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. Author manuscript; available in PMC: 2011 Aug 1.
Published in final edited form as: Neuropeptides. 2010 Apr 28;44(4):323–331. doi: 10.1016/j.npep.2010.04.002

Multiple neurotrophic effects of VEGF on cultured neurons

Alma Sanchez 1, Suchin Wadhani 1, Paula Grammas 1,*
PMCID: PMC2879433  NIHMSID: NIHMS202051  PMID: 20430442

Abstract

A large literature demonstrates the multifunctional nature of vascular endothelial growth factor (VEGF). Though initially characterized as an endothelial cell-specific factor, recent studies reveal that VEGF has numerous effects on diverse cell types in the brain including neurons. The objective of this study is to examine the effects of VEGF in cultured cortical neurons on survival, p38 mitogen-activated protein kinase (p38 MAP kinase) activity, pro- and anti-apoptotic protein expression and on release of neurotrophic and neurotoxic factors. The results show that VEGF dose-dependently enhances the survival of neurons in culture. VEGF decreases active caspase 3 levels and increases expression of the anti-apoptotic protein Bcl-2. VEGF decreases phosphorylated p38 MAP kinase level and activity in cortical neurons. In addition to modulating survival/death pathways in cortical neurons, VEGF also regulates release of proteins that affect neuronal viability. VEGF causes a dose-dependent release of the neurotrophic protein pigment epithelial-derived factor (PEDF), while significantly decreasing release of the neurotoxic protein amyloid beta. The VEGF-mediated decrease in amyloid beta is dependent on a functional Flt-1 receptor and is inhibited by dicoumarol, a multifunctional inhibitor of stress activated protein kinase (SAPK)/JNK and NFkappaB pathways. Taken together, these data demonstrate that the neurotrophic effects of VEGF are likely mediated directly by increasing survival and decreasing apoptotic proteins and signals as well as indirectly by modulating release of proteins that affect neuronal viability.

Keywords: PEDF, amyloid beta, apoptosis, survival, p38 MAP kinase, Bcl-2, caspase 3

Introduction

A large literature demonstrates the multifunctional nature of vascular endothelial growth factor (VEGF) (Ng et al., 2006; Nieves et al., 2009). Initially identified as a vascular permeability factor, VEGF is also a potent mitogen for endothelial cells and a key regulator of angiogenesis (Leung et al., 1989; Dvorak et al. 1995; Yancopoulos et al., 2000). VEGF promotes vasculogenesis during development and physiological angiogenesis that occurs in wound repair (Swift et al. 1999, Grazul-Bilska et al. 2003) and inflammation (Dvorak et al. 1995; Detmar et al. 1998) in the adult. This multifaceted protein also plays a role in pathologic angiogenesis associated with tumor growth (Kim et al. 1993; Dvorak et al. 1995; Ferrara and Davis-Smyth 1997) and age-related macular degeneration (Bhisitkul and Rutar, 2006).

Though initial studies over a decade ago indicate that VEGF is an endothelial cell-specific factor, more recent findings reveal that VEGF has direct effects on the nervous system on neuronal growth, axonal outgrowth and neuroprotection. Application of VEGF causes axonal outgrowth (Sondell et al. 2000; Khaibullina et al. 2004) and protects neurons against ischemic, hypoxic, and excitotoxic injury (Jin et al. 2000a, 2001; Matsuzaki et al. 2001). Genetic studies show that mice with reduced VEGF develop adult-onset motor neuron degeneration, reminiscent of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS), and that VEGF overexpression delays neurodegeneration and prolongs survival in ALS mice (Oosthuyse et al., 2001; Wang et al., 2007). VEGF is up-regulated in the brain after stroke (Issa et al.,1999) and has been implicated in other neurological disorders such as diabetic and ischemic neuropathy, nerve degeneration, Parkinson’s disease, Alzheimer disease, and multiple sclerosis (Storkebaum et al., 2004; Ruiz de Almodovar et al., 2009)

In endothelial cells the signaling cascades that mediate VEGF-induced changes in proliferation, migration, permeability, and cell survival have been extensively documented (Kumar et al., 2004; Youn et al., 2009). In contrast, the underlying mechanisms that contribute to VEGF’s neuroprotective functions remain to be defined. In neuronal cells, the neuroprotective effects of VEGF have been attributed to its binding primarily to VEGF Flk-1 receptor (Sondell et al. 2000) and activation downstream of phosphatidylinositol 3’-kinase/protein kinase B (PI3/Akt), the mitogen activated protein kinase kinase/extracellular signal-regulated protein kinases (MAPK/ERK 1/2) or both (Jin et al. 2000b; Matsuzaki et al. 2001; Wick et al. 2002; Kilic et al. 2006a; 2006b). In the retina, overexpression of VEGF reduces phosphorylation of p38 MAP kinase, a stress-activated enzyme that can initiate apoptosis in neurons (Hou et al., 2002; Jeohn et al., 2002), and protects against neurodegeneration (Kilic et al. 2006b). The role of p38 MAP kinase, in VEGF’s neuronal signaling remains unclear.

Many signaling pathways that affect neuronal survival/death decisions converge downstream on common targets. Among the most well studied of these are the anti-apoptotic protein Bcl-2 and the pro-apoptotic protease caspase 3 (Yuan and Yankner, 2000; Antonsson and Martinou, 2000; Sadowski-Debbing et al., 2002). We have previously shown in cultured neurons that acetaminophen-mediated protection against oxidant injury is associated with an increase in Bcl-2 and a decrease in caspase 3 (Tripathy and Grammas, 2009). Whether these pathways are regulated by VEGF in neurons is unknown.

VEGF could also promote neuronal survival indirectly by interacting with other proteins with neurotrophic or neurotoxic properties. In this regard, expression of pigment epithelium-derived factor (PEDF), a neurotrophic protein widely distributed in the CNS (Barnstable and Tombran-Tink, 2004), has been shown to be correlated with VEGF in the striatum of patients with Parkinson’s disease (Yasuda et al., 2007). However, whether VEGF affects expression of PEDF in neurons is unknown. Similarly, a recent study suggests that VEGF can affect processing of the amyloid precursor protein in brain slice cultures derived from transgenic Alzheimer’s disease mice (Bürger et al., 2009). The ability of VEGF to affect amyloid beta (Aβ) levels may contribute to the multi-factor nature of VEGF-mediated neuroprotection.

The objective of this study is to examine in primary cultured neurons the effects of VEGF on neuronal survival, p38 MAP kinase, pro- and anti-apoptotic protein expression and on release of neurotrophic and neurotoxic factors.

Materials and methods

Primary neuronal cultures and culture treatments

Cell culture reagents and media were purchased from Invitrogen (Carlsbad, CA). Rat cerebral cortical cultures were prepared from cortices of 18-day gestation fetuses, as previously described (Grammas et al., 1999; Reimann-Philipp et al., 2001). The cells were seeded on 6-well poly-L-lysine coated plates at a density of 3–5 × 105 cells per ml and incubated in Neurobasal medium containing B-27 supplement, antibiotic/antimycotic, glutamine (0.5 mM) and 5-fluoro-2′-deoxyuridine (20 μg/ml) to inhibit proliferation of glial cells. On day 5, fresh medium without 5-fluoro-2′-deoxyuridine was added. Neuronal cultures were used for experiments after 8–9 days in culture. The primary cerebral cortical cultures utilized were enriched for neurons (>95%). The neuronal identity of the cultures was confirmed using immunofluroescent labeling for beta-3 tubulin, a neuron-specific protein (Promega, Madison, WI) and glial fibrillary acidic protein (Promega).

Recombinant rat VEGF (CRV017A) was obtained from Cell Sciences (Canton, MA). VEGF was reconstituted in sterile, deionized water frozen until use and diluted in treatment media to attain the experimental doses (0–100 ng/ml). The p38 MAP kinase inhibitor SB203580 was obtained from EMD Biosciences (La Jolla, CA) and used at a final concentration of 100 μM in treatment media (neurobasal media containing N-2 supplement, 0.5 mM glutamine, and antibiotic/antimycotic). The p38 MAP kinase activator anisomycin was also purchased from EMD Biosciences and used at a final concentration of 7.5 μM. VEGF receptor Flt-1 and Flk-1 neutralizing antibodies were from R&D Systems (Minneapolis, MN). VEGF receptor antibodies were used together with VEGF (100 ng/ml) at a final concentration of 4 μg/ml. The MAP kinase kinase (MEK) 1 and MEK2 inhibitor (U0126), phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) and the c-Jun N-terminal kinase (JNK) inhibitor (JNK Inhibitor II) were purchased from Calbiochem (La Jolla, CA) and used at final concentrations of 5 μM, 5 μM, and 10 μM, respectively. Tyrosine kinase (Genistein) and protein kinase C (PKC) (bisindolylmaleimide) inhibitors were purchased from Sigma (St. Louis, MA) and used at 100 μM and 10 μM final concentrations, respectively. Dicoumarol was obtained from Calbiochem and used at a final concentration of 50 μM.

Assessment of cell viability

Cell viability was assayed using the MTT assay (Promega) as follows. Treatment medium was replaced with fresh treatment medium containing 25 μl/ml of the Cell Titer 96 Aqueous One Solution and incubated for 10 min at 37°C after which optical density was measured at 490 nm using a microplate reader. The quantity of soluble formazan product, as measured by the amount of absorbance, was directly proportional to the number of viable cells. The optical density of untreated controls was set at 100%. The number of viable cells after treatment was determined by measuring optical density and expressing viability as percent of untreated controls.

Quantitation of proteins in culture media by ELISA

An indirect ELISA procedure was used to measure the amyloid beta1–42 (Aβ1–42) and pigment epithelium-derived factor (PEDF) proteins. After treatments, cell media were collected and used for ELISA. ELISA plates (96-well flat bottom) were coated overnight at 4°C with 100 μl cell media mixed with 100 μl of coating buffer (0.1 M sodium bicarbonate buffer, pH 9.5), blocked with 1% bovine serum albumin solution at 37°C for 45 min and then incubated with the primary antibody (for Aβ1–42, AB5758P, Chemicon, Temecula, CA; for PEDF 07–280, Upstate Cell Signaling Solutions, Lake Placid, NY) diluted in sodium bicarbonate buffer (1:1000 dilution). The plate was incubated with the peroxidase-conjugated secondary antibody (1:1000) (BioRad, Hercules, CA) for 1 h and washed three times. Color reaction was developed by addition of 200 μl/well of o-phenylene diamine H2O2 (Thermoscientific, Rockford, IL). Optical density was measured at 450 nm using a microplate reader. The Aβ1–42 (03–111) and PEDF (01-211) peptides used for standard calculation were from Biosource (Camarillo, CA USA), and Upstate Cell Signaling Solutions, respectively.

Western blot analysis

Total protein was extracted from neurons using lysis buffer containing 0.1% SDS, 1% Triton X-100 and 0.5% phenylmethyl sulfonylfluoride. Protein was determined by the Bradford method using Bio-Rad protein reagents. Equal amounts of protein were run on a 12% polyacrylamide gel, transferred on to a PVDF membrane, blocked with 5 % milk solution (non-fat dry milk in Tris-buffered saline Tween-20) and immuno-blotted with primary antibodies. Primary antibodies from Cell Signaling Technology (Danvers, MA) include cleaved form of caspase 3 (#9664, 1:200), total p38 MAPK (#9212, 1:200), and phospho-p38 MAP kinase (#9211, 1:200). The Bcl-2 (ab7973, 1:300) antibody was from AbCam (Cambridge, MA). Membranes were washed with Tris-buffered saline Tween-20 and incubated with peroxidase- conjugated secondary antibodies. After extensive washing to remove unbound antibodies, membranes were developed with chemiluminescence reagents. Band intensities were quantified using Quantity One software (Bio-Rad) and graphically expressed as intensity units which reflects the average intensity over the area of the band. Densitometric measurements of bands were normalized to corresponding GAPDH levels with control samples set to 100 and treatment values expressed as percent of control.

p38 MAP kinase assay

Activity of p38 MAP kinase was determined using the nonradioactive p38 MAP kinase assay kit from Cell Signaling (#9820). After treatments, cell lysates were collected in cell lysis buffer and used for kinase assay according to manufacturer’s protocol. p38 MAP kinase was immunoprecipitated overnight from samples (100 μg) using phospho p38 (Thr180/Try184) MAP kinase monoclonal antibody immobilized to agarose hydrozide beads. The immunoprecipitated phospho p38 MAP kinase pellet was resuspended in kinase buffer containing 2 μg ATF-2 fusion protein and 200 μM cold ATP. The reaction mix was incubated at 30°C for 30 min and the reaction was terminated by addition of sample buffer. Activity of phospho p38 MAP kinase was determined by detecting ATF-2 phosphorylation using phospho-ATF-2 (Thr71) antibody through western blotting and chemiluminescent detection.

Statistical Analysis

Data from each experiment were expressed as mean ± standard error (SEM). Comparisons between control and treatment groups were conducted using the one-way ANOVA followed by Bonferroni’s multiple comparison test for multiple samples. Statistical significance was determined at p<0.05.

Results

VEGF improves neuronal survival in vitro

Exposure of eight-day old neuronal cultures to various concentrations (10 -100 ng/ml) of VEGF for 24 h increased neuronal survival (Fig. 1A). This increase was significant (p<0.05) at 25 ng/ml and highly significant at 50 ng/ml (p<0.001). At 100 ng/ml there was a 45% increase in neuronal cell survival compared to untreated cell cultures.

Figure 1.

Figure 1

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Eight-day old cultured neurons were treated with increasing doses of VEGF for 24 h. A. Cell survival was determined by MTT assay. Data are mean ± SEM expressed as percent of control (untreated cells) from 4 separate experiments.

*p<0.05 vs. control; ***p<0.001 vs. control.

B. Total protein was extracted and western blot analysis performed using specific antibodies for cleaved caspase 3. Data are normalized to GAPDH and expressed as percent of control (untreated). Relative expression determined from densitometric scans from 2 separate experiments is shown below representative western blot.

**p<0.01 vs. control; ***p<0.001 vs. control.

C. Western blot analysis was performed using specific antibodies for Bcl-2 and relative expression determined from densitometric scans from 2 separate experiments is shown below representative western blot.

*p< 0.05 vs. control; **p<0.01 vs. control.

To determine whether the neuroprotective effect of VEGF on primary cortical neurons was mediated by changes in cell death machinery/mediators we determined if VEGF blocked the activation of caspase 3. Neuron cultures were exposed to increasing doses of VEGF (10–100 ng/ml) for 24 h and western blots were performed using an antibody specific for the 17 kDA active (cleaved) form of caspase 3. Figure 1B shows that exposure to VEGF significantly (p<0.01–0.001) reduced immunoreactivity for the active form of caspase 3.

Experiments to determine other possible targets in the apoptotic cascade that might be affected by VEGF showed that the level of Bcl-2, an anti-apoptotic protein upstream of caspase 3, was also altered. In contrast to the suppression of active caspase 3 by VEGF, the expression of the anti-apoptotic protein Bcl-2 was significantly (p<0.05–0.01) increased in a dose-dependent manner by VEGF (Fig. 1C).

VEGF decreases phosphorylated p38 MAP kinase level and activity in cortical neurons

Since the p38 MAP kinase pathway is linked to caspase activation (Junn and Mouradian 2001; Choi et al. 2004) and apoptosis induced by Aβ (Zhu et al. 2005), we examined the role of p38 MAP kinase in VEGF signaling. Neuronal cultures were exposed to increasing doses of VEGF (10–100 ng/ml) for 24 h and western blots were performed using an antibody specific for the phosphorylated form of p38 MAP kinase. Figure 2A shows that VEGF treatment had no significant effect on total p38 MAP kinase but increasing concentrations of VEGF reduced immunoreactivity for the phosphorylated form of p38 MAP kinase (Fig 2A).

Figure 2.

Figure 2

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Eight day old neurons were treated with increasing dose of VEGF for 24 h. A. Total protein was extracted and western blot analysis performed using specific antibodies for phosphorylated p38 MAP kinase, total p38 MAP kinase and GAPDH. Data are normalized to total p38 MAP kinase and expressed as percent of control (untreated). Relative expression determined from densitometric scans from 4 separate experiments is shown below representative western blot.

*p< 0.05 vs. control; **p<0.01 vs. control.

B. Cell lysates were used to measure p38 MAP kinase activity by measuring ATF-2 phophorylation in vitro. Activity of phospho p38 MAP kinase was determined by western blot using antibody to phosphorylated ATF-2. The figure is representative of 2 experiments.

Experiments to determine the activity of phophorylated p38 MAP kinase were performed using the ATF-2 fusion protein as a substrate and its level detected by western blot analysis. The data showed that increasing doses of VEGF (10–100 ng/ml) caused a decrease in phosphorylation of ATF (Fig. 2B).

Dissociation of VEGF effects on p38 MAP kinase activity and on neuronal survival

We examined the ability of VEGF to affect p38 MAP kinase activity, as indicated by ATF-2 phosphorylation (Fig. 3A), and neuronal survival (Fig. 3B) in the presence of a p38 MAP kinase inhibitor (SB203580) or the p38 MAP kinase activator anisomycin. Addition of VEGF (100 ng/ml) caused a decrease in p38 MAP kinase activity as did addition of the p38 MAP kinase inhibitor SB203580 (100 μM). The combination of VEGF and SB203580 completely inhibited p38 MAP kinase activity to levels below detection (Fig. 3A). Addition of the p38 MAP kinase activator anisomycin (7.5 μM) increased p38 MAP kinase activity. Co-incubation of VEGF with anisomycin significantly (p<0.001) reduced p38 MAP kinase activity compared to anisomycin alone (Fig. 3A).

Figure 3.

Figure 3

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Eight-day old neurons were treated for 24 h as follows: Control, VEGF (100 ng/ml), SB203580 (100 μM), SB203580+VEGF, anisomycin (7.5 μM) and anisomycin+VEGF. A. Cell lysates were used to determine p38 MAP kinase activity by measuring ATF-2 phophorylation in vitro. Activity of phospho p38 MAP kinase was determined by western blot using antibody to phosphorylated ATF-2. Relative expression determined from densitometric scans from 3 separate experiments is shown below representative western blot.

***p<0.001 vs control; ap<0.05 vs SB20358+VEGF; bp<0.001 vs anisomycin.

B. Cell survival was determined by MTT assay. Data are mean ± SEM expressed as percent of control (untreated cells) from 2 separate experiments.

***p<0.001 vs control; *p< 0.05 vs control; ap<0.05 vs SB20358+VEGF; cp<0.001 vs VEGF.

In contrast the effects of VEGF in combination with SB203580 or anisomycin on neuronal cell survival were complex. Incubation of neuronal cultures with either VEGF or SB203580 alone caused a significant (p<0.001) increase in cell survival. However, survival of cultures exposed to VEGF plus SB203580, although higher than untreated cultures (p<0.05), was significantly (p<0.05) less than either agent alone (Fig. 3B). Addition of anisomycin caused a pronounced (72%) and highly significant (p<0.001) decrease in neuronal survival. Neuronal survival was similar in cultures exposed to anisomycin alone and in cultures treated with anisomycin plus VEGF and significantly (p<0.001) less than VEGF alone (Fig. 3B).

VEGF affects release of neutoxic and neurotrophic factors from cultured neurons

To determine other mediators/pathways that may be involved in VEGF’s neurotrophic effects, we examined the ability of VEGF to alter the release of the neurotoxic peptide Aβ1-42 and the neurotrophic protein PEDF from cultured neurons. Analysis of media by indirect ELISA from cultured neurons at 24 h showed release of Aβ1-42 (Fig. 4A). Neuronal cultures exposed to increasing concentrations of VEGF (5–100 ng/ml) showed a strong, dose-dependent decrease in the level of Aβ1-42 detectable in culture supernatants. The decrease was significant (p<0.05) at 50 ng/ml VEGF and 100 ng/ml VEGF caused a 51% decrease in released Aβ1-42 (Fig. 4A).

Figure 4.

Figure 4

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Primary neuronal cultures were treated with increasing doses of VEGF for 24 h, media collected and analyzed by indirect ELISA for levels of (A) Aβ1-42 or (B) PEDF. Data are mean + SEM from 5 separate experiments. *p<0.05 vs. control; ***p<0.001 vs. control.

In contrast, exposure of neuronal cultures to similar concentrations of VEGF for 24 h resulted in a dose-dependent increase in the level of PEDF released into the supernatant. VEGF was less potent in its ability to evoke PEDF release compared to the inhibition of Aβ1-42 release. The increase in PEDF was significant (p<0.05) at 50 ng/ml VEGF (Fig. 4B).

VEGF-mediated decrease in Aβ1-42 is mediated by Flt-1 receptor and is blocked by dicoumarol

In an effort to understand the mechanism behind the VEGF-mediated decrease in release of Aβ1-42, cultured neurons were exposed to VEGF in combination with neutralizing antibodies for the VEGF receptors Flk-1 and Flt-1. Incubation of neurons with an antibody to the Flk-1 receptor during VEGF treatment did not affect the decrease in Aβ1-42 release induced by VEGF. In contrast, in neuronal cultures exposed to a Flt-1 receptor antibody the ability of VEGF to inhibit Aβ 1-42 release was significantly (p<0.001) diminished (Figure 5).

Figure 5.

Figure 5

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Neuron cultures were exposed to media (control), VEGF (100 ng/ml), VEGF + Flt-1 or Flk-1 neutralizing antibodies (4 μg/ml) for 24 h and Aβ1-42 released into media assessed by ELISA. Data are mean ± SEM from 3 separate experiments. *** p<0.001 vs control.

Co-incubation of neurons with VEGF and single-target kinase inhibitors of MEK 1/2, PI3K, JNK, PKC or tyrosine kinase did not alter the effect of VEGF on Aβ1-42 release (Table 1). In contrast, the use of VEGF in combination with dicoumarol, a multifunctional inhibitor of several signaling pathways including stress activated protein kinase (SAPK)/JNK and NFkappaB (Cross et al., 1999), abolished the VEGF-induced decrease in Aβ1-42 (Table 1).

Table 1.

Effects of signaling inhibitors on VEGF-mediated Aβ1-42 decrease.

graphic file with name nihms202051f6.jpg
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Neuronal cultures were treated as indicated for 24 h and Aβ1-42 levels in the media determined by ELISA. Data are mean ± SEM from 4 separate experiments.

***p<.001 vs VEGF

Discussion

Considerable literature now supports the idea that VEGF, a key angiogenic factor, also plays a crucial role in the nervous system. This protein can regulate neuronal migration in the CNS (Schwarz et al., 2004) and stimulate axonal outgrowth of cortical neurons and retinal ganglion cells (Sondell et al., 2000; Böcker-Meffert et al., 2002; Khaibullina et al. 2004). VEGF has been implicated in adult neurogenesis (Cao et al., 2004). The notion that VEGF may be a central mediator of vascular-neuronal interactions is supported by data showing that neuropilins are shared co-receptors between axon guidance molecules (semaphorins) and VEGF (Salikhova et al., 2008; Sköld and Kanje, 2008). Also, VEGF shares common receptor signaling with the neuronal guidance molecule SEMA3A and thus could link the coordinated patterning of the developing vascular and nervous systems (Rosenstein and Krum, 2004).

In the normal adult brain VEGF is expressed at relatively low levels; primarily in neurons (Merrilll and Oldfield, 2005). The physiologic functions of this basal expression are unknown. In the current study we document VEGF dose-dependently enhances the survival of uninjured neurons in culture. These data suggest that VEGF has neurotrophic effects as well as the widely documented ability to protect neurons from exogenous injury. VEGF can inhibit or reduce neuronal cell death evoked in vitro by hypoxia, glutamate, or serum-deprivation (Wick et al., 2002; Tolosa et al., 2008; 2009). In vivo, VEGF induces neuritic growth and provides neuroprotection, particularly after ischemia or spinal cord injuries (Widenfalk et al., 2003; Kilic et al., 2006a). Also, VEGF is a modifier of motor neuron degeneration in humans and in a mouse model of ALS (Storkebaum et al., 2004). Despite these protective effects, VEGF overexpression contributes to the phenotype associated with many CNS disorders. In this regard, marked upregulation of VEGF is observed in traumatic injury, stroke, aneurysms, and AD (Skirgaudas et al., 1996; Kalaria et al., 1998; Shore et al., 2004; Storkebaum et al., 2004; Merrilll and Oldfield, 2005). This increase could be a compensatory response to injury or could directly relate to disease pathogenesis. Because VEGF has a potentiating effect on CNS inflammation and increases vascular permeability (Merrilll and Oldfield, 2005), continuous upregulation of VEGF, at sites of brain injury or inflammation, may contribute to the pathogenesis of chronic neuroinflammatory disease. Defining the pathways and secondary mediators regulated by this multifaceted protein is important for developing targeted therapeutics that best harness the neurotrophic/protective effects of VEGF and minimize untoward effects.

VEGF has been shown to affect expression/activity of numerous mediators in neurons that are part of the apoptotic cascade. VEGF protects motor neurons from serum deprivation-induced down regulation of the anti-apoptotic protein Bcl-2 (Tolosa et al., 2009). Chronic glutamate excitotoxicity in spinal cord neurons induces down regulation of Bcl-2 that is prevented by VEGF (Tolosa et al., 2008). We have previously shown the induction of Bcl-2 in cultured cortical neurons by the drug acetaminophen enhances neuronal survival when cells are exposed to oxidant stress (Tripathy and Grammas, 2009). In the current study, we document a dose-dependent increase in Bcl-2 expression in cultured neurons exposed to VEGF. Increased expression of Bcl-2 may be a common and important target of neuroprotective/trophic proteins in neurons. In mouse cortical neuronal cultures subjected to hypoxia, the neuroprotective effects of VEGF also involve suppression of cell death pathways mediated by caspase 3. In those cultures VEGF triggers the PI3K/Akt signaling cascade leading to inhibition of caspase 3 (Jin et al., 2001). VEGF reduces ischemic brain infarct and decreases hypoxic neuronal death via inhibition of caspase 3 (Shen et al., 2006). In the current study we document that VEGF dose-dependently inhibits expression of the cleaved (active) form of caspase 3 in uninjured neurons. Thus, in neurons VEGF improves cell survival both by enhancing pro-survival signals (Bcl-2) as well as inhibiting pro-apoptotic proteins (caspase 3).

VEGF has been shown to activate several signaling cascades in neurons. The ability of VEGF to protect motor neurons from chronic glutamate excitoxicity is mediated by activation of PI3K/Akt (Tolosa et al., 2008). VEGF protects motor neurons from serum deprivation-induced cell death through PI3K mediated p38 MAP kinase inhibition (Tolosa et al., 2009). In cerebral ischemia VEGF increases phosphorylation of Akt and ERK-1/2 and decreases phosphorylation of c-Jun NH2-terminal kinase (JNK)-1/2 and p38 MAP kinase (Kilic et al., 2006a). After axotomy, VEGF transgenic animals demonstrate, compared to control mice, increased phosphorylated form of ERK-1/2 and Akt and reduced phosphorylated p38 MAP kinase and protection from neuronal degeneration (Kilic et al., 2006b). Here we show VEGF- mediated effects on phosphorylation of p38 MAP kinase in uninjured neurons. However, our experiments with a specific inhibitor (SB203580) and stimulator (anisomycin) of p38 MAP kinase reveal a partial dissociation between VEGF’s effects on p38 MAP kinase activity and on neuronal survival. Incubation of neuronal cultures with either VEGF or SB203580 alone causes a significant increase in cell survival. However, survival of cultures exposed to VEGF plus SB203580, is significantly less than either agent alone. The mechanism underlying this observation is unclear. However, it is possible that the combination of both SB203580 and VEGF may result in a high, possibly noxious level of VEGF. In this regard, SB203580 has been shown to increase VEGF secretion in fibroblast-like cells, and furthermore, in tumors p38 MAP kinase itself can stimulate VEGF secretion (Yoshino et al., 2006; Lee et al., 2009). Alternatively, the extreme suppression of p38 MAP kinase evoked by the combination of SB203580 and VEGF treatment may abrogate functions of p38 MAP kinase recently reported to play a role in initiation/maintenance of cell cycle checkpoints and survival (Thornton and Rincon, 2009). Addition of the p38 MAP kinase stimulator anisomycin causes a pronounced decrease in neuronal survival. Neuronal survival is similar in cultures exposed to anisomycin alone and in cultures treated with anisomycin plus VEGF. Thus, VEGF is unable to mitigate the toxic effect of anisomycin on neuronal cultures. This suggests that other signals/pathways evoked by anisomycin have a greater influence over neuronal survival than p38 MAP kinase. Taken together these data suggest that the trophic effect of VEGF on neurons is complex and likely involves multiple mechanisms and/or mediators.

In addition to the direct effects of VEGF on neurons, VEGF could affect neuronal survival indirectly by inhibiting release of neurotoxic factors, promoting release of neurotrophic proteins, or both. In this regard, there could be a heretofore underexplored relationship between VEGF and the neurotoxic protein amyloid beta (Aβ). Data in the literature suggests that there is an interaction between VEGF and Aβ protein. In the neurodegenerative disorder Alzheimer’s disease, VEGF is co-localized in senile plaques with Aβ (Yang et al., 2004). Structural interactions between these two proteins have been reported in vitro (Yang et al., 2005). VEGF receptor (Flt-1) is increased in Alzheimer’s disease and in rat hippocampi injected with Aβ1-42 (Ryu et al., 2009). In the current study we show, for the first time, that treatment of cultured neurons with VEGF results in a dose-dependent decrease in the release of Aβ1-42 into the media. This effect, unlike other neurotrophic effects documented for VEGF that utilize the Flk-1 receptor, is dependent on a functional Flt-1 receptor.

The intracellular mechanisms that mediate the VEGF response are numerous and complex and thus likely to be unaffected by single-target kinase inhibitors. In this regard, we find that inhibitors that block either MEK 1/2, PI3K, JNK, PKC, or tyrosine kinase are unable to affect the action of VEGF on Aβ secretion. In contrast, use of dicoumarol, a pharmacologic inhibitor of of several signaling systems including stress activated protein kinase (SAPK)/JNK and NFkappaB (Cross et al., 1999) completely blocks the VEGF-mediated decrease of Aβ release. Furthermore, dicoumarol, a long-established anticoagulant, has multiple modes of action that involve redox perturbations including inhibition of heat shock proteins and effects on protein degradation (Seanor et al., 2003; Hernandez et al., 2008; Alard et al., 2009).

Finally, another mechanism that may underlie VEGF’s effect on Aβ1-42 is endocytosis. VEGF affects endocytosis (Salikhova et al., 2008), a process required for amyloidogenic processing of amyloid precursor protein and subsequent release of Aβ (Schneider et al., 2008). This mechanism may be especially relevant for our observation that VEGF mediates a decrease in Aβ1-42 levels in light of a recent report using brain slices from Alzheimer’s disease transgenic mice that shows that VEGF exposure reduces the formation of soluble, SDS-extractable Aβ1-42. The VEGF-mediated decrease in Aβ-42 in that study is accompanied by a transient decrease in β-secretase activity indicating that VEGF affects the processing of amyloid precursor protein (Bürger et al., 2009).

Another protein that may play a role in VEGF’s trophic effect is PEDF. Interestingly, although both PEDF and VEGF are neurotrophic, PEDF has opposite functions from VEGF in terms of angiogenesis and permeability. For example, it has been shown that intradermal injections of PEDF in nude mice block VEGF- induced vascular hyperpermeability (Yamagishi et al., 2007). The dissociation between VEGF’s neurotrophic and permeability effects is important. Indeed, the use of VEGF as a neuroprotectant therapeutic has been limited by concerns that neuroprotective and permeability effects are both mediated by activation of PI3K/Akt. Thus, the demonstration that VEGF can release the neurotrophic protein PEDF from neurons and that this protein antagonizes the permeability effects of VEGF suggests that further dissecting the mechanisms/mediators regulated by the versatile protein VEGF in the brain will lead to more targeted approaches that take advantage of the beneficial actions while minimizing potentially toxic side effects.

Acknowledgments

Sources of support: This work was supported in part by grants from the National Institutes of Health (AG15964, AG020569 and AG028367). Dr. Grammas is the recipient of the Shirley and Mildred Garrison Chair in Aging. The authors gratefully acknowledge the secretarial assistance of Terri Stahl.

Footnotes

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