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Vascular endothelial growth factor rescues hippocampal neurons from glutamate-induced toxicity: signal transduction cascades ¹

HIDEO MATSUZAKI^*,, MICHIO TAMATANI^*,2, ATSUSHI YAMAGUCHI^*,, KAZUHIKO NAMIKAWA, HIROSHI KIYAMA^,, MICHAEL P. VITEK, NORIAKI MITSUDA^*, and MASAYA TOHYAMA^*,

^* Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, 2–2 Yamadaoka, Suita, Osaka 565-0871, Japan;
Department of Anatomy, Asahikawa Medical College, 4–5-3–11, Nishikagura, Asahikawa, Hokkaido 078-8510, Japan;
Division of Neurology, Duke University Medical Center, Durham, NC 27710, USA; and
CREST, Japan Science and Technology

²Correspondence: Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, 2–2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: tamatani{at}h8.dron.ne.jp

SPECIFIC AIM

Although vascular endothelial growth factor (VEGF) is well known as an endothelial cell mitogen, its increased presence in the brain after an ischemic event suggested to us that VEGF could play a neuroprotective role. Using primary neuronal cultures, we have investigated whether VEGF could protect against glutamate-induced neurotoxicity and examined the molecular and cellular mechanisms, focusing on the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the mitogen-activated protein kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK) pathways through specific receptor(s).

PRINCIPAL FINDINGS

1. VEGF protects neurons from glutamate-induced toxicity
Using lactate dehydrogenase (LDH) release as a measure of cell survival, 50 µM glutamate treatment for 24 h reduced cell survival to approximately 40% of untreated controls (Fig. 1 B). Six hours of pretreatment with VEGF before exposure to glutamate resulted in a dose-dependent rescue of cell viability with 100 ng/ml VEGF treatment, increasing cell survival to about 75% of untreated controls (Fig. 1B ). VEGF also dose-dependently inhibited glutamate-induced up-regulation of caspase-3-like activity as detected spectrophotometrically by cleavage of 7-amino-4-methylcoumarin-DEVD with extracts of treated cells. Consistent with these results, pretreatment with VEGF (100 ng/ml) attenuated DNA laddering induced by glutamate.

View larger version (29K): [in this window] [in a new window]   Figure 1. VEGF protects hippocampal neurons through activation of PI3-K/Akt and MEK/ERK pathways against glutamate toxicity. Neuronal cultures incubated for 6 h with or without wortmannin (W; 100 nM), LY294002 (LY; 1 µM), and/or U0126 (U; 50 µM) in the presence of VEGF were exposed to 50 µM glutamate. A) Before glutamate exposure, the cells were lysed and the blots were probed with antibodies to Akt, phospho-Akt, ERK, and phospho-ERK. B, C) 24 h after glutamate treatment, neuronal viability as measured by LDH release (B) and caspase-3-like activity as measured by peptide cleavage (C) were determined. Cells preincubated with vehicle and not exposed to glutamate were defined as control cells (Cont). *P < 0.05 vs. VEGF plus glutamate and **P < 0.05 vs. wortmannin or U0126 in the presence of VEGF and glutamate.

2. Neuroprotective effect of VEGF is associated with activation of PI3-K/Akt and MEK/ERK pathways
Several studies have indicated that PI3-K/Akt and/or MEK/ERK signaling pathways are involved in the effect of neurotrophic factors. To examine the mechanism of VEGF’s neuroprotective action, we initially analyzed phosphorylation of kinases from the PI3-K/Akt and/or MEK/ERK pathways. VEGF increased the levels of phosphorylated-Akt and of phosphorylated-ERK (Fig. 1A ). Pretreatment of cells with either of the PI3-K inhibitors, wortmannin or LY294002, inhibited VEGF-induced phosphorylation of Akt without affecting ERK; conversely, the MEK inhibitor U0126 blocked activation of ERK without affecting Akt (Fig. 1A ). Similarly, treatment with these inhibitors reduced the ability of VEGF to protect from glutamate-mediated neurotoxicity (Fig. 1B ) or to reduce caspase-3-like activation (Fig. 1C ). Addition of both wortmannin and U0126 additively inhibited the effect of VEGF on glutamate-induced neurotoxicity and caspase-3-like activation (Fig. 1B , C ). These results strongly suggest that Akt and ERK are located in different VEGF-activated pathways and that activation of PI3-K/Akt and MEK/ERK pathways is required for the effect of VEGF on glutamate-induced neuronal death and on caspase-3-like activation. The roles of Akt and ERK in neuroprotection were further confirmed by the observation that adenovirus-mediated expression of either activated Akt or activated MEK inhibited glutamate-induced neuronal death and caspase-3-like activation and that these effects are additively enhanced by simultaneous expression of the activated forms of both kinases.

3. Reduction of VEGF receptor levels reduces VEGF-mediated neuroprotection.
VEGF biological activity is mediated through its binding to at least two receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase (Flk-1). To examine which receptor mediated the neuroprotective effects of VEGF, antisense oligonucleotide (ODN) for Flt-1 or Flk-1 or a scrambled ODN sequence were used. Treatment of neurons daily for 4 days with the Flt-1 or Flk-1 antisense ODN (1 µM), but not with the scrambled ODN (1 µM), almost completely blocked the basal expression and VEGF-induced phosphorylation of the Flt-1 or Flk-1 receptor, respectively (Fig. 2A , B ). Treatment of neurons with the Flk-1 antisense ODN completely blocked VEGF’s neuroprotective effect against glutamate toxicity, whereas the Flt-1 antisense or the scrambled ODN were without significant effect (Fig. 2C ). Similar to the effects on neuroprotective activity, the Flk-1 antisense ODN, but not the Flt-1 antisense or the scrambled ODN, almost completely blocked VEGF-induced phosphorylation of both Akt and ERK (Fig. 2D ).

View larger version (36K): [in this window] [in a new window]   Figure 2. Inhibition of Flk-1 expression by antisense ODN blocks the effects of VEGF. Cells were untreated or treated with 1 µM of Flt-1 antisense ODN (Flt-AS), Flk-1 antisense ODN (Flk-AS), or scrambled ODN (SCR) for 4 days. Cells were then stimulated with 100 ng/ml VEGF in the presence of the ODN. A) Before VEGF stimulation, cells were lysed and extracts of neurons were subjected to Western blot analysis for Flt-1 and Flk-1 expression. B) Extracts from neuronal cultures after 15 min stimulation with VEGF were immunoprecipitated with antibody against phosphorylated tyrosine, followed by Western blot analysis with antibodies against Flt-1 or Flk-1. C) After 6 h incubation with vehicle (saline) or 100 ng/ml VEGF in the presence or absence of Flt-1 or Flk-1 antisense ODN, cells were exposed for 24 h to 50 µM glutamate. Neuronal survival was then determined by LDH assay. *P < 0.05 vs. VEGF plus glutamate. D) Extracts from cells after 6 h incubation with vehicle or VEGF were subjected to SDS-PAGE and the blots were probed with antibodies to phospho-Akt, Akt, phospho-ERK, or ERK.

CONCLUSIONS

The present study provides the first evidence that VEGF, assumed to specifically affect endothelial cells, is a survival factor for central nervous system neurons and elucidates the molecular mechanisms involving PI3-K/Akt and MEK/ERK signaling pathways in VEGF-mediated neuroprotective action against glutamate toxicity. We also found that the effects of VEGF are mediated primarily through the Flk-1 receptor.

VEGF is known as a selective endothelial cell mitogen that promotes angiogenesis and increases blood vessel permeability. In the nervous system, only diffuse expression of VEGF has been observed in adult brain, with the exception of specialized cell types such as epithelial cells in the choroid plexus. However, hypoxia/ischemia results in marked increase in VEGF and its receptors at both the mRNA and protein level. It has been reported that topical application of VEGF after transient cerebral artery occlusion significantly reduced ischemic brain damage. These results lead us to speculate that VEGF induced by ischemia functions to prevent ischemic damage via acting directly on neurons as well as other types of cells such as endothelial and glial cells. The present study demonstrated that VEGF directly acted on hippocampal neurons and protected neurons from cell death induced by glutamate, which is thought to be a main mediator of ischemic neuronal death.

The mechanism for VEGF’s neuroprotective activity appears to begin with VEGF binding to the Flk-1 receptor, but not with the Flt-1 receptor. Each receptor appears to be coupled to unique signal transduction cascades that culminate in unique biological responses. For example, VEGF binding to Flk-1 on mouse endothelial cells initiates a signal transduction cascade that culminates in VEGF-mediated angiogenesis in developing and adult animals. As another receptor for VEGF, Flt-1 appears to be primarily involved in endothelial cell morphogenesis during embryonic development. When specific pathways were evaluated, cells expressing recombinant Flk-1 receptors bound VEGF, which then promoted binding of the Shc and Nyc adaptors, Grb-2 binding, and ERK activation. ERK was not activated by VEGF, however, in cells expressing recombinant Flt-1 in two separate studies. Similarly, VEGF binding to the Flk-1 receptor activated Akt and enhanced endothelial cell survival. Like these actions on other types of cells, we show that VEGF-mediated activation of Flk-1, but not of Flt-1, is able to activate Akt and ERK that serves to protect hippocampal neurons from glutamate toxicity.

We found that VEGF inhibits glutamate-induced neuronal death by two independent pathways: the PI3-K/Akt pathway and the MEK/ERK pathway. Singularly blocking each of these pathways significantly inhibited VEGF’s protective action against glutamate-induced toxicity. Singular blocking incompletely inhibited VEGF’s protective action while simultaneous blocking of both pathways produced a greater effectthat apparently mimicked the glutamate-mediated death seen in the absence of VEGF. Treatment of cells with the PI3-K inhibitors inhibited VEGF-induced activation of Akt without affecting ERK; conversely, the MEK inhibitor blocked activation of ERK without affecting Akt. In addition, adenovirus-mediated overexpression of activated form of either Akt or MEK alone significantly protects neurons from cell death, the effects being enhanced by simultaneous expression of both kinases. These results support the presence and function of two independent anti-apoptotic pathways that are mediated by VEGF.

In summary, VEGF has an effect on hippocampal neurons to protect them against glutamate-induced toxicity. This effect is mediated primarily by the Flk-1 receptor through the PI3-K/Akt and the MEK/ERK signaling pathways. These findings imply that VEGF-like agents may be useful for the treatment of neurodegenerative disorders, in addition to its well-documented beneficial actions in angiogenesis.

View larger version (11K): [in this window] [in a new window]   Figure 3. VEGF signals neuroprotection through the Flk-1 receptor in association with the PI3-K/Akt and the MEK/ERK pathways. Although VEGF interacts with both Flt-1 and Flk-1 receptors, VEGF activation of Flk-1 stimulates the PI3-K/Akt or the MEK/ERK pathways, each of which function independently to protect against glutamate-mediated neurotoxicity.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0495fje ; to cite this article, use FASEB J. (March 12, 2001) 10.1096/fj.00-0495fje

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