# Enhanced angiogenesis and reduced infarct size by vascular endothelial growth factor D is not translated to behavioral outcome in a rat model of ischemic stroke

Jukka Jolkkonen, Pavel Yanev, Kari J Airenne, Seppo Ylä-Herttuala, Thomas Wirth

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Jukka Jolkkonen, Pavel Yanev, Kari J Airenne, Seppo Ylä-Herttuala, Thomas Wirth. Enhanced angiogenesis and reduced infarct size by vascular endothelial growth factor D is not translated to behavioral outcome in a rat model of ischemic stroke. Journal of Experimental Stroke & Translational Medicine, 2010. hal-04363813

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# Enhanced angiogenesis and reduced infarct size by vascular endothelial growth factor D is not translated to behavioral outcome in a rat model of ischemic stroke

Pavel Yanev<fnref n="1" />,<fnref n="2" />; Jukka Jolkkonen<fnref n="2" />,<fnref n="3" />*; Kari J. Airenne<fnref n="1" />; Seppo Ylä-Herttuala<fnref n="1" />; Thomas Wirth<fnref n="1" />

## Abstract

Vascular endothelial growth factors (VEGF) induce angiogenesis in experimental stroke models. Robust angiogenic response by VEGF, however, initially creates leaky vessels leading to edema, which may compromise functional recovery. The aim of the present work was to develop adenoviral and baculoviral gene delivery of VEGF-D, a novel member of the VEGF family, to enhance long-term angiogenesis and functional recovery in a photothrombotic model of stroke in rats. Treatment effects were assessed by histology and behavioral testing. The lesion size in the adeno-VEGF-D<sup>ΔNΔC</sup> (p< 0.01) and adeno-VEGF-D (p< 0.05) groups was substantially reduced compared to vehicle controls. The short form of VEGF-D significantly increased the number of newly formed vessels in the medial (p< 0.001) and lateral cortex (p< 0.05) compared to vehicle controls. Baculoviral VEGF-D<sup>ΔNΔC</sup> treated rats showed significant effect in the medial cortex (p< 0.01). The limb placing test showed transient impairment in all virus treated groups compared to the vehicle group at the acute phase 2 and 4 days after operation (p< 0.05). The cylinder test revealed a significant limb-use asymmetry in all ischemic animals at day 7, 14 and 21 after ischemia, which was not affected by VEGF-D treatment. In conclusion, viral mediated gene transfer of VEGF-D induced angiogenesis and reduced infarct size, which were, however, not translated to improved functional outcome in the stroke model used.

**Keywords:** Therapeutic vascular growth; VEGF-DΔNΔC; cerebral ischemia; neuroprotection; rat.

## 1. Introduction

Cerebral ischemia remains one of the main causes of death, which is surpassed only by heart disease and cancer. The World Health Organization estimates that in 2002 there were over 20.5 million strokes worldwide, 5.5 million of which were fatal. More than 50% of stroke patients are left with a motor disability and stroke causes more loss of quality-adjusted life years (QALY) than any other disease in the western countries (Donnan et al. 2008). The current aging population will result in a dramatic increase in these numbers in the next two decades.

Stroke has long been considered untreatable and there is no effective drug therapy to help stroke patients during the acute phase except thrombolysis, which however is available only for a small fraction of patients. A different approach for helping stroke patients

is to promote functional recovery. Various degrees of spontaneous recovery following weeks or months after an ischemic event are observed in clinical practice and have been related to brain repair mechanisms such as neuronal sprouting, neurogenesis and angiogenesis (Martinez-Vila et al. 2005). Stimulating the formation of new blood vessels opens a new horizon for neurorestoration that aims to enhance brain perfusion, rescuing hypoxic neurons and promoting plasticity and neurological recovery (Chen and Chopp 2006).

Angiogenesis is induced following cerebral ischemia through a variety of growth factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) (Ruiz de Almodovar et al. 2009; Shibuya 2009; Storkebaum et al. 2004). All members of the VEGF

<footnote n="1">¹Department of Molecular Medicine and Biotechnology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland</footnote>

<footnote n="2">²Institute of Clinical Medicine - Neurology, University of Eastern Finland</footnote>

<footnote n="3">³Brain Research and Rehabilitation Center Neuron, Kortejoki, Kuopio</footnote>

<footnote>* Correspondence should be sent to:</footnote>

<footnote>Dr. Jukka Jolkkonen, Institute of Clinical Medicine - Neurology, University of Eastern Finland, P. O. Box 1627, Yliopistonranta 1 C, 70211 Kuopio, Finland. Tel: +358-40-3552519; FAX +358-17-162048; Email: [Jukka.Jolkkonen@uef.fi](mailto:Jukka.Jolkkonen@uef.fi)</footnote>
family exert their effects by binding to tyrosine ki-
nase receptors (VEGFR) on the cell surface. Particu-
larly, VEGF-A is a powerful mediator to increase an-
giogenesis and neovascularization by binding to its
primary receptor VEGFR-2 (Tammela et al. 2005).
However, vessels induced by VEGF-A overexpres-
sion are leaky, which leads to edema or hemorrhage,
that may compromise functional recovery (Jolkkonen
et al. 2007; Yu et al. 2007). Thus, other members of
the VEGF family such as VEGF-D may be safer for
therapeutic angiogenesis after stroke.

VEGF-D is a secreted glycoprotein that induces both
angiogenesis and lymphangiogenesis. It consists of a
central domain, containing binding sites for VEGFR-2
and VEGFR-3, and N- and C-terminal propeptides
(Achen et al. 1998). The natural cell product of
VEGF-D is a homodimer of the full-length form that
can be proteolytically processed to remove the pro-
peptides into VEGF-D<sup>ANAC</sup> (Brockington et al. 2004;
Sun et al. 2003). The mature form of VEGF-D has
many times greater affinity for VEGFR-2 binding than
the unprocessed one (McColl et al. 2007). While pro-
teolytically processed VEGF-D has high affinity for
binding to both VEGFR-2 and VEGFR-3, the full-
length VEGF-D binds with high affinity only to
VEGFR-3.

The aim of the present study was to evaluate the an-
giogenic potential of VEGF-D, a member of the
VEGF gene family, after differential transduction of
the brain by adenoviral and baculoviral gene delivery,
in a photothrombotic model of stroke in rats. Delayed
expression of VEGF-D and particularly its short
processed form was expected to improve the forma-
tion of functional and stable blood vessels, which
translates to behavioral improvement.

## 2. Methods

2.1 Animals

Eighty-four male Wistar rats (6 months, 385-503 g) were used in the study. The animals had free access to food and water and were housed in individual cages in a temperature-controlled environment (20±1°C) with lights on from 7.00 to 19.00 h. Experimental procedures were conducted in accordance with the European Community Council directives 86/609/EEC and the study was approved by the Ethics Committee of the University of Eastern Finland and the Provincial Government of Kuopio.

## 2.2 Cortical photothrombosis

Cortical photothrombosis was induced by focusing
light to the sensorimotor cortex in Rose Bengal
treated rats (Watson et al. 1985). Briefly, the rats
were anesthetized with 5% halothane in 30% O₂/70%
N₂O and placed in a stereotactic frame. The anesthe-
sia was maintained during the operation with 1-2%

halothane delivered by a nose mask and the body
temperature was kept at 37 °C by a rectal probe and
heating pad. The skull was exposed and a cold white
light (Olympus, Denmark) with a 4 mm aperture was
positioned onto the exposed skull 0.5 mm anterior to
the bregma and 3.7 mm lateral to the midline over the
right motor cortex. The photochemical dye Rose
Bengal (Sigma) was infused into the femoral vein via
a microinjection pump within 2 min (20 mg/kg), after
which the light was turned on for 10 min. The skull
surface temperature was monitored with a probe
placed between the skull and the light source, and
kept constant by cool air flow. Sham-operated ani-
mals were treated similarly, but the light was not
turned on. The rats were removed from the frame,
sutured, and allowed to wake up in an incubator
(32 °C) before being returned to their home cages.

## 2.3 Vector preparation and characterization

*Adenovirus.* Replication-deficient human clinical-
grade E1- partial E3-deleted first-generation adenovi-
ruses encoding human VEGF-D<sup>ANAC</sup> and lacZ under
the cytomegalovirus (CMV) promoter were con-
structed by homologous recombination and produced
in 293 cells as described previously (Hiltunen et al.
2000; Laitinen et al. 1998; Mäkinen et al. 2002).

*Baculovirus.* All entry clones were completely sequenced to verify the sequences before cloning of VEGF-D<sup>ANAC</sup> into a pBVboostFGII expression vector using the BVboost system LR-reaction (Laitinen et al. 2005). Avidin displaying baculoviruses (Räty et al. 2004) encoding for VEGF-D<sup>ANAC</sup> (BAAVI-VEGF-D<sup>ANAC</sup>) were generated by using an improved Bac-to-Bac method (Airenne et al. 2003). For the Baculo-lacZ virus, a nuclear targeted β-galactosidase (β<sub>nt</sub>-Gal) cassette with a CMV promoter was inserted into the Stul site of transfer vector pFASTBac1 (pFB) (Gibco BRL, Life Technologies), generating plasmid pFBCMV- β<sub>nt</sub> (Airenne et al. 2000). Purification, concentration, and titration of viral particles were performed as described previously (Airenne et al. 2000). Virus preparations were tested for sterility and analyzed for lipopolysaccharide and mycoplasma contamination.

2.4 Gene transfer

After photothrombotic operation a burr hole was
drilled and 10 µl of viral suspensions (Adeno-VEGF-D
(n=10), Adeno-VEGF-D<sup>ANAC</sup> (n=13), Adeno-LacZ
(n=10), Baculo-VEGF- D<sup>ANAC</sup> (n=15), Baculo-LacZ
(n=14)) containing 2x10<sup>10</sup> p.f.u/ml were injected into
the left ventricle (AP: -0.8 mm, L: 1.5 mm, V: 3.5 mm)
at a rate 0.1 µl per minute. The needle was with-
drawn after 25 minutes of injection. Control animals
received the same amount of saline.

2.5 Behavioral testing
The behavioural tests selected for the study are sensitive to detect treatment effects. The animals were tested before operation and on postoperative days 2, 4, 7, 14 and 21. Behavioural testing was carried out by a person blind to treatment.

**Limb-placing test.** The limb-placing test was used to assess the sensorimotor integration of fore- and hindlimb responses to tactile and proprioceptive stimulation (De Ryck et al. 1989; Jolkkonen et al. 2000). The test has seven limb placing tasks, which are scored: 2 points, the rat performs normally; 1 point, the rat performs with a delay (> 2 sec) and/or incompletely and 0 points, the rat does not perform normally. The both sides of the body are tested. In the first task the rat is suspended 10 cm above the table surface. Non-lesioned rats stretch both fore-limbs towards the table. On the second task the rat is held facing towards the table, resting its forelimbs on the table edge. The forelimb is gently pulled down, from the table, and subsequent retrieval and limb placement is checked. Non-lesioned rats replace both limbs on the table. The third task is the same as the second, except that the rat is not able to see the table or make vibrissal contact, since the head is held upward at a 45° angle. The rats are next placed along the table edge to check the lateral placing of the fore- (the fourth task) and hindlimbs (the fifth task). The limbs are pulled down, as described in task 2, and limb retrieval is scored accordingly. In the sixth task the rat are placed with their rear end at the edge of the table, with the hindlimbs resting on the table edge. The hindlimbs, 1 at a time, are gently pulled down and from the table. If necessary, retrieval of the limb to the original resting place on the table edge can be stimulated by pushing the animal towards the table edge. In the seventh task the rat is placed at the table edge, facing away from the table surface. The forelimbs of the rat are placed on the edge of the table, and the rat is gently pushed from behind toward the edge. Injured rats cannot keep their grip and the injured limb slips off the edge.

**Cylinder test.** The cylinder test was used to assess imbalances between the impaired and non-impaired forelimb use (Schallert and Woodlee 2005). For the test, the rat is placed in a transparent cylinder (ø 20 cm) and videotaped during the light part of the light:dark cycle before operation and at postoperative days 7, 14, and 21. A mirror was placed at 45° angle beneath the cylinder so that behavior could be filmed from below the cylinder. Exploratory activity for 1 to 3 min was analyzed by using a video recorder with slow motion capabilities. The number of contacts by both forelimbs and by either the impaired or nonimpaired forelimb was counted. The cylinder score for the impaired forelimb was calculated as: (impaired forelimb + ½ x both forelimbs)/(total contacts) x 100%.

## 2.6 Immunohistochemistry

Rats were perfused transcardially 21 days after operation with 0.9 % NaCl followed by 4 % paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were removed from the skulls, postfixed, and cryoprotected. Frozen sections (35 µm) were cut with a microtome and stored in a cryoprotectant tissue-collection solution at -20 °C. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) was stained as a marker for blood vessels from five randomly selected rats in each experimental group. The sections were pretreated for 30 minutes with hot (85 °C) citrate buffer after which the sections were transferred to a solution containing the primary antibody (rabbit anti-PECAM-1 at 1:1000, Santa Cruz) and Tris-buffered saline with 5 % normal goat serum (NGS) and 0.5 % Triton X-100 (TBS-T). Following 18 hours of incubation in this solution on a shaker table at room temperature (20 °C) in the dark, the sections were rinsed three times in 5 % NGS and TBS-T and transferred to a solution containing the secondary antibody (goat anti-rabbit*biotin at 1:500, Vector). After 2 hours, the sections were rinsed three times with 2 % NGS and TBS-T and transferred to a solution containing mouse ExtrAvidin® (Sigma) for 1 hour, and then incubated for approximately 3 minutes with Ni-enhanced diaminobenzidine.

## 2.7 Evaluation of neovascularization

In order to quantify angiogenesis after cortical photothrombosis, images (x 20 magnification) of the cortex lateral and medial the lesion and striatum were acquired using Olympus BX40 microscope and Olympus digital camera DP50-CU and image acquisition software Viewfinder (Pixera Corporation). Images were digitally processed using Adobe® Photoshop® CS3. In each section the corresponding areas from the contralateral hemisphere were used as a control (Fig. 1). PECAM-1 immunoreactivity in each image was determined using two image analysis systems. Images were analyzed initially with ImageJ, a Java-based image processing program, but in order to get reliable values, custom made software was adopted. It incorporated the color segmentation method in a MathWorksTM Matlab® 7.5 environment, modified for 3 channels so that vessels could be better distinguished from the background. In comparison, ImageJ uses binary basis for quantification. The values corresponding to total vessel coded color areas were averaged and expressed as the mean percentage of stained vessels per 100 µm².

## 2.8 Assessment of infarct volumes

Nissl-stained sections were used for assessment of infarct volumes. Estimation of the infarct area in the cortex was performed on the sections digitalized under a × 20 objective (Olympus BX40) using a 3-CCD
color video camera (Sony DXC-970MD) interfaced
with an image analysis system (MCID, Imaging Re-
search). The image of each section was taken as
1280 x 1024 matrix of calibrated pixel units. The digi-
tal image was then displayed on a video screen and
the lesion area was manually outlined. The total vo-
lume was calculated by multiplying the infarct area by
the distance between the sections and summing to-
gether the volumes for each brain.

## 2.9 Statistical analysis

SPSS software was used for statistical analyses. Sta-
tistical differences in infarct volumes in the cortex and
blood vessel counts between groups were analyzed
using one-way analysis of variance (ANOVA) fol-
lowed by a post hoc test (Scheffe) when necessary.
Differences in the limb-placing scores between ex-
perimental groups were analyzed by Kruskal-Wallis
followed by Mann-Whitney U-tests. Cylinder data for
the overall group effect were analyzed using ANOVA
for repeated measures. Comparisons between the
groups were then made using one-way ANOVA fol-
lowed by a post hoc test (Scheffe).

## 3. Results

### 3.1 Survival of rats

The cortical photothrombosis is generally characte-
rized by a very low mortality. In the present experi-
ment only two rats had to be sacrificed due to com-
plications during the recovery period. There was no
significant difference in body weight between sham-
operated and ischemic control rats or between
treated ischemic rats and vehicle controls.

### 3.2 Ischemic lesion

The cortical infarct was located in the frontal cortex
(Fr1 and Fr2) and extended to the corpus callosum
with no evidence of damage to the underlying stria-
tum (Fig. 1A). Resolution of the necrotic tissue re-
sulted in the formation of a partly fluid-filled cyst by
the end of the 3 week follow-up period. In the current
settings the average size of the cortical lesion in ve-
hicle controls was 33.6±2.4 mm³. The infarct sizes in
the adeno-VEGF-D^ΔNΔC^ (20.4±6.1 mm³) (p< 0.01) and
adeno-VEGF-D (22.4±5.5 mm³) (p< 0.05) groups
were substantially reduced compared to vehicle con-
trols (Fig. 2). Baculo-VEGF-D^ΔNΔC^ group (26.9±2.4
mm³) was not different from vehicle controls.

<figure><img src="image_2.png" /><figcaption>Fig. 1. Pseudo-colored image from the rat subjected to cortical photothrombosis and representation of areas in the ipsilateral and contralateral cortex selected for the analysis of angiogenesis (A). A micrograph of PECAM staining in the ipsilateral (B) and contralateral medial cortex (C) from the Ad-VEGF- D^ΔNΔC^ treated rat. Scale bar 100 µm.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Fig. 2. Infarct volumes (mm³) in rats subjected to cortical photothrombosis followed by treatment with viral delivered VEGF-D. Data are mean±S.D. The number of rats in each group is in parenthesis. Statistical significance: \* p< 0.05; \*\*\* p< 0.001 (from vehicle).</figcaption></figure>

### 3.3 Quantification of angiogenesis

In order to determine whether intraventricular administration of different forms of VEGF-D enhances cerebral angiogenesis, photothrombotic ischemic rats were treated with the viral constructs 30 minutes after ischemia. It was expected that delayed production of VEGF-D would surpass the peak of brain excitotoxicity and edema and show therapeutic effect on angiogenesis. Previous studies with VEGF-A have showed a peak of newly formed vessels 12 days after administration of viral vectors (Dellian et al. 1996). For vessel quantification, 7 distinct non-overlapping areas were selected from the ipsilateral hemisphere and the corresponding areas from the contralateral hemisphere (Fig. 1A). The ratio of the two was then compared to that of the control group. The program

used for vessel density quantification is novel in the sense that it distinguishes vessels much more accurately from the background staining as it incorporates the 3 color segmentation mechanism as compared for example to conventional NIH Image® software for Image Analysis (National Institutes of Health).

Compared to the vehicle controls, the short form of VEGF-D<sup>ΑΝΑC</sup> significantly increased the number of newly formed vessels in the medial (p< 0.001) and lateral cortex (p< 0.05) (Fig. 3). Interestingly, baculo-VEGF-D<sup>ΑΝΑC</sup> did show significant effect only in the medial cortex (p< 0.01). The other vector construct, adeno-VEGF-D, which is a far less potent angiogenic factor than the proteolytically processed VEGF-D did not increase the angiogenic response significantly.
<figure><img src="image_4.png" /><figcaption>Fig. 3. Angiogenesis as assessed by PECAM staining in the ipsilateral and contralateral cortex and striatum in rats subjected to cortical photothrombosis followed by treatment with viral delivered VEGF-D. The ratio between the ipsilateral and contralateral cortex was calculated for statistical analysis. Data are mean±S.D. The number of rats in each group is in parenthesis. Statistical significance: \* p< 0.05; \*\*p< 0.01; \*\*\*p< 0.001 (from vehicle).</figcaption></figure>

### 3.4 Behavioral testing

To determine whether VEGF-D and consequential angiogenesis improve functional recovery after photothrombotic stroke, the behavioral performance of rats was tested during the 21 day follow-up. Rats in all the ischemia operated groups showed a significant sensorimotor impairment at the acute phase 2 days after stroke. Partial spontaneous recovery was observed in all groups.

The limb placing test showed impaired performance in all virus treated rats compared to vehicle controls at 4 days (p< 0.05), however, this was transient and

no difference was detected at 21 days except in the case of baculo-VEGF-D<sup>ΔNΔC</sup> (Fig. 4), which indicates that this was most likely due to some adverse effects of the viral treatment at the peak of gene expression (e.g., inflammatory response). All ischemic groups showed a reduced use of the impaired forelimb in the cylinder test at day 7, 14 and 21 after the infarct (Fig. 5). ANOVA for repeated measures showed a significant group effect (p< 0.01) and a significant time x group interaction (p< 0.01). These were explained by the difference between sham-operated rats and ischemic groups.
<figure><img src="image_5.png" /><figcaption>Fig. 4. Forelimb use during lateral exploration in rats subjected to cortical photothrombosis followed by treatment with viral delivered VEGF-D. The score was calculated as (impaired forelimb use + ½ both forelimb use/total contacts) x 100%. Data are mean±S.D. The number of rats in each group is in parenthesis. There were no statistically significant differences among ischemic rats.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 5. Limb-placing scores in rats subjected to cortical photothrombosis followed by treatment with viral delivered VEGF-D. Initial severe impairment is followed by spontaneous recovery during the follow-up. Data are median±S.D. The number of rats in each group is in parenthesis. Statistical significance: \*p\< 0.05; \*\*p\< 0.001</figcaption></figure>
(Baculo-VEGF- D^ANΔC vs. vehicle); #p< 0.05 (Baculo-LacZ vs. vehicle); §p< 0.05 (Ad-VEGF-D/Ad-LacZ/Ad-VEGF- D^ANΔC vs. vehicle).

4. Discussion

In the present study we evaluated the therapeutic
potential of VEGF-D after ischemic stroke.

To our knowledge, there are no studies in the litera-
ture that have evaluated the therapeutic potential of
VEGF-D induced angiogenesis in experimental
stroke. Several studies have suggested that VEGF-D
and particularly its processed form are powerful angi-
ogenic factors in muscle and heart tissues. Adenovir-
al VEGF-D^ΔΝΔC gene transfer in pig myocardium has
been shown to induce a dose dependent protein pro-
duction and promote angiogenic effects much more
diffusely throughout the myocardial tissue as com-
pared to VEGF-A₁₆₅ (Rutanen et al. 2004). This dif-
ference could be attributed to the soluble and freely
diffusible VEGF-D^ΔΝΔC while VEGF-A₁₆₅ has good
matrix-binding properties. In a rabbit hindlimb ische-
mia model, VEGF-D^ΔΝΔC produces the strongest an-
giogenic effect among the members of the VEGF
family (Rissanen et al. 2003). VEGF-D^ΔΝΔC upregu-
lates VEGFR-2 and αvβ₃ integrin expression in endo-
thelial cells, which suggest that angiogenic signaling
mechanisms are very similar to those of VEGF-A
(Ferrara 1999). With a higher dose of both VEGF-
D^ΔΝΔC and VEGF-A there is substantial edema forma-
tion. However, VEGF-A induces greater edema as
compared to VEGF-D^ΔΝΔC after intramyocardial gene
transfer (Rutanen et al. 2004), and might therefore be
better as a pro-angiogenic candidate in therapeutic
use.

4.1 Different transduction profiles of adenovirus and baculovirus in the CNS

Easy production and high gene deliver efficiency into
immune-privileged sites such as those of the CNS
make adenovirus and baculovirus positive candidates
for a therapeutic delivery of VEGF-D. Moreover, they
have different transduction patterns in the brain (Leh-
tolainen et al. 2002). Baculoviruses were found to
transduce the cuboid epithelium of the choroid plexus
very efficiently (76.8±14%) whereas adenoviruses
showed a marked preference for the ventricular
ependymal cell lining (71.4±9%) and corpus callosum
(83.5±11%). Transgene expression with ade-
no/baculovirus starts as early as 6 h after transduc-
tion and reaches peak at 3-6 days depending on the
construct and the vector (Airenne et al. 2009; Russel
2000). Both viral vector types lead also to expression
of the transgene in the brain microvessel endothelial
cells (Lehtolainen et al. 2002). In accordance, a dif-
ference in the angiogenic response between adenovi-

rus and baculovirus was detected also in the present
study.

4.2 Angiogenesis and VEGF-D

Here we demonstrated that adenovirus and baculovi-
rus induced expression of VEGF-D^ΔΝΔC significantly
enhances cerebral angiogenesis ipsilaterally in a rat
photothrombotic stroke model. The virus itself did not
enhance angiogenesis, as there was no difference in
vessel density ratios compared to the sham and ve-
icle treated animals. The advantage of transducing
the ependymal cell lining and the choroid plexus by
adenovirus and baculovirus, respectively, is that
VEGF-D is a soluble protein and will be secreted into
the cerebrospinal fluid. The vectors were not injected
into the ischemic core, because the cells are affected
by necrotic death and extensive edema a few hours
after the onset of ischemia (Siesjö 1981; Taoufik and
Probert 2008). There have been many studies in
which the method of application of the therapeutic
agent has been chosen to be direct parenchymal
administration close to the lesion (Gunnett and Heis-
tad 2001). Although this was a feasible delivery ap-
proach especially in the photothrombotic model of
stroke where the position of infarcted tissue is more
or less constant, we expected that forced overpro-
duction of VEGF-D would increase the burden on the
already compromised brain environment in the peri-
infarct site.

In our paradigm, VEGF-D protein disseminates
through the cerebrospinal fluid circulation in the brain.
Abundant VEGF-D delivery may also cause angioge-
nesis in remote areas of the brain and possibly in the
contralateral hemisphere. Nevertheless elevated le-
vels of VEGFs, within a certain range, are not suffi-
cient to produce a significant angiogenic response in
the non-affected brain tissue. The formation of stable
new blood vessels requires activation of brain repair
mechanisms around the lesion as well as in some
parts of the ipsilateral hemisphere functionally con-
nected with the injury. For example, numerous deve-
lopmental factors are re-expressed in the peri-infarct
regions, which promote neurogenesis, synaptogene-
sis and angiogenesis (Chopp and Li 2006). In this
sense the density of VEGFR-2, which plays the major
role in angiogenesis, increases within 48 h post-
ischemia (Marti et al. 2000) and this is predominantly
on ischemic neurons (Beck et al. 2002) and astro-
cytes (Choi et al. 2007). Growth factors such as
VEGF-A stimulate further overexpression of the
VEGFR-2 (Wang et al. 2005).

It is interesting to point out that while VEGFR-3, the
primary receptor for long unprocessed form of VEGF-
D, was long believed to be responsible only for lym-
phangiogenesis, new reports have emerged showing the compensatory function of VEGFR-3 signaling in angiogenesis (Tammela et al. 2008). Studies show that signaling through it promotes proliferation and survival of endothelial cells in culture (Goldman et al. 2007). Moreover, blocking this receptor appears to most prominently affect the number of sprouting and branching points of new blood vessels. In the experiments on tumor anti-angiogenesis, it became evident that VEGFR-3 can drive angiogenesis even in conditions where VEGFR-2 is targeted therapeutically to suppress its angiogenic signals. In our case the long form of VEGF-D did not produce a significant angiogenic response, but it reduced infarct size.

### 4.3 Reduction of infarct size by VEGF-D

The production of VEGF-D starts at 6 h post transduction, yet the formation of the first new stable blood vessels takes at least a few days, a time window surpassing the rapid period in which the necrotic core expands (Chen et al. 1994). Thus, it is likely that the smaller lesion size in VEGF-D<sup>ΔNΔC</sup> treated rats is a result of a mechanism other than true neuroprotection. One possibility is that VEGF-D reduces the brain injury by protecting the vascular system in the peri-infarct site against ischemia. Indeed, VEGF-A has an important role in maintaining or restoring the integrity of endothelial cells (Radisavljevic et al. 2000). A second possibility is that VEGF-D has a potent growth and survival effect on neurons and vascular cells similar to VEGF-A (Chavakis et al. 2002; Nishijima et al. 2007; Rosenstein et al. 2003). Although there are some differences in the reports of the temporal course of gene induction as well as the identification of expressing cells, VEGF-A is increased after injury in the peri-infarct region as early as 1-3 h (Beck and Plate 2009; Hermann and Zechariah 2009) and persists for up to 1 month after ischemia (Hai et al. 2003). VEGFR-1 and VEGFR-2 expression is also elevated in neurons and endothelial cells of penumbral areas beginning at 3 h, peaking at 24 h, and persisting for more than 7 days after ischemia (Brockington et al. 2004). The VEGFR-2 signaling pathway that results in cAMP response element-binding protein (CREB) phosphorylation is the shared mechanism that underlies the preconditioning-mediated protection of neurons and vascular endothelial cells in the neonatal rat brain (Lee et al. 2009). A further study on the matter suggested that VEGFR-2/CREB signaling is critical for VEGF-A-mediated protection in neurons and cerebral vascular endothelial cells (Lee et al. 2010). This could also contribute to our results. Inflammatory response as indicated by the increase of body temperature in all virus treated rats (data not shown), might also alter lesion size. And last, reduction of infarct volume may be due to an enhanced

removal process of debris due to angiogenesis induced by VEGF-D treatment.

### 4.4 Functional recovery

This is the first report on the effect of VEGF-D on functional recovery after focal cerebral ischemia in rats. Our study indicates that VEGF-D<sup>ΔNΔC</sup>-induced peri-lesional angiogenesis is not obviously translated to functional recovery as assessed by sensorimotor tests. The recent literature on the possible therapeutic effects of VEGF in stroke is mixed (Manoonkitiwongsa et al. 2004; Yang et al. 2010). Different experimental models, timing of treatment, administration route, doses and outcome measures are likely to contribute to this. Inflammation caused by viral vectors may compromise beneficial treatment effects in the present study. In addition, in all the groups there is already a spontaneous functional recovery. Because the current model produces only mild motor function impairment, it may be that the VEGF-D treatment is not sufficient to further boost the existing recovery mechanisms. Those effects on neurological outcome could be much better demonstrated in a model with more severe brain infarction such as MCAO. Another limitation of the current model is that the photothrombotic lesion does not leave enough salvageable brain tissue where the angiogenic factor could exert its restorative potential fully. Indeed, the penumbral area here is substantially smaller, and although there is expansion of the necrotic core, it is not as profound as in the model of MCAO (Watson et al. 1995; Witte et al. 2000). Another speculation for the behavioral test results could be that the timing of increased angiogenesis and of increased vessel density may not be sufficient for a robust amelioration of neurological deficit. Angiogenesis is elevated in the peri-infarct area in the beginning of the recovery process, but it may need much more time to promote neurogenesis and plasticity in a functionally relevant manner. Although this seems a possible explanation, prolonged expression of angiogenic factors such as VEGF-D and VEGF-A could also be harmful. A more advanced approach to the problem would utilize Tet-controlled expression, where the transgene is expressed only in the presence of doxycycline (Gosden et al. 1995).

## 5. Conclusion

Here, we have tested the restorative potential of VEGF-D through angiogenesis in the area surrounding an evolving brain infarct in an experimental stroke model. Increased angiogenesis and reduced infarct size were observed by viral gene delivery of VEGF-D, but this was not translated to behavioral outcome during the three week follow-up. Thus, in the acute and subacute phases after stroke, angiogenesis may
have predominantly other functions such as removal of edema and/or necrotic tissue in the ischemic border zone rather than promoting direct functional improvement. Further research is needed to understand the mechanisms and the therapeutic value of angiogenic factors in enhancing functional recovery after stroke.

## Acknowledgment

This study was supported by Finnish Academy, the Finnish Funding Agency for Technology and Innovation and Ark Therapeutics Ltd.

## Conflict of Interest

None

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