Deficiency of vasodilator-stimulated phosphoprotein (VASP) increases blood-brain-barrier damage and edema formation after ischemic stroke in mice.

Deficiency of Vasodilator-Stimulated Phosphoprotein
(VASP) Increases Blood-Brain-Barrier Damage and Edema
Formation after Ischemic Stroke in Mice
Peter Kraft1, Peter Michael Benz2,3, Madeleine Austinat1, Marc Elmar Brede4, Kai Schuh2,3, Ulrich Walter2,
Guido Stoll1, Christoph Kleinschnitz1*
1Department of Neurology, University of Wu¨rzburg, Wu¨rzburg, Germany, 2 Institute for Clinical Biochemistry and Pathobiochemistry, University of Wu¨rzburg, Wu¨rzburg,
Germany, 3Department of Physiology, University of Wu¨rzburg, Wu¨rzburg, Germany, 4Department of Anesthesiology, University of Wu¨rzburg, Wu¨rzburg, Germany
Abstract
Background: Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of
neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown.
Vasodilator-stimulated phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial
barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular
leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic
brain in vivo awaits clarification.
Methodology/Principal Findings: Focal cerebral ischemia was induced in Vasp2/2 mice and wild-type (WT) littermates by
transient middle cerebral artery occlusion (tMCAO). Evan’s Blue tracer was applied to visualize the extent of blood-brain-
barrier (BBB) damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride
(TTC)-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters
relevant for edema formation and stroke outcome. BBB damage (p,0.05) and edema volumes (1.7 mm360.5 mm3 versus
0.8 mm360.4 mm3; p,0.0001) were significantly enhanced in Vasp2/2 mice compared to controls on day 1 after tMCAO.
This was accompanied by a significant increase in infarct size (56.1 mm3617.3 mm3 versus 39.3 mm3610.7 mm3,
respectively; p,0.01) and a non significant trend (p.0.05) towards worse neurological outcomes.
Conclusion: Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic
modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema
formation in ischemic brain damage.
Citation: Kraft P, Benz PM, Austinat M, Brede ME, Schuh K, et al. (2010) Deficiency of Vasodilator-Stimulated Phosphoprotein (VASP) Increases Blood-Brain-Barrier
Damage and Edema Formation after Ischemic Stroke in Mice. PLoS ONE 5(12): e15106. doi:10.1371/journal.pone.0015106
Editor: Sven G. Meuth, University of Muenster, Germany
Received September 20, 2010; Accepted October 21, 2010; Published December 3, 2010
Copyright: � 2010 Kraft et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany, SFB 688, TP A2 (to UW), A13 (to CK), and B1 (to GS) and
Wilhelm Sander-Stiftung, Mu¨nchen, Germany (2009.017.1 to CK). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: Christoph Kleinschnitz is a PLoS ONE Academic Editor.
* E-mail: christoph.kleinschnitz@mail.uni-wuerzburg.de
Introduction
Disruption of the blood-brain barrier (BBB) and successive
edema formation are pathological hallmarks of many neurological
diseases and can dramatically deteriorate clinical symptoms
especially in patients with ischemic stroke [1,2]. So far no
medication, e. g. steroids or hyperosmolaric solutions, has proven
to effectively reduce brain edema in acute stroke [3–5] and the
molecular mechanisms underlying edema formation are largely
unknown.
The vascular endothelium controls the transition of fluids and
cells between blood vessels and the interstitium of most organs
including the brain [6]. Efficient barrier function requires stable
cell-cell- and cell-matrix-interactions and paracellular permeability
is regulated by a complex interplay of transmembrane adhesion
molecules, tight junctions and cytoskeletal proteins [7–10].
Impairment of any of these interactions can increase endothelial
leakage and result in excessive edema formation [9]. Tight
junctions represent the most apical of these cell-cell contacts and
are of major importance for sealing vascular barriers. While earlier
studies mainly focused on the relevance of transmembrane
adhesion molecules for tight junction functionality, there is a
growing body of evidence pointing towards a critical role of actin
cytoskeleton dynamics in modulating the permeability of the BBB
[11] and other endothelia [12,13].
Vasodilator-stimulated phosphoprotein (VASP) is the founding
member of the Enabled/vasodilator-stimulated phosphoprotein
(Ena/VASP) protein family [14]. In mammals, this family
comprises three molecules: mammalian Ena (Mena), VASP, and
Ena/VASP-like (EVL). Ena/VASP proteins are important
mediators in actin cytoskeleton control and participate in a variety
of actin-based processes such as cell-adhesion, -spreading, and -
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shape change [14]. Only recently the fundamental role of VASP
for maintaining endothelial barrier functions has been established.
Several in vitro studies verified the expression of VASP at
endothelial cell-cell and cell-matrix contacs in different vascular
beds [15–19]. Moreover, VASP was shown to initiate perijunc-
tional actin filament assembly thereby stabilizing cell-cell contacts
and decreasing endothelial permeability [18]. Accordingly,
increased vascular leakage was observed in the inflamed skin of
Vasp2/2 mice [18] and mice deficient in Mena, Vasp, and EVL
develop spontaneous and generalized edema due to severe vessel
texture defects [20]. Although VASP is expressed in primary brain
capillary endothelial cells (BCECs) [21], little is known about the
significance of VASP for regulating vascular permeability in the
central nervous system (CNS) especially under pathological
conditions in vivo.
We here show that BBB leakage and edema formation are
increased in Vasp2/2 mice in a model of brain ischemia/
reperfusion(I/R)-injury.
Results
Systemic blood pressure, cerebral blood flow and the
brain vasculature are unchanged in Vasp2/2 mice
First of all we collected critical physiological and anatomical
parameters that could possibly influence stroke outcome and
edema formation in genetically altered Vasp2/2 mice. A complete
Circle of Willis was identified in Vasp2/2 and Vasp+/+ mice upon
macroscopic assessment and the distribution of the middle cerebral
artery (MCA) trunk and branches appeared to be identical (Figure
1A, left panel). Collateralisation via the posterior communicating
arteries (PComAs) can affect the susceptibility for brain ischemia in
transgenic mice [22]. We therefore assessed the development of
PComAs in Vasp2/2 mice and littermate controls using a
quantitative score [23]. No differences in PComAs scores were
found between the both groups (1.960.6 versus 1.760.6; p.0.05)
(Figure 1A, right panel).
In the present study tMCAO was used to induce focal brain
ischemia. After advancing the filament to the origin of the MCA
the decrease in regional cerebral blood flow (rCBF) was similar
between WT mice and Vasp2/2 mice (17.7%63.2% versus
18.7%64.0%; p.0.05) (Figure 1B). Ten minutes after removal
of the filament (reperfusion) rCBF in the MCA territory was
reconstituted to .60% of baseline levels and again did not
significantly differ between the two mouse groups (62.7%610.4%
versus 60.3%68.3%; p.0.05) (Figure 1B). These findings exclude
preformed alterations in rCBF related to the Vasp2/2 genotype
and prove that MCA occlusion and reperfusion were sufficient in
our model.
Changes in arterial blood pressure can directly influence final
stroke sizes and the magnitude of BBB disruption [24]. We
therefore compared systemic blood pressure and heart rate
between the two groups. Again, no significant differences were
observed (systolic blood pressure: 115 mm Hg 69 mm Hg versus
104 mm Hg 615 mm Hg; diastolic blood pressure 73 mm Hg
68 mm Hg versus 70 mm Hg 69 mm Hg; heart rate: 405 min21
631 min21 versus 487 min21 611 min21; p.0.05) (Figure 1C).
Vasp deficiency increases infarct size, blood-brain-barrier
damage and edema formation after ischemic stroke
We next subjected Vasp2/2 mice to tMCAO and, after 24 h,
assessed infarct volumes by staining brain sections with 2,3,5-
triphenyltetrazolium chloride (TTC) (Figure 2A, upper panel).
Infarct volumes were significantly larger, by approximately 45%,
in Vasp-deficient mice than in WT controls (56.1 mm3617.3 mm3
versus 39.3 mm3610.7 mm3, respectively; p,0.001) (Figure 2A,
lower panel). Although mice without Vasp tended to develop more
severe neurological deficits after stroke, the difference was not
statistically significant (Bederson score: 1.860.9 versus 1.560.5,
respectively; p.0.05) (Figure 2B, upper panel). In line with these
results, mortality rates were similar in both groups (p.0.05)
(Figure 2B, lower panel). Thus, our observations corroborate
previous reports on a poor correlation between infarct size and
neurological outcome in rodents [25–27].
Next we sought to elucidate the underlying mechanisms of this
VASP-specific stroke protection. Increased edema formation was
recently reported in the inflamed skin of Vasp2/2 mice [18] and
hypoxia has been shown to foster vascular leakage by downreg-
ulation of VASP in vitro [28]. Therefore, we injected mice the
vascular tracer Evan’s Blue to investigate whether VASP is also
involved in stroke-induced BBB damage and edema formation.
Evan’s Blue staining of the brain parenchyma was absent in
healthy Vasp2/2 and Vasp+/+ mice as well as sham-operated
controls of either genotype suggesting that VASP is of minor
importance for the regulation of vascular permeability under basal
conditions (not shown). 24h after tMCAO BBB leakage, i.e. Evan’s
Blue extravasation was more pronounced in Vasp2/2 mice
compared with littermate controls (39.1 mm3617.2 mm3 versus
19.5 mm367.9 mm3, respectively; p,0.05) (Figure 2C). Accord-
ingly, Vasp2/2 mice developed significantly more brain edema
(0.8 mm360.4 mm3 versus 1.7 mm360.5 mm3, respectively;
p,0.0001).
Discussion
We here demonstrate that VASP is crucial for maintaining
vascular integrity in the ischemic brain. Vasp deficiency resulted in
enhanced BBB disruption, edema formation and neuronal damage
after experimental stroke in mice.
There is accumulating evidence that VASP is critically involved
in stabilizing endothelial barriers. Several studies could demon-
strate that VASP co-localizes with cell-cell contacts (e.g. the tight
junction marker zonula occludens protein-1) and focal adhesion
sites (e.g. VE-cadherin) in endothelial cell cultures [15,18,19].
Linkage of intercellular contacts and focal adhesions to the
intracellular actin cytoskeleton is important for sufficient sealing
[29]. VASP has been shown to regulate actin organization at
cadherin-adhesive contacts [30] and stabilize endothelial barrier
function by promoting actin polymerization and relaxation of the
actin cytoskeleton [18,19]. Consequently, transendothelial perme-
ability was significantly increased in endothelial cells from Vasp2/2
mice [15,18] and Ena/VASP triple null mice [20].
Most of the data underscoring the significance of VASP for
preserving vascular integrity have so far been derived from in vitro
studies which in addition were mainly conducted in microvascular
endothelial cell lines from myocardium, lung or skin under
physiological conditions [15,16,19,28]. We here confirm and
further extend these findings by demonstrating that VASP also
prevents BBB damage and edema formation in the brain after
tMCAO in mice, a well established in vivo model of ischemic stroke.
Hypoxemia is a potent trigger of vascular leakage [31–33] and
downregulation of VASP by hypoxia-inducible factor (HIF) possibly
participates in this process at least in cell culture systems [28].
Because HIF is also strongly induced in the murine brain after
tMCAO [34–36], HIF-dependent degradation of VASP could also
be functionally relevant for edema formation in ischemic stroke in
vivo. Interestingly, inhibition of HIF-1 using small interfering RNA
reduced Evan’s Blue extravasation and brain ischemia-reperfusion
injury in rats [36]. Two recent in vitro studies investigated a possible
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implication of VASP for hypoxia-induced BBB disruption. Davis
and co-workers found that hypoxia-induced VEGF expression
increased BBB permeability and correlated with VASP phosphor-
ylation, which was in part mediated through the VEGF receptor 2
[37]. In the second study, the subcellular distribution of VASP in
immortalized brain endothelial cells was significantly altered under
hypoxic conditions [38].
Inflammation is another well-established trigger of increased
vascular permeability [12]. In a model of LPS-induced acute lung
injury, Vasp2/2 mice showed increased pulmonary damage,
neutrophil infiltration and vascular leakage compared with wild-
type animals [39]. Benz and co-workers [18] recently reported that
Vasp2/2 mice develop more skin edema upon subcutaneous
injections of the proinflammatory peptide hormone bradykinin,
the end product of the kallikrein/kinin-system. In line with these
findings, we [40,41] and others [42] could show that endogenous
bradykinin also fosters edema formation in ischemic stroke and
traumatic brain injury. However, the exact molecular interplay
Figure 1. Vasp deficiency does not alter anatomical and physiological parameters relevant for stroke outcome. (A) (left) A complete
Circle of Willis (arrows) was present in wild-type (WT) and Vasp2/2 mice and the trunk and branches of the middle cerebral artery (MCA) were similar
in both groups as depicted by ink perfusion. (right) The formation of the posterior communicating arteries (PComAs) was quantitatively assessed
under a microscope in both mouse groups. The PComAs score did not differ between Vasp2/2 mice and WT controls (n = 5/group), p.0.05; unpaired,
two-tailed Student’s t-test compared with WT mice. (B) rCBF in the MCA territory was measured by Laser Doppler flowmetry before (baseline) and
immediately after MCAO (ischemia), and again 10 min after removal of the occluding filament (reperfusion). No significant differences in rCBF were
observed at any time point between WT and Vasp2/2 mice (n = 5/group); p.0.05. Bonferroni-corrected 2-way ANOVA compared to baseline rCBF. (C)
Systolic and diastolic blood pressure (RR) (left) as well as heart rates (right) are similar in Vasp2/2 mice and WT controls, p.0.05; unpaired, two-tailed
Student’s t-test compared with WT mice. ns: not significant.
doi:10.1371/journal.pone.0015106.g001
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Figure 2. Vasp deficiency increases infarct volumes, BBB damage and edema formation after ischemic stroke. (A) (top) Representative
2,3,5-triphenyltetrazolium chloride (TTC) stains of three corresponding coronal brain sections of wild-type (WT) and Vasp2/2 mice on day 1 after
tMCAO. The ischemic infarctions appear white. (bottom) Brain infarct volumes as measured by planimetry without correction for edema (direct
volumes) in WT (n = 15) and Vasp2/2 mice (n = 17) on day 1 after tMCAO, **p,0.01; unpaired, two-tailed Student’s t-test compared with WT mice. (B)
Neurological Bederson score (top) and mortality rates (bottom) of WT mice (n = 15) and Vasp2/2 mice (n = 17) on day 1 after tMCAO, p.0.05; non-
parametric Mann Whitney test (for Bederson score) or Fisher`s exact contingency test (for mortality) compared with WT mice. (C) (top, left)
Representative coronal brain sections from Vasp2/2 and WT mice on day 1 after tMCAO and injection of the vascular tracer Evan’s blue. (bottom, left)
Volume of Evan’s blue (EB) extravasation as determined by planimetry (n = 5/group). (right) Brain edema volumes as calculated from direct and
indirect infarct volumes on day 1 after tMCAO in WT mice (n = 15) and Vasp2/2 mice (n = 17), *p,0.05, ***p,0.0001; unpaired, two-tailed Student’s t-
test compared with WT mice.
doi:10.1371/journal.pone.0015106.g002
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between the kallikrein/kinin-system and VASP in the context of
hypoxia, inflammation and vascular leakage needs to be further
established.
Taken together, our study indentifies VASP as critical regulator
of BBB maintenance and fluid hemostasis during cerebral
ischemia. Interference with VASP or VASP-dependent signalling
pathways could become a promising strategy to treat excessive
brain edema in stroke and possibly other neurological diseases
afflicted with severe BBB disruption.
Materials and Methods
Animals
A total of 89 mice were used in this study. Animal experiments
were approved by legal state authorities (Bezirksregierung of
Unterfranken, approval number 54-2531.01-25/06) and conduct-
ed according to recent recommendations for research in basic
stroke studies including blinded evaluation of the results,
randomization of animals, predefinition of exclusion criteria, and
power calculations (see below) [43]. The generation and extensive
characterization of Vasp2/2mice is described elsewhere [44]. 6–8
week old male and female Vasp2/2mice were used and wild-type
(WT) littermates (Vasp+/+) matched for age and sex served as
controls.
Stroke model
The transient middle cerebral artery occlusion (tMCAO) model
was applied to induce focal cerebral ischemia as described
elsewhere [45,46]. Briefly, mice were anesthetized with 2%
isoflurane in a 70% N2O/30% O2 mixture. A servo-controlled
heating blanket was used to maintain core body temperature close
to 37uC throughout surgery. Following a midline neck incision a
standardized silicon rubber-coated 6.0 nylon monofilament (60-
1720RE; Doccol, Redlands, CA, USA) was inserted into the right
common carotid artery and advanced via the internal carotid
artery to occlude the origin of the MCA. After 60 min mice were
re-anesthetized and the occluding filament was removed to allow
reperfusion. Operation time per animal did not exceed 15 min
and operators (PK, CK and MA) were blinded for the respective
genotypes throughout the study.
The exclusion criteria were as follows:
a) Death within 24h after tMCAO
b) Subarachnoid hemorrhage (SAH; as macroscopically assessed
during brain sampling)
c) Bederson score (see below) = 0 (24 h after tMCAO)
3 out of 43 WT mice (6.9%) and 4 out of 46 Vasp2/2 mice
(8.7%) met at least one of the exclusion criteria (2 deaths and 1
non-fatal SAH in the WT group and 3 deaths and 1 non-fatal
SAH in the Vasp2/2 group, respectively). The excluded animals
were used only for mortality analysis (Figure 2B). 82 out of 89 mice
(92.1%) were included for final analysis.
Anatomical assessment of the cerebral vasculature
For assessment of the cerebral vasculature Vasp-deficient mice
and controls (n = 5/group) were deeply anesthetized with CO2 and
transcardially perfused with 4% paraformaldehyde (PFA), followed
by 3 ml black ink diluted in 4% PFA (1:5 v/v). Brains were
carefully removed, fixed in 4% PFA overnight at 4uC and the
Circle of Willis and major arteries were examined under a
microscope. To further quantitatively examine the vascular
structures, we graded the development of the posterior commu-
nicating arteries (PComAs), which can affect brain sensitivity to
ischemia [23], according to the following score: 0, absent; 1,
capillary anastomosis; 2, small truncal vessel; 3, patent.
Regional cerebral blood flow measurement
Laser-Doppler flowmetry (Moor Instruments, U.K.) was used to
monitor regional cerebral blood flow (rCBF) in Vasp2/2 mice and
WT controls before surgery (baseline), immediately after MCA
occlusion, and 10 minutes after removal of the occluding
monofilament (reperfusion) (n = 5/group) [47]. For this procedure
a small incision was made in the skin overlying the temporal
muscle, and a 0.7 mm flexible laser-Doppler probe (model P10)
was positioned perpendicular on the superior portion of the
temporal bone (6 mm lateral and 2 mm posterior from bregma).
This position corresponds to the core of the ischemic territory.
Invasive hemodynamics
For invasive hemodynamics Vasp2/2 mice and controls (n = 4/
group) were anesthetized with 2.5% isoflurane and catheterized
via the right carotid artery with a high-fidelity 1.4 F Millar
microtip catheter as described [40]. Hemodynamic data (blood
pressure and heart rate) were digitized via a MacLab system (AD
Instruments, Castle Hill) connected to an Apple G4 PowerPC
computer and analyzed.
Assessment of functional outcome
After recovery from anesthesia and again after 24h, neurological
function was assessed by two investigators unaware of the
genotype according to the Bederson score (n = 15 in the wild-
type and n= 17 in the Vasp2/2group, respectively) [48] with: 0, no
deficit; 1, forelimb flexion; 2, as for 1, plus decreased resistance to
lateral push; 3, unidirectional circling; 4, longitudinal spinning; 5,
no movement. In addition, the mortality rate was monitored until
24h after tMCAO.
Determination of infarct size and edema volumes
Animals were sacrificed 24h after tMCAO. Brains were quickly
removed and cut in three 2-mm thick coronal sections using a
mouse brain slice matrix (Harvard Apparatus, Holliston, MA,
USA). The slices were stained for 20 min at 37uC with 2% 2,3,5-
triphenyltetrazolium chloride (TTC; Sigma-Aldrich, Taufkirchen,
Germany) in PBS to visualize the infarctions [46,49].
Direct, i.e. without correction for brain edema, and indirect, i.e.
corrected for brain edema, infarct volumes (n = 15 in the wild-type
and n= 17 in the Vasp2/2group, respectively) were calculated by
volumetry (ImageJ software, National Institutes of Health, USA)
according to the following equations:
Vdirect (mm3)~(Area TTC section 1 (mm2)|2 mm)
z(Area TTC section 2 (mm2)|2 mm)
z(Area TTC section 3 (mm3)|2 mm)
Vindirect (mm3)~VInfarct|(1{(VI{VC)7VC)
where the term (VI{VC)represents the volume difference
between the ischemic hemisphere and the control hemisphere and
(VI{VC)7VC expresses this difference as a percentage of the
control hemisphere.
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