SEARCH

SEARCH BY CITATION

Keywords:

  • astrocytes;
  • brain injury;
  • endothelin-1;
  • VEGF

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
Thumbnail image of graphical abstract

Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin-1 (ET-1) were examined in rat cultured astrocytes. Expressions of VEGF-R1 and -R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF-R3 and Tie-2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF-R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF-E, a selective VEGF-R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF165 were reduced by selective antagonists for VEGF-R1 and -R2. Treatment with ET-1 increased VEGF-R1 mRNA and protein levels. The effects of ET-1 on VEGF-R1 mRNA were mimicked by Ala1,3,11,15-ET-1, a selective agonist for ETB receptors, and inhibited by BQ788, an ETB antagonist. ET-1 did not affect the mRNA levels of VEGF-R2, -R3, and Tie-2. Pre-treatment with ET-1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET-1 induces up-regulation of VEGF-R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues.

To clarify the regulatory mechanisms of vascular endothelial growth factor (VEGF) receptors, the effects of endothelin-1 (ET-1) were examined in rat cultured astrocytes. Effects of selective VEGF-R1 and R2 agonist showed that these receptors were linked to focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Treatment with ET-1 increased expression of VEGF-R1, which was mediated by ETB receptors. Pre-treatment with ET-1 potentiated the VEGF-R1-mediated activations of FAK and ERK1/2. These findings suggest that ET-1 induces up-regulation of VEGF-R1 receptors in astrocytes.

Abbreviations used
CHX

cycloheximide

ERK

extracellular signal regulated kinase

ET

endothelin

G3PDH

glyceraldehyde-3-phosphate dehydrogenase

MEM

minimum essential medium

PLGF

placenta growth factor

SDS

sodium dodecyl sulfate

VEGF

vascular endothelial growth factor

FAK

focal adhesion kinase

The vascular endothelial growth factor (VEGF) protein family plays pivotal roles in the regulation of vascular remodeling during repair processes of damaged tissues (Roy et al. 2006; Roskoski 2008). Some VEGF ligands are present in the brain. Expressions of brain VEGF ligands are increased after brain ischemia (Papavassiliou et al. 1997, Cobbs et al. 1998) and head trauma (Sköld et al. 2005). Recent studies have suggested that such increases in brain VEGFs were involved in the pathophysiological responses of damaged nerve tissues (Shibuya 2009; Wittko-Schneider et al. 2013). Administration of VEGFs attenuated neuronal damages in animal models of brain ischemia and neurodegenerative diseases (Manoonkitiwongsa et al. 2004; Azzouz et al. 2004; Yasuhara et al. 2004). Besides promotion of angiogenesis in damaged brain, VEGFs have direct effects on neuronal viability (Jin et al. 2000), neurite outgrowth (Rosenstein et al. 2003), and neurogenesis (Jin et al. 2002). In contrast, Zhang et al. (2000) reported that early post-ischemic administration of VEGFs increased the leakage of blood–brain barrier and neuronal damage in rat brain ischemia. Administration of a neutralizing antibody against VEGF reduced brain edema formation and infarct volume in a rat brain ischemia model (Kimura et al. 2005). Based on these studies, excess amounts of VEGFs may show detrimental actions on damaged brain by inducing breakdown of the blood–brain barrier and vasogenic brain edema (Nag et al. 2011). Thus, modulation of VEGF signals in neurological disorders may be significant for protection and repair of damaged nerve tissues (Wittko-Schneider et al. 2013).

The biological actions of VEGFs are mediated by three different types of receptors: VEGF-R1 (Flt-1), VEGF-R2 (Flk-1/KDR), and VEGF-R3 (Flk-4). These VEGF receptor subtypes belong to tyrosine-kinase-linked receptors, but have different affinities for VEGF ligands (Roy et al. 2006; Roskoski 2008). Previous studies have shown different intracellular signal mechanisms and functions among these VEGF receptors, which have been well characterized in vascular endothelial cells (Shibuya 2006). Histological observations of adult brains showed that these VEGF receptor subtypes were present in astrocytes (Krum et al. 2008; Shin et al. 2010, Koyama et al. 2011), suggesting that VEGFs are involved in regulation of astrocytic functions.

In response to brain pathologies, astrocytes turn their phenotypes to reactive astrocytes. Reactive astrocytes are characterized by hypertrophy of the cell body and become proliferative. Hyperplasia of reactive astrocytes often results in glial scar formation at the damaged areas (Pekny and Nilsson 2005; Sofroniew 2009). As it has been shown in vascular endothelial cells (Shibuya 2006) and neuronal progenitors (Jin et al. 2002; Xiao et al. 2007), VEGFs have mitogenic activity on astrocytes: VEGFs stimulated proliferation of cultured astrocytes (Freitas-Andrade et al. 2008; Schmid-Brunclik et al. 2008; Koyama et al. 2012). In brain injury models, inhibition of VEGF signals reduced the induction of reactive astrocytes at the damaged areas (Krum and Khaibullina 2003; Krum et al. 2008), indicating an important role of VEGFs in astrocytic proliferation. VEGF signals in astrocytes are regulated not only by ligand production but also by expression levels of VEGF receptors. In animal models of brain ischemia and traumatic injury, expressions of astrocytic VEGF-R1 and -R2 receptors were increased (Krum and Rosenstein 1998; Choi et al. 2007a,b; Wang et al. 2005). Increased expressions of astrocytic VEGF-R3 receptors were observed after brain ischemia and neuroinflammatory injury (Shin et al. 2008; Park et al. 2013). Together with the increases in VEGF ligands, up-regulations of VEGF receptor subtypes suggest that regulation of astrocytic functions by VEGF signals becomes more prominent in neurologic disorders. However, in spite of the regulatory roles on astrocytic functions, which subtypes of VEGF receptors are predominantly found in astrocytes remains still unknown. In addition, extracellular signals inducing up-regulations of astrocytic VEGF receptor subtypes in neurological disorders have not been examined.

Endothelins (ETs), which were originally found as a vasoconstrictor peptide family, are present in the brain. Productions of brain ETs are increased by several brain pathologies (Ostrow and Sachs 2005). Receptors for ETs are classified as ETA or ETB types. In the brain, the ETB type is highly expressed in astrocytes (Peters et al. 2003; Rogers et al. 2003; Wilhelmsson et al. 2004). Administrations of an ETB agonist into rat brain increased the number of reactive astrocytes (Ishikawa et al. 1997; Koyama et al. 2003), while in animal brain injury models, ETB antagonists reduced astrocytic proliferation in damaged areas (Koyama et al. 1999; Gadea et al. 2008). Further examinations showed that activation of ETB receptors stimulated functional alterations of astrocytes, which included the conversion to reactive astrocytes, and the associated proliferation, morphological changes, and productions of bioactive substances (Koyama and Michinaga 2012; Koyama 2013). From these findings, ETs may serve as regulators of astrocytic pathophysiological responses in damaged brains.

To explore VEGF receptor signals in astrocytes, we examined, (i) signal transduction mechanisms of astrocytic VEGF receptor subtypes and (ii) the effects of ETs on their expressions in rat cultured astrocytes. We found that VEGF-R1 and R2 receptors were linked to focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2), which mediated receptor signals for astrocytic proliferation (Teixeira et al. 2000; Koyama et al. 2004). Moreover, treatment with ETs increased the expressions of VEGF-R1 receptors, and enhanced VEGF-R1-mediated signals in cultured astrocytes.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

Preparation of primary cultured astrocytes from rat brain

All experimental protocols conformed to the Guiding Principles for the Care and Use of Animals by the Japanese Pharmacological Society, and were approved by the Animal Experiment Committee of Osaka Ohtani University. Astrocytes were prepared from the cerebra of 1- to 2-day-old Wistar rats as described previously (Koyama et al. 2004). The isolated cells were seeded at 1 × 104 cells/cm2 in 75 cm2 culture flasks and grown in minimal essential medium (MEM) supplemented with 10% fetal calf serum. To remove small process-bearing cells (mainly oligodendrocyte progenitors and microglia from the protoplasmic cell layer), the culture flasks were shaken at 250 rpm overnight, 10–14 days after seeding. The monolayer cells were trypsinized and seeded on six-well culture plates. At this stage, approximately 95% of the cells showed immunoreactivity for glial fibrillary acidic protein.

Treatment of cultured astrocytes with ETs and VEGFs

Before treatment with ETs and VEGFs, astrocytes in six-well culture plates were incubated in serum-free MEM for 48 h. ETs and VEGFs were then added to the cells in fresh serum-free MEM. Then, astrocytes were treated for the time indicated at 37°C. Antagonists of ET and VEGF receptors were included in the medium 30 min before additions of ET-1 and VEGF165. After treatments with ETs and VEGFs, cultured astrocytes were rinsed with ice-cold phosphate-buffered saline and used for preparations of total RNA and cell lysates.

Measurement of mRNA Levels by Quantitative RT-PCR

Total RNA in cultured astrocytes was extracted by an acid-phenol method as described previously (Koyama et al. 2011). First-strand cDNA was synthesized from total RNA (1 μg) using MMLV reverse transcriptase (200 U; Invitrogen, Carlsbad, CA, USA), random hexanucleotides (0.2 μg; Invitrogen), and an RNase inhibitor (20 U; Takara, Tokyo, Japan) in 10 μL of a buffer supplied by the enzyme manufacturer. The mRNA levels of VEGFs and angiopoietins in each sample were determined by quantitative PCR using SYBR Green fluorescent probes. Each reverse transcription product was added to Sybr Green Master Mix (Toyobo, Tokyo, Japan) along with the primer pairs, and the mixture was placed in a thermal cycler (Opticom 2; MJ Research, Waltham, MA, USA). The following primer pairs were used: rat membrane type VEGF-R1, 5′-CGACACTCTTTTGGCTCCTTCTAAC-3′ and 5′-TGACAGGTA GTCCGTCTTTACTTCG-3′; VEGF-R2, 5′- CAGCATCACCAGCAGTCAG-3′ and 5′-CAAGAACTCCATGCCCTTA-3′; VEGF-R3, 5′-TGGTACCGGCTCAACCTCTC-3′ and 5′-CACGTTCTTG CAGTCGAGCA-3′; Tie-2, 5′-TGCCACCATCACTCAATACC-3′ and 5′-AAACGCCAATAGCACGGTGA-3′; G3PDH, 5′-CTCATGACCACAGTCCATGC-3′ and 5′-TACATTGGGGGTAGGAACAC-3′.

As a standard for the copy numbers of PCR products, serial dilutions of each amplicon were amplified in the same manner. The amount of cDNA was calculated as the copy number of each reverse transcription product equivalent to 1 μg of total RNA, and normalized to the value for glyceraldehyde-3-phosphate dehydrogenase.

Measurement of protein levels by immunoblotting

Cultured astrocytes in six-well culture plates were lysed in 100 μL of ice-cold homogenization buffer (20 mM Tris/HCl pH 7.4, 1% sodium dodecyl sulfate, 2 mM ethylene-diaminetetraacetic acid, 2 mM phenylmethylsulfonyl fluoride, 20 μg/mL aprotinin) at 4°C. The lysates were centrifuged at 15 000 g for 10 min, and the protein contents of the supernatants were measured. The cell lysates were applied to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. For detection of phosphorylated proteins, the membranes were first probed with phosphorylated protein-specific antibodies. The following antibodies were used; rabbit anti-phospho-FAK (1 : 4000 dilution, Millipore, Temecula, CA, USA), rabbit anti-phospho-ERK1/2 (1 : 4000 dilution; Cell Signal Tech Inc., Danver, MA, USA), rabbit anti-phosho-VEGF-R1 (1 : 10 000 dilution; Millipore), and rabbit anti-phospho-VEGF-R2 (1 : 2000 dilution, ab38473; Abcam, Tokyo, Japan). Then, membranes were incubated with peroxidase-conjugated secondary antibodies. The exposed X-ray films were scanned, and the densities of the protein bands were measured using Image J 1.45 (NIH, Bethesda, MD, USA). After detection of the phosphorylated proteins, the membranes were re-probed with respective antibodies to detect total protein levels: rabbit anti-FAK (1 : 2000 dilution, C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-ERK1/2 (1 : 4000 dilution, Cell Signal Tech Inc.), rabbit anti-VEGF-R1 (1 : 2000 dilution, C-17, Santa Cruz Biotechnology), and rabbit anti-VEGF-R2 (1 : 2000 dilution, ab39256; Abcam). Total protein levels were measured as described above. Levels of protein phosphorylations were indicated as a ratio of phosphorylated protein to total protein. For examinations of VEGF-R1 and -R2 protein levels, blotted polyvinylidene difluoride membranes were first probed with rabbit anti-VEGF-R1 (C-17; Santa Cruz Biotechnology) and rabbit anti-VEGF-R2 (ab39256; Abcam), respectively. After determinations of VEGF receptor levels, membranes were re-probed with primary antibodies for a mouse anti-β-actin primary antibody (1 : 4000 dilution; Chemicon, Temecula, CA, USA). Expression levels of VEGF-R1 proteins were indicated as a ratio of VEGF-R1 to β-actin proteins.

Statistical analysis

Based on preliminary results in each examination, experimental numbers for meaningful results were estimated by using G*Power 3.1 (Heinrich-Heine-Universität Düsseldorf, Germany). All results were presented as means ± SEM. Results were analyzed by one-way anova followed by Dunnett's test or Fisher's test. The p-values were calculated by using Ekuseru-Toukei2010 (Social Survey Research Information Co., Ltd, Tokyo, Japan).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

Expressions of functional VEGF-R1 and R2 receptors in rat cultured astrocytes

Histological observations of adult brains have shown the presence of receptor subtypes for VEGFs, i.e., VEGF-R1, VEGF-R2, and VEGF-R3, in astrocytes. At first, we determined the mRNA copy numbers of these VEGF receptor subtypes in non-treated rat cultured astrocytes (Table 1). The mRNA copy number of VEGF-R1 was the highest among VEGF receptor subtypes. The expression level of astrocytic VEGF-R2 mRNA was smaller, but in a similar order to those of VEGF-R1. VEGF-R3 mRNA was detected in astrocytes, but at a much lower level with copy numbers about 1/30 of VEGF-R1 mRNA. Angiopoietins, another angiogenic protein family, coordinately regulate functions of vascular endothelial cells with VEGFs. The copy number of Tie-2, a receptor for angiopoietins, was about 1/25 of VEGF-R1 mRNAs in cultured astrocytes.

Table 1. Comparison of the mRNA copy numbers of receptors for VEGFs and angiopoietins in rat cultured astrocytes
 mRNA copy number (× 103/μg total RNA)
  1. Cultured astrocytes were prepared from the cerebra of Wistar rats, and total RNA was extracted. The mRNA copy numbers of receptors for VEGFs and angiopoietins were determined by quantitative RT-PCR. The copy numbers of G3PDH mRNA in the same samples were also determined as an internal standard. The data are the means ± SEM of 6–12 different preparations and are presented as ×103 copy numbers/μg total RNA.

VEGF-R179.00 ± 16.10
VEGF-R220.98 ± 3.22
VEGF-R32.39 ± 0.36
Tie-23.13 ± 0.46
G3PDH29 890.97 ± 2146.40

In some types of cells including vascular endothelial cells, VEGF-R1 and -R2 receptors can link to FAK and ERK1/2 (Rousseau et al. 2000; Abu-Ghazaleh et al. 2001; Matsumoto et al. 2002; Kazi et al. 2004; Swendeman et al. 2008). We tested the effects of VEGF ligands on astrocytic FAK and ERK1/2 activation. VEGF165, which is a major form of VEGF-A and binds to both VEGF-R1 and -R2 receptors, increased phosphorylated (activated) forms of FAK, while total protein levels of FAK were not affected (Fig. 1a). VEGF165 also increased phosphorylated ERK1/2 without affecting total ERK1/2 levels. Treatment for 20–120 min with 50 ng/mL placenta growth factor (PLGF), a selective agonist for the VEGF-R1 receptor, increased phosphorylated FAK and ERK1/2 in cultured astrocytes (Fig. 1b and c). VEGF-E, a selective agonist for VEGF-R2, stimulated phosphorylations of FAK and ERK1/2 at 50 ng/mL. The increased FAK and ERK1/2 phosphorylations returned to the control levels in treatments with PLGF and VEGF-E for 3–6 h (data not shown). The VEGF165-induced increases in phosphorylations of FAK and ERK1/2 were reduced by the anti-Flt-peptide (H-Gly-Asp-Gln-Trp-Phe-Ile-OH, 20 μM) (Bae et al. 2005). Similarly, the VEGF-R2/KDR antagonist (H-Ala-Thr-Trp-Leu-Pro-Pro-Arg-OH, 20 μM) (Binétruy-Tournaire et al. 2000) reduced the effects of VEGF165 on FAK and ERK1/2 phosphorylations (Fig. 2).

image

Figure 1. Effects of vascular endothelial growth factor (VEGF) ligands on phosphorylations of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2) in cultured rat astrocytes. (a) Effects of VEGF165: Cultured astrocytes were treated with 100 ng/mL VEGF165 for the time indicated. Phosphorylated FAK and ERK1/2 were detected by immunoblotting. After detection of phosphorylated proteins, the same blots were re-probed with anti-FAK or anti-ERK1/2 antibody to detect total levels of these proteins. Typical immunoblot images among three experiments with similar results are presented. (b) Effects of VEGF receptor selective agonists: Cultured astrocytes were treated with 50 ng/mL placenta growth factor (PLGF) or 50 ng/mL VEGF-E for the time indicated. Then, phosphorylated FAK and ERK1/2 were detected. After detection of phosphorylated proteins, total FAK and ERK1/2 proteins were detected. (c) Quantification of VEGF agonist-induced phosphorylations of FAK and ERK1/2 proteins: After detections of both phosphorylated and total proteins, the protein bands in X-ray films were subjected to densitometry analyses. The results are means ± SEM of four experiments and are presented as ratios of phosphorylated/total proteins. (●): 50 ng/mL PLGF, (○): 50 ng/mL VEGF-E, *< 0.05, **< 0.01 versus 0-time by one-way anova followed by Dunnett's test.

Download figure to PowerPoint

image

Figure 2. Effects of vascular endothelial growth factor (VEGF) receptor antagonists on VEGF165-induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). (a) Cultured astrocytes were treated with 50 ng/mL VEGF165 for 20 min. Anti-Flt-peptide (H-Gly-Asp-Gln-Trp-Phe-Ile-OH, 20 μM) or VEGF-R2/KDR antagonist (H-Ala-Thr-Trp-Leu-Pro-Pro-Arg-OH, 20 μM) was included in the treatment medium 30 min before addition of VEGF165. Typical immunoblots of FAK and ERK1/2 are shown. (b) Quantification of VEGF-induced phosphorylations of FAK and ERK1/2 proteins in the presence of VEGF antagonists: After detections of both phosphorylated and total proteins, the protein bands in the X-ray films were subjected to densitometry analyses. The results are the means ± SEM of six experiments, and are presented as ratios of phosphorylated/total proteins. **< 0.01 versus non-treatment (no VEGF165 in the absence of VEGF antagonists); #< 0.05, ##< 0.01 versus VEGF165 without an antagonist, by one-way anova followed by Fisher's test.

Download figure to PowerPoint

Effects of ETs on the expression of VEGF receptors in cultured astrocytes

Treatment with 100 nM ET-1 for 2–6 h increased VEGF-R1 mRNA levels in cultured astrocytes; this increase was 300% of non-treated cells after 2 h (Fig. 3). Ala1,3,11,15-ET-1, a selective agonist for ETB receptors, also increased astrocytic VEGF-R1 mRNA levels with a similar time course of ET-1. Expressions of astrocytic VEGF-R2, VEGF-R3, and Tie-2 mRNAs were not affected by treatment with ET-1 and Ala1,3,11,15-ET-1. The effects of ET-1 on VEGF-R1 mRNA levels were dose dependent and significant effects were obtained at 10 nM (Fig. 4a). The increases in VEGF-R1 mRNA by ET-1 were inhibited by 1 μM BQ788, an ETB antagonist, but the ETA antagonist, FR139317 at 1 μM, had no effect (Fig. 4b). Immunoblot analysis showed that treatments with 100 nM ET-1 increased the expression levels of VEGF-R1 proteins in cultured astrocytes (Fig. 5), but ET-1 did not affect astrocytic VEGF-R2 protein levels (data not shown).

image

Figure 3. Effects of endothelins (ETs) on vascular endothelial growth factor (VEGF) and angiopoietin receptor mRNA expressions in cultured rat astrocytes. Cultured astrocytes were treated with 100 nM ET-1 (●) or 100 nM Ala1,3,11,15-ET-1 (○) for the times indicated. The expression levels of VEGF-R1, -R2, -R3, and Tie-2 mRNAs were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The results are expressed as means ± SEM of 9–22 experiments. *< 0.05, **< 0.01 versus. 0-time by one-way anova followed by Dunnett's test.

Download figure to PowerPoint

image

Figure 4. (a) Dose responses of endothelin (ET)-induced changes in vascular endothelial growth factor (VEGF)-R1 mRNA levels in cultured astrocytes: Cultured astrocytes were treated with the indicated concentrations of ET-1 for 6 h. The results are the means ± SEM of 12–14 experiments. *< 0.05 versus control by one-way anova followed by Dunnett's test. (b) Effects of ET receptor antagonists on the ET-induced increases in VEGF-R1 mRNA levels: Astrocytes were treated with 10 nM ET-1 for 6 h. BQ788 (1 μM) or FR139317 (1 μM) was added to the medium 30 min before treatment with ET-1. The expression levels of VEGF-R1 mRNAs were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The results are expressed as means ± SEM of nine experiments. **< 0.01 versus non-treatment (no ET-1 in the absence of ET antagonists), ##< 0.01 versus no antagonist by one-way anova followed by Fisher's test. NS, not significant.

Download figure to PowerPoint

image

Figure 5. (a) Effects of endothelin-1 (ET-1) on expression of vascular endothelial growth factor (VEGF)-R1 protein: Astrocytes were cultured in serum-free minimum essential medium (MEM) with 100 nM ET-1 for the times indicated. The expression levels of VEGF-R1 protein were measured by immunoblotting. After detection of VEGF-R1 proteins, blots were re-probed by an anti-β-actin antibody to confirm that equal amounts of protein were loaded in each lane. (b) Quantification of astrocytic VEGF-R1 protein levels: After detection of both VEGF-R1 proteins and β-actin by immunoblotting, the protein bands in X-ray films were subjected to densitometry analyses. The results are the means ± SEM of four experiments, and are presented as ratios of VEGF-R1/β-actin proteins. **< 0.01 versus 0-time by one-way anova followed by Dunnett's test.

Download figure to PowerPoint

Potentiation of VEGF-R1 receptor signals by pre-treatment with ET-1

ET-1 treatments stimulated the expression of VEGF-R1 receptors in cultured astrocytes (Figs 3 and 5). We then examined whether the increased expression of VEGF-R1 affected the receptor signals. In non-treated astrocytes, PLGF increased phosphorylation of FAK and ERK1/2 in a dose-dependent manner (Fig. 6). Pre-treatment with 100 nM ET-1 for 16 h had no obvious effect on the phosphorylations of FAK and ERK1/2. Pre-treatment with ET-1 enhanced the effects of PLGF on phosphorylations of FAK; the potentiation was significant at 1–20 ng/mL PLGF (Fig. 6a). Phosphorylations of ERK1/2 at the submaximal concentrations of PLGF, i.e., 1 and 5 ng/mL, were also enhanced by pre-treatment with ET-1 (Fig. 6b). In contrast, phosphorylations of FAK and ERK1/2 by VEGF-E were not enhanced by pre-treatment with ET-1.

image

Figure 6. Effects of pre-treatment with endothelin-1 (ET-1) on vascular endothelial growth factor (VEGF) agonist-induced phosphorylations of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). (a) FAK: Cultured astrocytes were treated with 100 nM ET-1 for 16 h in serum-free minimum essential medium (MEM). After ET-1 pre-treatment, fresh serum-free MEM replaced the ET-1-containing MEM and the astrocytes were further incubated for 3 h. Then, astrocytes were treated by placenta growth factor (PLGF) and VEGF-E for 20 min at the concentrations indicated. Phosphorylated and total FAK proteins were detected by immunoblotting and quantified. The phosphorylation of FAK is presented as ratios of phosphorylated/total proteins. The results are the means ± SEM of six experiments. *< 0.05, **< 0.01 versus 0 ng/mL PLGF or VEGF-E; #< 0.05, ##< 0.01 versus no ET-1 treatment by one-way anova followed by Fisher's test. (b) ERK1/2: Cultured astrocytes were pre-treated with 100 nM ET-1 as described above. Then, cultured astrocytes were treated by PLGF and VEGF-E for 20 min at the concentrations indicated. Phosphorylated and total ERK1/2 proteins were detected by immunoblotting and quantified. The phosphorylation of ERK1/2 is presented as ratios of phosphorylated/total proteins. The results are the means ± SEM of six experiments. *< 0.05, **< 0.01 versus 0 ng/mL PLGF or VEGF-E; #< 0.05, ##< 0.01 versus no ET-1 treatment by one-way anova followed by Fisher's test. NS, not significant.

Download figure to PowerPoint

VEGF receptors can be activated by stimulation of some G-protein-coupled receptors in coronary endothelial cells and in melanoma (Miura et al. 2003; Spinella et al. 2013). However, ET-1 did not increase tyrosine-phosphorylated (activated) forms of VEGF-R1 and -R2 receptors in cultured astrocytes, while VEGF165 induced their phosphorylations (Fig. 7). Cycloheximide (CHX), a protein synthesis inhibitor, prevented the increases in astrocytic VEGF-R1 proteins by pre-treatment with ET-1 (Fig. 8a). In the presence of CHX, pre-treatment with ET-1 did not cause potentiation of FAK and ERK1/2 phosphorylations induced by PLGF (Fig. 8b).

image

Figure 7. Effects of endothelin-1 (ET-1) on phosphorylations of astrocytic vascular endothelial growth factor (VEGF)-R1 and VEGF-R2 receptors. Cultured astrocytes were treated with 100 nM ET-1 for the time indicated. Phosphorylated and total VEGF receptor proteins were detected by immunoblotting. As a positive control, cultured astrocytes were treated with 100 ng/mL VEGF165 for 20 min. Typical immunoblot images from four experiments with similar results are presented.

Download figure to PowerPoint

image

Figure 8. Cycloheximide inhibited endothelin (ET)-induced vascular endothelial growth factor (VEGF)-R1 expression and enhancement of VEGF signals. (a) VEGF-R1 expression: Cultured astrocytes were treated with 100 nM ET-1 for 16 h in the presence or the absence of 10 μg/mL cycloheximide (CHX). Expression levels of VEGF-R1 protein were measured by immunoblotting. After detection of VEGF-R1 proteins, blots were re-probed by an anti-β-actin antibody to confirm that equal amounts of protein were loaded in each lane. The quantitative results are the means ± SEM of six experiments and are presented as ratios of VEGF-R1/β-actin proteins. *< 0.05 versus non-treatment (no ET-1 in the absence of CHX), ##< 0.01 versus no CHX by one-way anova followed by Fisher's test. (b) Enhancement of VEGF signals: Cultured astrocytes were pre-treated with 100 nM ET-1 for 16 h in the presence or the absence of 10 μg/mL CHX. After ET-1 pre-treatment, the media were replaced by fresh serum-free minimum essential medium (MEM) and further incubated for 3 h. Then, cultured astrocytes were treated with 5 ng/mL placenta growth factor (PLGF) for 20 min. Phosphorylated and total proteins of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2) were detected by immunoblotting and the protein levels were quantified. The results are the means ± SEM of four to six experiments, and are presented as ratios of phosphorylated/total proteins. *< 0.05, **< 0.01 versus 0 ng/mL PLGF in each condition, #< 0.05 versus no CHX by one-way anova followed by Fisher's test. NS, not significant.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

VEGFs activate astrocytic FAK and ERK1/2 through VEGF-R1 and -R2 receptors

The receptors for VEGF are classified into VEGF-R1, -R2, and -R3 receptors, which belong to tyrosine-kinase-linked receptors (Roy et al. 2006; Roskoski 2008). Immunohistochemical observations on nerve tissues showed presence of VEGF receptor subtypes in astrocytes (Krum et al. 2008; Shin et al. 2010, Koyama et al. 2011), suggesting regulation of astrocytic functions by VEGFs. In fact, VEGFs stimulated proliferation and migration of cultured astrocytes (Freitas-Andrade et al. 2008; Schmid-Brunclik et al. 2008; Koyama et al. 2012). However, few studies have examined the VEGF receptor subtypes responsible for these actions on astrocytes. A comparison of mRNA levels among VEGF receptor subtypes showed similar expression levels of VEGF-R1 and -R2, whereas the expression of VEGF-R3 was lower (Table 1). In several cell types, FAK and ERK1/2 mediated intracellular signals triggered by VEGF-R1 and -R2 receptors (Rousseau et al. 2000; Abu-Ghazaleh et al. 2001; Matsumoto et al. 2002; Kazi et al. 2004; Swendeman et al. 2008). Also, in these cells, VEGF165 increased phosphorylated forms of FAK and ERK1/2 in cultured astrocytes (Fig. 1). We found that these effects of VEGF165 were partly inhibited by selective antagonists against VEGF-R1 and -R2 receptors (Fig. 2). In addition, the VEGF-R1 and -R2 selective agonists stimulated phosphorylations of astrocytic FAK and ERK1/2 with a similar potency (Fig. 1b and c). These results indicate that cultured astrocytes have levels of VEGF-R1 and -R2 receptors that are high enough to induce biological actions in response to VEGFs.

Increases in astrocytic VEGF-R1 receptor expression by ET-1

Expressions of VEGF receptor subtypes in astrocytes were shown to be up-regulated in several brain pathologies. For example, increased expressions of astrocytic VEGF-R1 and -R2 receptors were observed after ischemia and traumatic injury in animal models (Krum and Rosenstein 1998; Choi et al. 2007a,b; Wang et al. 2005). Increased expression of astrocytic VEGF-R3 was observed after brain ischemia and neuroinflammatory injury (Shin et al. 2008; Park et al. 2013). The up-regulations in VEGF receptor subtypes suggest that actions of VEGF signals on astrocytes become prominent in the damaged brain. However, signal molecules regulating the expression of astrocytic VEGF receptors in damaged brain have not been examined.

In this study, treatment with ET-1 increased expression of VEGF-R1 receptors in cultured astrocytes without affecting the expression levels of VEGF-R2 and -R3 receptors (Figs 3 and 5). The effect of ET-1 on VEGF-R1 receptor expression was mimicked by Ala1,3,11,15-ET1, a selective agonist for ETB receptors (Fig. 3), and reduced by an ETB receptor antagonist (Fig. 4b). These findings indicate that ET-1 up-regulated astrocytic VEGF-R1 receptor expression through ETB receptors. In brain pathologies, production of ET-1 is stimulated at damaged areas (Ostrow and Sachs 2005). Because ETB receptors are highly expressed in astrocytes (Peters et al. 2003; Rogers et al. 2003; Wilhelmsson et al. 2004), astrocytes are a likely target for brain ET-1. Activation of ETB receptors is involved in regulations of several astrocytic pathophysiological responses to brain damage (Koyama and Michinaga 2012; Koyama 2013). Thus, the ET-induced VEGF-R1 receptor expressions in cultured astrocytes suggest that ET-1 is a factor-stimulating astrocytic VEGF-R1 receptor expression after brain injuries.

Enhancement of astrocytic VEGF-R1 signal by pre-treatment with ET-1

In the examinations to show whether the up-regulation of VEGF-R1 receptors affects its receptor signals, PLGF-induced activations of FAK and ERK1/2 were enhanced by pre-treatment with ET-1 (Fig. 6). In contrast, pre-treatment with ET-1 did not enhance the actions of VEGF-E on these kinases. PLGF and VEGF-E are selective ligands for VEGF-R1 and VEGF-R2, respectively (Roy et al. 2006). Considering this specific enhancement of PLGF-induced signals, it is likely that pre-treatment with ET-1 did not activate signal mechanisms commonly shared by both VEGF-R1 and -R2, but selectively affected VEGF-R1 receptor-mediated signals.

In some types of cells, activations of tyrosine-kinase-linked growth factor receptors by G-protein-coupled receptor signals have been shown; this interaction is called ‘transactivation’. As for transactivation of VEGF receptor subtypes, stimulations of bradykinin B2 and ETB receptors activated VEGF receptors in vascular endothelial cells and melanoma (Miura et al. 2003; Spinella et al. 2013). However, stimulation of astrocytic ET receptors did not activate VEGF-R1 and -R2 receptors in the absence of VEGF ligands (Fig. 7), indicating that transactivation of VEGF receptors is not involved in the ET-induced enhancement of astrocytic VEGF signals. In contrast, CHX, an inhibitor of de novo protein synthesis, prevented ET-induced increases in VEGF-R1 protein levels (Fig. 8a), and also reduced enhancement of VEGF-R1 signals by ET-1 (Fig. 8b). Although, at present, an involvement of other mechanisms cannot be excluded, these findings suggest that increases in VEGF-R1 receptor expressions underlie the ET-induced enhancement of VEGF-R1 signals in cultured astrocytes.

Pathophysiological significance of ET-induced astrocytic VEGF-R1 receptor expression

Productions of VEGF ligands are stimulated in several brain pathologies, such as brain ischemia and head trauma, where astrocytes are a main source of brain VEGFs (Papavassiliou et al. 1997; Cobbs et al. 1998; Sköld et al. 2005). Increased brain VEGF ligands stimulate vascular endothelial cells and neuronal progenitors to promote angiogenesis and neurogenesis in the repair of damaged nerve tissues. In addition to producing VEGF, astrocytes have receptors for VEGFs. Thus, VEGFs are likely autocrine regulators of astrocytic pathophysiological responses. In damaged nerve tissues, brain ETs are increased and activate ETB receptors in astrocytes (Ostrow and Sachs 2005). We previously showed that ETs stimulated production of astrocytic VEGF-A through ETB receptors (Koyama et al. 2012). Together with the increased production of VEGF-A, the up-regulation of astrocytic VEGF-R1 receptors indicates an enhancement of VEGF-mediated autocrine regulation in ET-stimulated astrocytes. VEGFs stimulated proliferation of cultured astrocytes by promoting the G1/S phase cell cycle progression (Schmid-Brunclik et al. 2008; Koyama et al. 2012).

In this study, stimulation of VEGF-R1 receptors activated astrocytic FAK and ERK1/2 (Figs 1 and 2) and the effects were enhanced by pre-treatment with ET-1 (Fig. 6). Because FAK and ERK1/2 have pivotal roles in the G1/S phase progression of astrocytes (Cazaubon et al. 1997; Teixeira et al. 2000; Koyama et al. 2004), VEGF-R1 receptors might mediate the mitogenic action of VEGFs. Therefore, the enhancement of VEGF-R1 signals by ET-1 may be significant in astrocytic proliferation.

In response to brain damage, astrocytes turn their phenotype into a reactive phenotype. Accompanied by the conversion to the reactive phenotype, astrocytes become proliferative, and often, hyperplasia of reactive astrocytes results in glial scar formation (Pekny and Nilsson 2005; Sofroniew 2009). Krum and Khaibullina (2003) showed that inhibition of VEGF signals, including VEGF-R1 receptors, decreased the numbers of reactive astrocytes and prevented glial scar formation in traumatic brain injury models. These observations indicate an involvement of VEGFs in astrocytic proliferation in brain pathologies. Therefore, the ET-induced enhancement of VEGF-R1 signals may have a pathophysiological significance in the hyperplasia of reactive astrocytes in damaged brains.

However, this study cannot clarify whether the ET-induced enhancement of VEGF-R1 signals has beneficial or detrimental actions in damaged brain, because reactive astrocytes show both of these aspects depending on the type and severity of the brain disorders (Sofroniew 2009; Karimi-Abdolrezaee and Billakanti 2012). To clarify roles of the enhanced VEGF-R1 signals in damaged brain, examinations in individual brain injury models will be required.

Acknowledgments and conflict of interest disclosure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

This work was supported by a Grant-in-Aid for Scientific Research (C) from the JPSP (21590108). The authors have no conflicts of interest with this study.

All experiments were conducted in compliance with the ARRIVE guidelines.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  • Abu-Ghazaleh R., Kabir J., Jia H., Lobo M. and Zachary I. (2001) Src mediates stimulation by vascular endothelial growth factor of the phosphorylation of focal adhesion kinase at tyrosine 861, and migration and anti-apoptosis in endothelial cells. Biochem. J. 360(Pt 1), 255264.
  • Azzouz M., Ralph G. S., Storkebaum E., Walmsley L. E., Mitrophanous K. A., Kingsman S. M., Carmeliet P. and Mazarakis N. D. (2004) VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model. Nature 429, 413417.
  • Bae D. G., Kim T. D., Li G., Yoon W. H. and Chae C. B. (2005) Anti-flt1 peptide, a vascular endothelial growth factor receptor 1-specific hexapeptide, inhibits tumor growth and metastasis. Clin. Cancer Res. 11, 26512661.
  • Binétruy-Tournaire R., Demangel C., Malavaud B., Vassy R., Rouyre S., Kraemer M., Plouët J., Derbin C., Perret G. and Mazié J. C. (2000) Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis. EMBO J. 19, 15251533.
  • Cazaubon S., Chaverot N., Romero I. A., Girault J. A., Adamson P., Strosberg A. D. and Couraud P. O. (1997) Growth factor activity of endothelin-1 in primary astrocytes mediated by adhesion-dependent and -independent pathways. J. Neurosci. 17, 62036212.
  • Choi J. S., Kim H. Y., Cha J. H., Choi J. Y., Park S. I., Jeong C. H., Jeun S. S. and Lee M. Y. (2007a) Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 following acute spinal cord contusion in rats. J. Histochem. Cytochem. 55, 821830.
  • Choi J. S., Kim H. Y., Cha J. H., Choi J. Y., Chun M. H. and Lee M. Y. (2007b) Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 in rat hippocampus after transient forebrain ischemia. J. Neurotrauma 24, 521531.
  • Cobbs C. S., Chen J., Greenberg D. A. and Graham S. H. (1998) Vascular endothelial growth factor expression in transient focal cerebral ischemia in the rat. Neurosci. Lett. 249, 7982.
  • Freitas-Andrade M., Carmeliet P., Stanimirovic D. B. and Moreno M. (2008) VEGFR-2-mediated increased proliferation and survival in response to oxygen and glucose deprivation in PlGF knockout astrocytes. J. Neurochem. 107, 756767.
  • Gadea A., Schinelli S. and Gallo V. (2008) Endothelin-1 regulates astrocyte proliferation and reactive gliosis via a JNK/c-Jun signaling pathway. J. Neurosci. 28, 23942408.
  • Ishikawa N., Takemura M., Koyama Y., Shigenaga Y., Okada T. and Baba A. (1997) Endothelins promote the activation of astrocytes in rat neostriatum through ETB receptors. Eur. J. Neurosci. 9, 895901.
  • Jin K. L., Mao X. O. and Greenberg D. A. (2000) Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc. Natl Acad. Sci. USA 97, 1024210247.
  • Jin K., Zhu Y., Sun Y., Mao X. O., Xie L. and Greenberg D. A. (2002) Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc. Natl Acad. Sci. USA 99, 1194611950.
  • Karimi-Abdolrezaee S. and Billakanti R. (2012) Reactive astrogliosis after spinal cord injury-beneficial and detrimental effects. Mol. Neurobiol. 46, 251264.
  • Kazi A. S., Lotfi S., Goncharova E. A., Tliba O., Amrani Y., Krymskaya V. P. and Lazaar A. L. (2004) Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent. Am. J. Physiol. Lung Cell. Mol. Physiol. 286, L539L545.
  • Kimura R., Nakase H., Tamaki R. and Sakaki T. (2005) Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction. Stroke 36, 12591263.
  • Koyama Y. (2013) Endothelin systems in the brain: involvement in pathophysiological responses of damaged nerve tissues Biomol. Concepts 4, 335347.
  • Koyama Y. and Michinaga S. (2012) Regulations of astrocytic functions by endothelins: roles in the pathophysiological responses of damaged brains. J. Pharmacol. Sci. 118, 401407.
  • Koyama Y., Takemura M., Fujiki K., Ishikawa N., Shigenaga Y. and Baba A. (1999) BQ788, an endothelin ETB receptor antagonist, attenuates stab wound injury-induced reactive astrocytes in rat brain. Glia 26, 268271.
  • Koyama Y., Tsujikawa K., Matsuda T. and Baba A. (2003) Intracerebroventricular administration of an endothelin ETB receptor agonist increases expressions of GDNF and BDNF in rat brain. Eur. J. Neurosci. 8, 887894.
  • Koyama Y., Yoshioka Y., Shinde M., Matsuda T. and Baba A. (2004) Focal adhesion kinase mediates endothelin-induced cyclin D3 expression in rat cultured astrocytes. J. Neurochem. 90, 904912.
  • Koyama Y., Nagae R., Tokuyama S. and Tanaka K. (2011) I.c.v administration of an endothelin ETB receptor agonist stimulates vascular endothelial growth factor-A production and activates vascular endothelial growth factor receptors in rat brain. Neuroscience 192, 689698.
  • Koyama Y., Maebara Y., Hayashi M., Nagae R., Tokuyama S. and Michinaga S. (2012) Endothelins reciprocally regulate VEGF-A and angiopoietin-1 production in cultured rat astrocytes: implications on astrocytic proliferation. Glia 60, 19541963.
  • Krum J. M. and Khaibullina A. (2003) Inhibition of endogenous VEGF impedes revascularization and astroglial proliferation: roles for VEGF in brain repair. Exp. Neurol. 181, 241257.
  • Krum J. M. and Rosenstein J. M. (1998) VEGF mRNA and its receptor flt-1 are expressed in reactive astrocytes following neural grafting and tumor cell implantation in the adult CNS. Exp. Neurol. 154, 5765.
  • Krum J. M., Mani N. and Rosenstein J. M. (2008) Roles of the endogenous VEGF receptors flt-1 and flk-1 in astroglial and vascular remodeling after brain injury. Exp. Neurol. 212, 108117.
  • Manoonkitiwongsa P. S., Schultz R. L., McCreery D. B., Whitter E. F. and Lyden P. D. (2004) Neuroprotection of ischemic brain by vascular endothelial growth factor is critically dependent on proper dosage and may be compromised by angiogenesis. J. Cereb. Blood Flow Metab. 24, 693702.
  • Matsumoto Y., Tanaka K., Hirata G., Hanada M., Matsuda S., Shuto T. and Iwamoto Y. (2002) Possible involvement of the vascular endothelial growth factor-Flt-1-focal adhesion kinase pathway in chemotaxis and the cell proliferation of osteoclast precursor cells in arthritic joints. J. Immunol. 168, 58245831.
  • Miura S., Matsuo Y. and Saku K. (2003) Transactivation of KDR/Flk-1 by the B2 receptor induces tube formation in human coronary endothelial cells. Hypertension 41, 11181123.
  • Nag S., Kapadia A. and Stewart D. J. (2011) Molecular pathogenesis of blood-brain barrier breakdown in acute brain injury. Neuropathol. Appl. Neurobiol. 37, 323.
  • Ostrow L. W. and Sachs F. (2005) Mechanosensation and endothelin in astrocytes–hypothetical roles in CNS pathophysiology. Brain Res. Brain Res. Rev. 48, 488508.
  • Papavassiliou E., Gogate N., Proescholdt M., Heiss J. D., Walbridge S., Edwards N. A., Oldfield E. H. and Merrill M. J. (1997) Vascular endothelial growth factor (vascular permeability factor) expression in injured rat brain. J. Neurosci. Res. 49, 451460.
  • Park J. M., Shin Y. J., Cho J. M., Choi J. Y., Jeun S. S., Cha J. H. and Lee M. Y. (2013) Upregulation of vascular endothelial growth factor receptor-3 in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis. J. Histochem. Cytochem. 61, 3144.
  • Pekny M. and Nilsson M. (2005) Astrocyte activation and reactive gliosis. Glia 50, 427434.
  • Peters C. M., Rogers S. D., Pomonis J. D., Egnaczyk G. F., Keyser C. P., Schmidt J. A., Ghilardi J. R., Maggio J. E. and Mantyh P. W. (2003) Endothelin receptor expression in the normal and injured spinal cord: potential involvement in injury-induced ischemia and gliosis. Exp. Neurol. 180, 113.
  • Rogers S. D., Peters C. M., Pomonis J. D., Hagiwara H., Ghilardi J. R. and Mantyh P. W. (2003) Endothelin B receptors are expressed by astrocytes and regulate astrocyte hypertrophy in the normal and injured CNS. Glia 41, 180190.
  • Rosenstein J. M., Mani N., Khaibullina A. and Krum J. M. (2003) Neurotrophic effects of vascular endothelial growth factor on organotypic cortical explants and primary cortical neurons. J. Neurosci. 23, 1103611044.
  • Roskoski R., Jr (2008) VEGF receptor protein-tyrosine kinases: structure and regulation. Biochem. Biophys. Res. Commun. 375, 287291.
  • Rousseau S., Houle F., Kotanides H., Witte L., Waltenberger J., Landry J. and Huot J. (2000) Vascular endothelial growth factor (VEGF)-driven actin-based motility is mediated by VEGFR2 and requires concerted activation of stress-activated protein kinase 2 (SAPK2/p38) and geldanamycin-sensitive phosphorylation of focal adhesion kinase. J. Biol. Chem. 275, 1066110672.
  • Roy H., Bhardwaj S. and Ylä-Herttuala S. (2006) Biology of vascular endothelial growth factors. FEBS Lett. 580, 28792887.
  • Schmid-Brunclik N., Bürgi-Taboada C., Antoniou X., Gassmann M. and Ogunshola O. O. (2008) Astrocyte responses to injury: VEGF simultaneously modulates cell death and proliferation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 295, R864R873.
  • Shibuya M. (2006) Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis. J. Biochem. Mol. Biol. 39, 469478.
  • Shibuya M. (2009) Brain angiogenesis in developmental and pathological processes: therapeutic aspects of vascular endothelial growth factor. FEBS J. 276, 46364643.
  • Shin Y. J., Choi J. S., Lee J. Y., Choi J. Y., Cha J. H., Chun M. H. and Lee M. Y. (2008) Differential regulation of vascular endothelial growth factor-C and its receptor in the rat hippocampus following transient forebrain ischemia. Acta Neuropathol. 116, 517527.
  • Shin Y. J., Choi J. S., Choi J. Y., Hou Y., Cha J. H., Chun M. H. and Lee M. Y. (2010) Induction of vascular endothelial growth factor receptor-3 mRNA in glial cells following focal cerebral ischemia in rats. J. Neuroimmunol. 229, 8190.
  • Sköld M. K., von Gertten C., Sandberg-Nordqvist A. C., Mathiesen T. and Holmin S. (2005) VEGF and VEGF receptor expression after experimental brain contusion in rat. J. Neurotrauma 22, 353367.
  • Sofroniew M. V. (2009) Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci. 32, 638647.
  • Spinella F., Caprara V., Di Castro V., Rosanò L., Cianfrocca R., Natali P. G. and Bagnato A. (2013) Endothelin-1 induces the transactivation of vascular endothelial growth factor receptor-3 and modulates cell migration and vasculogenic mimicry in melanoma cells. J. Mol. Med. 91, 395405.
  • Swendeman S., Mendelson K., Weskamp G., Horiuchi K., Deutsch U., Scherle P., Hooper A., Rafii S. and Blobel C. P. (2008) VEGF-A stimulates ADAM17-dependent shedding of VEGFR2 and crosstalk between VEGFR2 and ERK signaling. Circ. Res. 103, 916918.
  • Teixeira A., Chaverot N., Strosberg A. D. and Cazaubon S. (2000) Differential regulation of cyclin D1 and D3 expression in the control of astrocyte proliferation induced by endothelin-1. J. Neurochem. 74, 10341040.
  • Wang W. Y., Dong J. H., Liu X., Wang Y., Ying G. X., Ni Z. M. and Zhou C. F. (2005) Vascular endothelial growth factor and its receptor Flk-1 are expressed in the hippocampus following entorhinal deafferentation. Neuroscience 134, 11671178.
  • Wilhelmsson U., Li L., Pekna M. et al. (2004) Absence of glial fibrillary acidic protein and vimentin prevents hypertrophy of astrocytic processes and improves post-traumatic regeneration. J. Neurosci. 24, 50165021.
  • Wittko-Schneider I. M., Schneider F. T. and Plate K. H. (2013) Brain homeostasis: VEGF receptor 1 and 2-two unequal brothers in mind. Cell. Mol. Life Sci. 70, 17051725.
  • Xiao Z., Kong Y., Yang S., Li M., Wen J. and Li L. (2007) Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation. Cell Res. 17, 7379.
  • Yasuhara T., Shingo T., Kobayashi K., Takeuchi A., Yano A., Muraoka K., Matsui T., Miyoshi Y., Hamada H. and Date I. (2004) Neuroprotective effects of vascular endothelial growth factor (VEGF) upon dopaminergic neurons in a rat model of Parkinson's disease. Eur. J. Neurosci. 19, 14941504.
  • Zhang Z. G., Zhang L., Jiang Q., Zhang R., Davies K., Powers C., Bruggen N. and Chopp M. (2000) VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J. Clin. Invest. 106, 829838.