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The phosphatidylinositol-3 kinase/Akt pathway mediates VEGF’s neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia

Ertugrul Kilic^*,1, Ülkan Kilic^*, Yaoming Wang^*,2, Claudio L. Bassetti^*, Hugo H. Marti and Dirk M. Hermann^*

^* Department of Neurology, University Hospital Zurich (USZ), Switzerland; and

Institute of Physiology and Pathophysiology, University of Heidelberg, Germany

¹Correspondence: Department of Neurology, University Hospital Zurich, Frauenklinikstr. 26, Zurich CH-8091, Switzerland. E-mail: ertugrul.kilic{at}usz.ch

SPECIFIC AIMS

Based on its trophic influence on neurons and vascular cells, vascular endothelial growth factor (VEGF) is a promising candidate for stroke treatment. VEGF’s survival-promoting effects are purchased at the expense of an increased blood brain barrier (BBB) permeability, which potentially compromises tissue survival. The mechanisms via which VEGF protects the brain against ischemia and induces BBB permeability remained unknown. This study aimed to characterize signal transduction pathways responsible for VEGF’s neuroprotection and BBB disturbances in our transgenic mouse line V1 that expresses human VEGF₁₆₅ in the brain under control of a neuron-specific enolase (NSE) promoter, which we subjected to 90 min of intraluminal middle cerebral artery (MCA) occlusion.

PRINCIPAL FINDINGS

1. VEGF receptor-2 (VEGFR-2) is expressed on ischemic neurons and astrocytes and is activated by human VEGF
To find out whether VEGFR-2 is expressed and activated in the ischemic brain, we combined immunohistochemical and immunoprecipitation studies. We show that VEGFR-2 is expressed on ischemic neurons and astrocytes at 24 h after reperfusion and that this receptor is phosphorylated (i.e., activated) by VEGF.

2. VEGF increases phosphorylated Akt and extracellular–regulated kinase (ERK)-1/-2 and reduces phosphorylated p38 and c-Jun NH2-terminal kinase (JNK)-1/-2
To elucidate how VEGF influences cell signaling, we prepared Western blots with tissue samples from ischemic brains. These blots revealed that phosphorylated (but not total, nonphosphorylated) Akt and ERK-1/-2 levels were increased, whereas phosphorylated p38 and JNK-1/-2 levels were reduced by VEGF (Fig. 1 ).

View larger version (24K): [in this window] [in a new window]   Figure 1. Expression and phosphorylation state of Akt, ERK-1/-2, MAP kinase/p38, and JNK-1/-2 in WT and V1tg mice subjected to focal cerebral ischemia. Animals were either untreated or i.c.v. treated with 2 µl of the solvent DMSO or DMSO containing Wortmannin, a PI3K/Akt inhibitor. Tissue samples were taken from the ischemic cortex and underlying striatum. Note that human VEGF increases phosphorylated (but not total) Akt and ERK-1/-2 and reduces phosphorylated p38 and JNK-1/-2 levels. Furthermore, note the reversal of p38, but not of ERK-1/-2 or JNK-1/-2 phosphorylation in animals treated with Wortmannin, in which Akt phosphorylation was suppressed. Our data suggested that Akt was involved in VEGF’s neuroprotective function and BBB permeability. Values are mean ± SD (n=3 samples/group), normalized with corresponding blots for ßbeta;-actin. *P < 0.05 compared with WT mice; #P < 0.05 compared with V1tg mice receiving DMSO.

3. VEGF down-regulates inducible NO synthase [inducible NOS (iNOS)] in the ischemic brain
To evaluate whether VEGF influences the expression of iNOS, which contributes to free radical formation in the reperfused brain, we performed immunohistochemistries. Robust expression of iNOS was noticed in ischemic tissue of wild-type (WT) mice (24.7±6.7 cells/ 62'500 µm² square; mainly NeuN+ neurons) that was decreased by VEGF (10.3±4.5 cells/ square, P<0.05).

4. The phosphatidyl inositol-3 kinase (PI3K)/Akt inhibitor Wortmannin reverses VEGF’s neuroprotective activity and restores BBB integrity
To clarify the role of the PI3K/Akt pathway in VEGF’s effects, we applied the solvent dimethyl sulfoxide (DMSO) or DMSO containing 0.1 mM of the PI3K/Akt inhibitor Wortmannin into the intracerebroventricular (i.c.v.) space of WT and V1tg mice subjected to focal cerebral ischemia, and compared these animals with animals not receiving i.c.v. injections. Whereas cerebral laser Doppler flow (LDF) did not differ between groups (Fig. 2 A), VEGF reduced infarct volume (Fig. 2B ), improved neurological abnormalities (Fig. 2C ), and increased BBB permeability (Fig. 2E ) without influencing macroscopic brain swelling (Fig. 2D ). Inhibition of PI3K/Akt completely abolished VEGF’s neuroprotection (Fig. 2B, C ), at the same time reversing BBB leakage to below levels in wt mice (Fig. 2D, E ).

View larger version (19K): [in this window] [in a new window]   Figure 2. The PI3K/Akt pathway mediates VEGF’s neuroprotective function and BBB permeability. Data from wt and V1tg mice that were either untreated or i.c.v. treated with DMSO or DMSO/Wortmannin. Note that LDF during ischemia does not differ between groups (A). Assessments of infarct vol. (B) and neurological deficits (C) reveal that human VEGF significantly reduces brain injury and ameliorates postischemic recovery. Brain swelling (D) is not influenced by human VEGF, whereas IgG extravasation (E) is moderately increased, indicating that VEGF expression promotes BBB leakage. Note that the neuroprotective effects of VEGF are reversed by PI3K/Akt inhibition (B, C), while brain swelling (D) and BBB permeability (E) are reduced, despite the exacerbation of brain infarcts. These data demonstrate that the PI3K/Akt pathway is responsible for VEGF’s neuroprotective function and vascular leakage. Values are means ± SD (n=5–11 animals/ group). *P < 0.05 compared with WT mice; #P < 0.05 compared with V1tg mice receiving DMSO.

5. Reversal of VEGF neuroprotection by Wortmannin is associated with restoration of p38, but not ERK-1/ -2 and JNK-1/-2
To analyze downstream effects of PI3K/Akt, we prepared additional Western blots for DMSO and DMSO/Wortmannin treated mice. Our data confirmed that Wortmannin indeed inhibited Akt phosphorylation (Fig. 1) . In contrast to phosphorylated ERK-1/-2 and JNK-1/-2, which were not influenced by the PI3K/Akt inhibitor, p38 phosphorylation was increased in Wortmannin treated animals that exhibited exacerbated brain infarcts (Fig. 1) . Our results indicate that the regulation of p38 by VEGF closely depends on the PI3K/Akt activation state.

6. PI3K/Akt inhibition does not reverse iNOS levels in ischemic neurons
To find out whether iNOS inhibition by VEGF occurs in a PI3K/Akt-dependent manner, we also examined DMSO and DMSO/Wortmannin treated animals by immunohistochemistry. In contrast to MAP kinase/p38, which was activated when PI3K/Akt was inhibited, neuronal iNOS levels were not reversed by Wortmannin (9.9±3.0 vs. 8.9±4.4 cells/square in DMSO and DMSO/Wortmannin-treated mice, respectively), demonstrating that iNOS inhibition by VEGF does not depend on PI3K/Akt.

CONCLUSIONS

Using our transgenic mouse line V1 that expresses human VEGF in the brain, we demonstrate that VEGF protects against focal cerebral ischemia and also induces BBB permeability via the PI3K/Akt pathway. Our data were obtained using: 1) in vivo experiments, in which brain injury was evaluated following 90 min of intraluminal MCA occlusions (24 h reperfusion); 2) immunohistochemical and immunoprecipitation studies using brain tissue samples, in which we examined the expression and phosphorylation (i.e., activation) of VEGF-receptor-2, Akt, MAP kinases (ERK-1/-2, p38, JNK-1/-2), and iNOS, as well as 3) additional in vivo experiments, in which we applied the pharmacological PI3K/Akt inhibitor Wortmannin into the i.c.v. space and evaluated effects of VEGF on ischemic injury and BBB permeability. By showing that VEGF: 1) activates VEGFR-2, which as we demonstrate is expressed on ischemic neurons and astrocytes; 2) stimulates Akt and ERK-1/-2; 3) inhibits p38 and JNK-1/-2; and 4) down-regulates iNOS, we identify a complex cellular signaling scenario, which VEGF activates in order to exert its neuroprotective function. Based on our finding that the PI3K/Akt pathway is responsible for VEGF’s neuroprotection and its BBB permeability, we predict that it may not easily be possible to make use of VEGF’s tissue survival without accepting its unfavorable consequence, the increased BBB leakage.

View larger version (37K): [in this window] [in a new window]   Figure 3. VEGF protects against ischemia and induces BBB leakage by activating the PI3K/Akt pathway. In ischemic brains of WT mice, both Akt and ERK-1/-2 activities are low, whereas p38 and JNK-1/-2 activities are high. As a consequence, ischemic neurons die and BBB remains functional. In mice constitutively expressing human VEGF (V1tg), on the other hand, Akt and ERK-1/-2 pathways are activated, whereas p38 and JNK-1/-2 pathways are inhibited. Thereby, ischemic neurons remain viable, at the expense of an enhanced BBB permeability. Inhibition of the PI3K/Akt pathway with Wortmannin completely abolishes the tissue protection induced by VEGF, at the same time reversing BBB function.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4829fje

² Present address: Buck Institute for Age Research, Novato, California, USA.

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4829fjev1 20/8/1185    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kilic, E. Articles by Hermann, D. M. Search for Related Content PubMed PubMed Citation Articles by Kilic, E. Articles by Hermann, D. M.