEGF gene activation. All of these factors are expressed in
the brain, become activated during cerebral ischemia,53
and thus may participate in VEGF gene activation. However, the close
temporal and spatial correlation between tissue hypoxia and induction
of VEGF gene expression strongly implies hypoxia as a crucial stimulus
for increased VEGF gene expression during stroke. Several reports deal
with the role of VEGF in focal ischemia. With permanent MCAO, VEGF was
detected in macrophages, endothelial cells, glial cells, and neurons.
Increased expression was sustained for up to 2 weeks and was observed
in both the infarcted core and the periphery.13,16
Our
studies revealed no VEGF gene expression in the ischemic core. However,
we found a close spatial and temporal correlation between VEGF gene
expression and the occurrence of hypoxic tissue, which was restricted
to the penumbra and the pia. After transient MCAO, the increase in VEGF
gene and protein expression in neurons and pial cells was generally
more rapid and declined earlier than in our model, which might depend
on reoxygenation processes14,15
While this work was in
progress, Plate et al reported up-regulation of VEGF mRNA in rats
specifically in microglial cells in the ipsilateral hemisphere starting
3 hours after the onset of MCAO and peaking after 24
hours.54
These data are in good agreement with our own
observations of strong VEGF mRNA induction between 6 and 24 hours after
MCAO.
Expression of VEGFRs was analyzed in three recent reports. Plate et al found an early up-regulation of VEGFR-1 mRNA in peri-ischemic endothelial cells starting 3 hours after MCAO which was sustained for 72 hours, but no expression of VEGFR-2 was found at all.54 On the other hand, Kovàcs et al found VEGFR-1 expression in endothelial cells in the infarcted core and periphery, with peak expression at 7 days after occlusion,13 and Lennmyr et al observed expression of VEGFR-1 not only in endothelial cells, but also in neurons and glial cells. Expression of VEGFR-2 was prominent in glial cells between days 1 and 3 after occlusion.16 Our data suggest that there might be a sequential temporal and spatial induction of both receptors, mainly in endothelial cells but also in glial cells and neurons. Gene expression of VEGFR-1 is directly inducible by hypoxia, as is the case for VEGF itself.6 Both of these events are mediated by HIF-1.55 In contrast, gene expression of VEGFR-2 is not directly induced by hypoxia.6 Accordingly, HIF-1 does not induce gene expression of VEGFR-2 in vitro.55 There are, however, several reports showing up-regulation of VEGFR-2 when endothelial cells were exposed to hypoxia in vitro, although this up-regulation does not occur at the level of transcription.56,57 Transcription of the VEGFR-2 gene, however, is clearly inducible by HIF-2 and by the ligand VEGF itself.29,58 Up-regulation of the VEGFR-2 gene induced by VEGF may represent an important positive feedback mechanism for VEGF action in ischemia-induced angiogenesis.59 As a result of cerebral ischemia, expression of both VEGFRs is initially increased in the hypoxic penumbra and the pia, followed by later expression in the ischemic core in vessel-like structures. This suggests that newly forming vessels, invading the core region, express VEGFRs.
Although VEGFR-2 expression is considered to be relatively confined to endothelial cells,60 VEGFR-2 expression has been described in neurons16 and neural progenitor cells of the retina.61 Interestingly, we found expression of VEGFR-2 in the hippocampus after cerebral ischemia, not only in the ipsilateral, but also in the contralateral hemisphere. Furthermore, increased expression levels were sustained for at least 7 days. The remote location of the hippocampus with respect to the ischemic damage suggests that a neuronal signal from the neocortex to the hippocampi, rather than ischemia itself, could have mediated the observed VEGFR-2 induction in this area. Considering that the collapsin/semaphorin receptor neuripilin-1 was recently identified as a coreceptor for VEGF,62 these results may implicate the VEGF/VEGFR system in neuronal guidance, neurogenesis, and even neuroprotection.
Twenty-four hours after hypoxia-induced VEGF gene expression and in close relation to the expression of the VEGFR-1 and VEGFR-2 genes, endothelial cells started to proliferate. Vessel-like structures were observed that emanate from pial and brain parenchyma vessels and invade the ischemic core. These results support the hypothesis that cerebral ischemia can cause active angiogenesis. Our data are supported by postmortem analysis of human brain tissue from stroke patients, which has revealed a significant increase in the number of microvessels in the ipsilateral hemisphere when compared to the contralateral normal side.2,63 Furthermore, in these patients higher blood vessel counts correlated with longer survival, suggesting a beneficial effect of this physiological response to ischemia.2 In addition, exposure to chronic hypoxia, without the occurrence of tissue damage, was sufficient to increase the vascular density in the brain.40 These findings suggest the presence of an intrinsic mechanism in the brain, the role of which is to protect the organ from hypoxic or ischemic damage.
Based on the aforementioned studies and on our results presented in
this study, we propose the following cascade of events for cerebral
ischemia: occlusion of a cerebral vessel leads to ischemia and
subsequent cell death. At the border between the infarction and the
healthy brain tissue, however, there is a zone that suffers from
hypoxia (Figure 3)
, the so-called penumbra. In this particular region,
hypoxia is a signal for induction of HIF-1 activity, which in turn
activates the expression for both VEGF (Figure 5)
and VEGFR-1 genes
(Figure 7)
. Subsequently, transcription of the VEGFR-2 gene also
becomes induced (Figure 8)
. The activation of the VEGF/VEGFR system
leads to endothelial cell proliferation and new vessel growth (Figure 1)
, from healthy tissue toward the penumbra and further into the core
region of the infarcted area. The development of new vessels may
therefore be regarded as an attempt to counteract the detrimental
effects of stroke by increasing perfusion and oxygenation of the
suffering tissue.
It is intriguing to speculate that exogenous support of this endogenous emergency response could be useful in enhancing survival after stroke. Atherosclerotic narrowing or occlusion of arteries in the heart or limb, leading to myocardial infarction and gangrene, respectively, are targets for therapeutic angiogenesis.64 Stimulation of physiological collateral growth and angiogenesis by exogenous VEGF has shown to be a promising strategy for the treatment of these diseases.65,66 This strategy may also hold a similar promise for the treatment of stroke. Two recent reports emphasize the validity of this concept. Infusion of VEGF via a minipump into the cortex of adult rats produced a remarkable, localized neovascularization,67 and topical application of VEGF to the surface of ischemic rat brain led to a significant reduction of infarct volume and limited the extent of neuronal damage.68 It remains to be established, however, whether the beneficial effect of VEGF is due to an angiogenic response or to a putative direct neuroprotective effect.
Treatment of stroke in human patients probably has to combine therapies at different levels of intervention. A combination of different strategies that include direct neuroprotection, stimulation of angiogenesis, and inhibition of inflammation may turn out to be the most successful. We have recently shown that the protective effect of erythropoietin in stroke can be attributed to a dual function of this growth factor as a direct neuroprotective agent and an angiogenic reagent.32 Recent reports of the synergistic protection achieved in stroke models by the combined use of caspase-inhibitory compounds and N-methyl-D-aspartate receptor antagonists lend further support to this concept.69,70
In conclusion, after cerebral ischemia, the penumbra suffers from hypoxia and responds with increased VEGF gene expression, mediated via the transcription factors HIF-1 and HIF-2. Subsequent expression of both VEGFR-1 and VEGFR-2 is induced, and finally an angiogenic reaction, characterized by proliferating endothelial cells and new vessel growth, originates from the penumbra and invades the ischemic core. Enforcing this physiological response by exogenous stimulation with VEGF may prove to have a beneficial outcome for human stroke patients.
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Acknowledgements |
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cDNA, Annette Damert (Göteborg, Sweden) for HIF-1 cDNA, and
Georg Breier (Bad Nauheim, Germany) for VEGFR-1 and -2 cDNAs. We
gratefully acknowledge Didier Divoux for excellent technical assistance
and Friedemann Kiefer, Hannes Drexler, and Chung-Wai Chow (all in Bad
Nauheim) for critically reading the manuscript. |
Footnotes |
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Supported by a fellowship from the Max Planck Society (to H. H. M.).
In memoriam: Werner Risau (1953–1998).
Accepted for publication November 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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regulates the VEGF expression and is potentially involved in lung and vascular development. Proc Natl Acad Sci USA 1997, 94:4273-4278 is regulated at the post-mRNA level. Kidney Int 1997, 51:560-563[Medline]
. Genes Dev 1998, 12:149-162 in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 1998, 394:485-490[Medline]
is required for solid tumor formation and embryonic vascularization. EMBO J 1998, 17:3005-3015[Medline]
-null embryos is not associated with VEGF deficiency but with mesenchymal cell death. Dev Biol 1999, 209:254-267[Medline]
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L. Sun, H. H. Marti, and R. Veltkamp Hyperbaric Oxygen Reduces Tissue Hypoxia and Hypoxia-Inducible Factor-1{alpha} Expression in Focal Cerebral Ischemia Stroke, March 1, 2008; 39(3): 1000 - 1006. [Abstract] [Full Text] [PDF]
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P. Thored, J. Wood, A. Arvidsson, J. Cammenga, Z. Kokaia, and O. Lindvall Long-Term Neuroblast Migration Along Blood Vessels in an Area With Transient Angiogenesis and Increased Vascularization After Stroke Stroke, November 1, 2007; 38(11): 3032 - 3039. [Abstract] [Full Text] [PDF]
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 O. Baranova, L. F. Miranda, P. Pichiule, I. Dragatsis, R. S. Johnson, and J. C. Chavez Neuron-Specific Inactivation of the Hypoxia Inducible Factor 1{alpha} Increases Brain Injury in a Mouse Model of Transient Focal Cerebral Ischemia J. Neurosci., June 6, 2007; 27(23): 6320 - 6332. [Abstract] [Full Text] [PDF]
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 D. McLeod A chronic grey matter penumbra, lateral microvascular intussusception and venous peduncular avulsion underlie diabetic vitreous haemorrhage Br. J. Ophthalmol., May 1, 2007; 91(5): 677 - 689. [Abstract] [Full Text] [PDF]
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 J. D.W. van der Bilt, M. E. Soeters, A. M.M.J. Duyverman, M. W. Nijkamp, P. O. Witteveen, P. J. van Diest, O. Kranenburg, and I. H.M. Borel Rinkes Perinecrotic Hypoxia Contributes to Ischemia/Reperfusion-Accelerated Outgrowth of Colorectal Micrometastases Am. J. Pathol., April 1, 2007; 170(4): 1379 - 1388. [Abstract] [Full Text] [PDF]
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A. T. McLaren, P. A. Marsden, C. D. Mazer, A. J. Baker, D. J. Stewart, A. K. Y. Tsui, X. Li, Y. Yucel, M. Robb, S. R. Boyd, et al. Increased expression of HIF-1{alpha}, nNOS, and VEGF in the cerebral cortex of anemic rats Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R403 - R414. [Abstract] [Full Text] [PDF]
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U. Kilic, E. Kilic, A. Jarve, Z. Guo, A. Spudich, K. Bieber, U. Barzena, C. L. Bassetti, H. H. Marti, and D. M. Hermann Human Vascular Endothelial Growth Factor Protects Axotomized Retinal Ganglion Cells In Vivo by Activating ERK-1/2 and Akt Pathways J. Neurosci., November 29, 2006; 26(48): 12439 - 12446. [Abstract] [Full Text] [PDF]
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 G. J. del Zoppo and R. Milner Integrin-Matrix Interactions in the Cerebral Microvasculature Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 1966 - 1975. [Abstract] [Full Text] [PDF]
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 E. Kilic, U. Kilic, Y. Wang, C. L. Bassetti, H. H. Marti, and D. M. Hermann The phosphatidylinositol-3 kinase/Akt pathway mediates VEGF's neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia FASEB J, June 1, 2006; 20(8): 1185 - 1187. [Abstract] [Full Text] [PDF]
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L. Schaffer, J. Vogel, C. Breymann, M. Gassmann, and H. H. Marti Preserved placental oxygenation and development during severe systemic hypoxia Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2006; 290(3): R844 - R851. [Abstract] [Full Text] [PDF]
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 A. W. Mould, S. A. Greco, M. M. Cahill, I. D. Tonks, D. Bellomo, C. Patterson, A. Zournazi, A. Nash, P. Scotney, N. K. Hayward, et al. Transgenic Overexpression of Vascular Endothelial Growth Factor-B Isoforms by Endothelial Cells Potentiates Postnatal Vessel Growth In Vivo and In Vitro Circ. Res., September 16, 2005; 97(6): e60 - e70. [Abstract] [Full Text] [PDF]
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R. Kimura, H. Nakase, R. Tamaki, and T. Sakaki Vascular Endothelial Growth Factor Antagonist Reduces Brain Edema Formation and Venous Infarction Stroke, June 1, 2005; 36(6): 1259 - 1263. [Abstract] [Full Text] [PDF]
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 A. Wagatsuma, H. Tamaki, and F. Ogita Capillary supply and gene expression of angiogenesis-related factors in murine skeletal muscle following denervation Exp Physiol, May 1, 2005; 90(3): 403 - 409. [Abstract] [Full Text] [PDF]
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R. Helton, J. Cui, J. R. Scheel, J. A. Ellison, C. Ames, C. Gibson, B. Blouw, L. Ouyang, I. Dragatsis, S. Zeitlin, et al. Brain-Specific Knock-Out of Hypoxia-Inducible Factor-1{alpha} Reduces Rather Than Increases Hypoxic-Ischemic Damage J. Neurosci., April 20, 2005; 25(16): 4099 - 4107. [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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 Y. Wang, E. Kilic, U. Kilic, B. Weber, C. L. Bassetti, H. H. Marti, and D. M. Hermann VEGF overexpression induces post-ischaemic neuroprotection, but facilitates haemodynamic steal phenomena Brain, January 1, 2005; 128(1): 52 - 63. [Abstract] [Full Text] [PDF]
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 S. M. Evans, K. D. Judy, I. Dunphy, W. T. Jenkins, W.-T. Hwang, P. T. Nelson, R. A. Lustig, K. Jenkins, D. P. Magarelli, S. M. Hahn, et al. Hypoxia Is Important in the Biology and Aggression of Human Glial Brain Tumors Clin. Cancer Res., December 15, 2004; 10(24): 8177 - 8184. [Abstract] [Full Text] [PDF]
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 C. Demougeot, M. Van Hoecke, N. Bertrand, A. Prigent-Tessier, C. Mossiat, A. Beley, and C. Marie Cytoprotective Efficacy and Mechanisms of the Liposoluble Iron Chelator 2,2'-Dipyridyl in the Rat Photothrombotic Ischemic Stroke Model J. Pharmacol. Exp. Ther., December 1, 2004; 311(3): 1080 - 1087. [Abstract] [Full Text] [PDF]
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 D.-H. Cho, Y.-M. Hong, H.-J. Lee, H.-N. Woo, J.-O. Pyo, T. W. Mak, and Y.-K. Jung Induced Inhibition of Ischemic/Hypoxic Injury by APIP, a Novel Apaf-1-interacting Protein J. Biol. Chem., September 17, 2004; 279(38): 39942 - 39950. [Abstract] [Full Text] [PDF]
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 T. Acker and H. Acker Cellular oxygen sensing need in CNS function: physiological and pathological implications J. Exp. Biol., August 15, 2004; 207(18): 3171 - 3188. [Abstract] [Full Text] [PDF]
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C. Michiels Physiological and Pathological Responses to Hypoxia Am. J. Pathol., June 1, 2004; 164(6): 1875 - 1882. [Abstract] [Full Text] [PDF]
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R. A. Nordal, A. Nagy, M. Pintilie, and C. S. Wong Hypoxia and Hypoxia-Inducible Factor-1 Target Genes in Central Nervous System Radiation Injury: A Role for Vascular Endothelial Growth Factor Clin. Cancer Res., May 15, 2004; 10(10): 3342 - 3353. [Abstract] [Full Text] [PDF]
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 K.-H. Jung, K. Chu, S.-W. Jeong, H.-K. Park, H.-J. Bae, and B.-W. Yoon Cerebral Cavernous Malformations With Dynamic and Progressive Course: Correlation Study With Vascular Endothelial Growth Factor Arch Neurol, November 1, 2003; 60(11): 1613 - 1618. [Abstract] [Full Text] [PDF]
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 W. J. Lukiw, A. Ottlecz, G. Lambrou, M. Grueninger, J. Finley, H. W. Thompson, and N. G. Bazan Coordinate Activation of HIF-1 and NF-{kappa}B DNA Binding and COX-2 and VEGF Expression in Retinal Cells by Hypoxia Invest. Ophthalmol. Vis. Sci., October 1, 2003; 44(10): 4163 - 4170. [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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H. J. Schoch, S. Fischer, and H. H. Marti Hypoxia-induced vascular endothelial growth factor expression causes vascular leakage in the brain Brain, November 1, 2002; 125(11): 2549 - 2557. [Abstract] [Full Text] [PDF]
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R. Veltkamp, N. Rajapakse, G. Robins, M. Puskar, K. Shimizu, and D. Busija Transient Focal Ischemia Increases Endothelial Nitric Oxide Synthase in Cerebral Blood Vessels Stroke, November 1, 2002; 33(11): 2704 - 2710. [Abstract] [Full Text] [PDF]
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M. Bernaudin, Y. Tang, M. Reilly, E. Petit, and F. R. Sharp Brain Genomic Response following Hypoxia and Re-oxygenation in the Neonatal Rat. IDENTIFICATION OF GENES THAT MIGHT CONTRIBUTE TO HYPOXIA-INDUCED ISCHEMIC TOLERANCE J. Biol. Chem., October 11, 2002; 277(42): 39728 - 39738. [Abstract] [Full Text] [PDF]
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 A. Tandar, G.M. Saperia, and D.H. Spodick Direct myocardial revascularization and therapeutic angiogenesis Eur. Heart J., October 1, 2002; 23(19): 1492 - 1502. [Full Text] [PDF]
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 M. Soderman, G. Rodesch, and P. Lasjaunias Transdural Blood Supply to Cerebral Arteriovenous Malformations Adjacent to the Dura Mater AJNR Am. J. Neuroradiol., September 1, 2002; 23(8): 1295 - 1300. [Abstract] [Full Text] [PDF]
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N. Miyamoto, M. Mandai, H. Takagi, I. Suzuma, K. Suzuma, S. Koyama, A. Otani, H. Oh, and Y. Honda Contrasting Effect of Estrogen on VEGF Induction under Different Oxygen Status and Its Role in Murine ROP Invest. Ophthalmol. Vis. Sci., June 1, 2002; 43(6): 2007 - 2014. [Abstract] [Full Text] [PDF]
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 T. Shoshani, A. Faerman, I. Mett, E. Zelin, T. Tenne, S. Gorodin, Y. Moshel, S. Elbaz, A. Budanov, A. Chajut, et al. Identification of a Novel Hypoxia-Inducible Factor 1-Responsive Gene, RTP801, Involved in Apoptosis Mol. Cell. Biol., April 1, 2002; 22(7): 2283 - 2293. [Abstract] [Full Text] [PDF]
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 B. P. Eliceiri, X. S. Puente, J. D. Hood, D. G. Stupack, D. D. Schlaepfer, X. Z. Huang, D. Sheppard, and D. A. Cheresh Src-mediated coupling of focal adhesion kinase to integrin {alpha}v{beta}5 in vascular endothelial growth factor signaling J. Cell Biol., April 1, 2002; 157(1): 149 - 160. [Abstract] [Full Text] [PDF]
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D. M. STROKA, T. BURKHARDT, I. DESBAILLETS, R. H. WENGER, D. A. H. NEIL, C. BAUER, M. GASSMANN, and D. CANDINAS HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia FASEB J, November 1, 2001; 15(13): 2445 - 2453. [Abstract] [Full Text] [PDF]
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 B. Yang, L. Graham, S. Dikalov, R. P. Mason, J. R. Falck, J. K. Liao, and D. C. Zeldin Overexpression of Cytochrome P450 CYP2J2 Protects against Hypoxia-Reoxygenation Injury in Cultured Bovine Aortic Endothelial Cells Mol. Pharmacol., August 1, 2001; 60(2): 310 - 320. [Abstract] [Full Text] [PDF]
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H. Beck, T. Acker, C. Wiessner, P. R. Allegrini, and K. H. Plate Expression of Angiopoietin-1, Angiopoietin-2, and Tie Receptors after Middle Cerebral Artery Occlusion in the Rat Am. J. Pathol., November 1, 2000; 157(5): 1473 - 1483. [Abstract] [Full Text] [PDF]
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 G. L. Semenza HIF-1 and human disease: one highly involved factor Genes & Dev., August 15, 2000; 14(16): 1983 - 1991. [Full Text]
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B. P. Eliceiri, X. S. Puente, J. D. Hood, D. G. Stupack, D. D. Schlaepfer, X. Z. Huang, D. Sheppard, and D. A. Cheresh Src-mediated coupling of focal adhesion kinase to integrin {alpha}v{beta}5 in vascular endothelial growth factor signaling J. Cell Biol., April 1, 2002; 157(1): 149 - 160. [Abstract] [Full Text] [PDF]
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