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The Journal of Neuroscience, July 15, 1999, 19(14):5731-5740
Vascular Endothelial Growth Factor Has Neurotrophic Activity and
Stimulates Axonal Outgrowth, Enhancing Cell Survival and Schwann Cell
Proliferation in the Peripheral Nervous System
Mariann
Sondell1,
Göran
Lundborg2, and
Martin
Kanje1
1 Department of Animal Physiology, University of Lund,
S-223 62 Lund, Sweden, and 2 Department of Hand Surgery,
Malmö General Hospital, University of Lund, S-20502 Malmö,
Sweden
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ABSTRACT |
Vascular endothelial growth factor (VEGF) is a mitogen for
endothelial cells, and it promotes angiogenesis in vivo.
Here we report that VEGF165 has neurotrophic actions on
cultured adult mouse superior cervical ganglia (SCG) and dorsal root
ganglia (DRG), measured as axonal outgrowth. Maximal effect was
observed at 10-50 ng/ml for SCG and 100 ng/ml for DRG. VEGF-induced
axonal outgrowth was inhibited by the mitogen-activated protein
kinase kinase inhibitor PD 98059 but not by the protein kinase
inhibitor K252a. VEGF also increased survival of both neurons and
satellite cells and the number of proliferating Schwann cells.
Immunocytochemistry and immunoblotting revealed that VEGF was expressed
in virtually all nerve cells in the SCG but only in a population of
small-diameter (<35 µm) neurons representing ~30% of the neurons
in DRG. Immunostaining showed that the VEGF receptor fetal liver kinase
receptor (flk-1) was found on nerve cell bodies in DRG and to a lesser
extent on neurons in SCG. Growth cones of regenerating axons from both
types of ganglia exhibited flk-1 immunoreactivity, as did Schwann
cells. We conclude that VEGF has both neurotrophic and mitogenic
activity on cells in the peripheral nervous system.
Key words:
axonal outgrowth; DRG; growth factor; mouse; nerve
regeneration; peripheral nerve; SCG; VEGF
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INTRODUCTION |
Vascular endothelial growth factor
(VEGF) is a potent and presumed selective endothelial cell mitogen that
promotes angiogenesis but also increases blood vessel permeability (for
review, see Ferrara and Davis-Smyth, 1997 ). VEGF is expressed in
epithelial cells of various capillary-rich tissues and is also found in
activated macrophages and in tumors (Berse et al., 1992). VEGF exists
in several isoforms because of alternative splicing of mRNA from a
single gene. So far, five isoforms have been isolated in humans, VEGF
206, 189, 165, 145, and 121 (Ferrara and Davis-Smyth, 1997; Poltorak et
al., 1997) and in murines, four different forms have been found, VEGF
188, 164, 120, and 115 (Breier et al., 1992; Sugihara et al., 1998).
VEGF exerts its action via high-affinity binding to two types of
phosphotyrosine kinase receptors: fms-like tyrosine kinase (flt-1) and
fetal liver kinase receptor (flk-1) (De Vries et al., 1992; Quinn et
al., 1993). Both receptor types are essential for development and
organization of endothelial cells and the disruption of the genes
encoding flk-1 and flt-1 results in defects of blood vessel formation
and an early death of homozygous mice embryo (Fong et al., 1995;
Shalaby et al., 1995). The human counterpart to the flk-1 receptor,
which activates the mitogen-activated protein kinase (MAPK)
pathway, is also known to induce reorganization of actin filaments,
chemotaxis, and give rise to a mitogen response in endothelial cells
(Waltenberger et al., 1994; Kroll and Waltenberger, 1997).
In the nervous system, VEGF mRNA has been found in neurons in the
capillary-rich areas of the brain, for instance in the pars distalis
cells of the pituitary gland but also in glial cells in the retina
after hypoxia (Ferrara et al., 1992; Stone et al., 1995). Furthermore,
VEGF expression is induced in astrocytes at the site of a spinal cord
injury (Bartholdi et al., 1997). The location of VEGF and pattern of
expression after injury in the nervous system thus appear consistent
with the role of VEGF as an angiogenic factor.
However, other angiogenic factors, including basic fibroblast growth
factor (bFGF) and platelet-derived growth factor (PDGF), have been
shown to be neurotrophic or exert growth-promoting activity (Rydel and
Greene, 1987; Davis and Stroobant, 1990; Smits et al., 1991; Fujimoto
et al., 1997). Because VEGF exhibits a low but significant structural
similarity to PDGF (Keck et al., 1989), we speculated that VEGF also
might have neurotrophic and/or growth-promoting activity on cells in
the nervous system.
Here we report that VEGF is expressed by neurons of the adult mouse
superior cervical ganglia (SCG) and dorsal root ganglia (DRG), and that
addition of VEGF to explanted ganglia promotes cell survival, axonal
outgrowth, and proliferation of Schwann cells. The VEGF receptor flk-1
was present on neurons in SCG and DRG and on Schwann cells. Blocking
the MAPK pathway inhibited the VEGF-induced axonal outgrowth,
suggesting that VEGF acts through stimulation of the flk-1 receptor.
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MATERIALS AND METHODS |
Animals. Five-week-old male NMRI mice weighing
~30 gm were obtained from B & K Universal AB. The mice were killed by
intraperitoneal injection of sodium pentobarbital (0.2 ml, 60 mg/ml)
followed by heart puncture and the removal of SCG and DRG (L4 and L5)
by dissection. A total number of 126 animals were used.
Test substances. For the axonal outgrowth experiments,
recombinant human VEGF165 (ICN Biochemicals, Costa Mesa,
CA) from Escherichia coli was used at final concentrations
of 1, 5, 10, 50, 100, and 200 ng/ml medium on SCG and 5, 10, 30, 50, 100, and 200 ng/ml on DRG.
Recombinant human  -nerve growth factor (-NGF) (PeproTech EC) was
used at concentrations of 50 and 1 ng/ml. Both VEGF and NGF were
dissolved in water. The MAPK kinase inhibitor PD 98059 (Calbiochem, La
Jolla, CA) and the protein kinase inhibitor K252a (LC Laboratories)
were dissolved in dimethylsulfoxide (DMSO).
Culture conditions. Explants of SCG and DRG were used for
studies of axonal outgrowth (Levi-Montalcini et al., 1954; Fenton, 1970; Tonge et al., 1998). The ganglia were mounted in 7 µl of Matrigel (Becton Dickinson, Mountain View, CA) in 35 mm plastic culture
dishes. For each pair of ganglia, one served as control, and the
contralateral ganglion was treated with growth factors and/or drug. Two
to three ganglia were mounted per culture dish. After a 5 min
incubation at 37°C to allow the Matrigel to polymerize, 2 ml
serum-free RPMI 1640 medium (Biochrome KG, Germany) supplemented with 1% antibiotic-antimycotic solution (Life Technologies),
was added. The ganglia were cultured at 37°C and maintained for 72 hr
in a humidified atmosphere of 95% O2 and 5%
CO2. In some additional experiments ganglia were maintained
in culture for 6 d to let the axons extend beyond the Matrigel and
reach the plastic surface of the culture dish. The same strategy but
for the difference that the ganglia were mounted on glass coverslips,
was used for visualization of the flk-1 receptor on axons and growth
cones. These structures become more accessible to the antibody outside the Matrigel than within it. Background fluorescence was diminished by
the use of glass coverslips.
Axonal outgrowth. Axonal outgrowth was evaluated in an
inverted phase-contrast microscope using a scaled ocular eyepiece to measure the length of the three longest axons at each principal outgrowth site after 48 hr in culture.
Axonal density was estimated by ocular microscopic observations of the
cultured SCG and DRG using a "ranked" sign test, where five plus
signs represented outgrowth in SCG exposed to 50 ng/ml of NGF, and one
plus sign represented outgrowth in the untreated DRG (see Figs.
2c and 4a for ranking).
Cell death. Cell survival in ganglia treated with VEGF were
studied by culturing the ganglia in plastic Eppendorf tubes. One ganglia in each tube was supplied with 250 µl medium ± VEGF and cultured for 48 hr as described above. SCG were cultured in the presence of 50 ng/ml of VEGF and DRG in 100 ng/ml of VEGF. The ganglia
were washed three times for 5 min each in PBS (0.01 M, pH 7.2) and then fixed in 4% formalin PBS for 3 hr at
room temperature followed by washing in PBS. They were then
cryoprotected by immersion in 20% sucrose in PBS at 4°C overnight.
The ganglia were mounted in TissueTek (Miles), and 10 µm sections
were obtained on a cryostat and collected on precoated Superfrost
slides (Menzel-Gläzer). The sections were allowed to dry for 20 min at room temperature and then stored at  20°C until use. A
modified method for terminal deoxynucleotidyl transferase-mediated
biotinylated UTP nick end labeling (TUNEL) was used to evaluate cell
survival (Ekström, 1995).
Immunocytochemistry. SCG and DRG were dissected and
immediately fixed followed by cryoprotection as described above. The
sections were collected on poly-L-lysine-coated glass
slides and allowed to dry for 20 min at room temperature. They were
then processed at once or stored at 20°C until use.
The slides were washed in 0.25% Triton X-100 (Packard, Meridian, CT)
in PBS two times for 10 min each. They were then treated with 0.5%
buffered Triton X-100 for 10 min at room temperature followed by 5%
dry milk in PBS for 30 min at room temperature to prevent nonspecific
antibody absorption. The sections were incubated with the primary
antibody polyclonal rabbit VEGF IgG (Santa Cruz Biotechnology, Santa
Cruz, CA) at a dilution of 1:500 in PBS containing 2% bovine serum
albumin (BSA) (w/v) at 4°C overnight. The sections were washed three
times for 5 min each in PBS and incubated 1 hr at room temperature in
darkness with fluorescein isothiocyanate (FITC)-conjugated swine
anti-rabbit antibodies (Dako, Glostrup, Denmark) diluted 1:80 in PBS.
After a final wash in PBS, the slides were mounted in glycerol in PBS
(1:2) and coverslipped.
Control studies included preabsorption of the primary antibody for 2 hr
at room temperature with an excess amount of VEGF165 (150 µg/ml undiluted antibody), and exclusion of primary antibody was
substituted with 2% BSA in PBS. No immunoreactivity was observed in
these controls.
Sections of freshly dissected ganglia, teased preparations of the mouse
sciatic nerve (Sondell et al., 1997), and ganglia cultured on glass
coverslips were processed for flk-1 receptor immunostaining as
described above, using anti rabbit flk-1 (Santa Cruz Biotechnology)
diluted 1:1000. The secondary biotin-conjugated antibody (Dako) was
diluted 1:300 followed by FITC-conjugated streptavidine 1:250. The
preparations were incubated with the biotin and streptavidine
antibodies for 1 hr at room temperature.
Control studies included exclusion of primary antibody substituted with
2% BSA in PBS. No immunoreactivity was observed in these controls. The
preparations were studied in an Olympus fluorescence microscope.
Staining of the Golgi apparatus. The method for staining of
the Golgi apparatus was based on a procedure developed by Lipsky and
Pagano (1985). Sections of SCG and DRG were rinsed in PBS three times
for 5 min each and then incubated with the fluorescent C6-NBD-ceramide
(Molecular Probes Europe BV) dissolved in DMSO and diluted to a final
concentration of 10 µM in PBS containing defatted BSA
(0.7 mg/ml, ICN Biochemicals). After 1 hr incubation at 37°C, the
sections were rinsed in PBS with defatted BSA three times for 5 min.
After the final wash, the sections were mounted in PBS and
coverslipped. The sections were studied in an Olympus fluorescence microscope.
Photography. Pictures were taken by an Eastman Kodak
(Rochester, NY) professional DCS 420 digital camera connected to an
Olympus microscope and a PowerMac computer. The public domain NIH Image software (written by Wayne Rasband at the United States National Institutes of Health and available from Internet by anonymous FTP from
zippy.nimh.nih.gov.) was used to count TUNEL-positive satellite cells
(particle analysis) and determine total area of the ganglia sections.
At a magnification of 100×, a satellite cell nuclei covered an area of
6-11 pixels. In both ganglia the overwhelming majority of
TUNEL-positive nuclei belonged to the satellite cells. For the particle
analysis, however, a minor contribution of small Schwann cells nuclei
cannot be excluded. The number of TUNEL-positive nerve cells were
manually counted at 200× magnification. The digital images of DRG were
processed in the software Adobe Photoshop 3.0 to allow determination of
the diameter of the VEGF-immunoreactive neuronal profiles and the total
number of neuronal profiles in the sections. Digital images of DRG and
SCG were also processed in Adobe Photoshop 3.0 to count
bromodeoxyuridine (BrdU)-labeled cells, and the section areas were
determined using the software mentioned above.
Electrophoresis and Western blotting. Six freshly dissected
SCG and 10 DRG were homogenized by sonication in 300 µl of sample buffer (0.0625 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol).
Samples of VEGF were prepared by diluting VEGF in sample buffer to a
final concentration of 19 ng/µl. -mercaptoethanol and bromphenol
blue were added to a final concentration of 5 and 4% (v/v),
respectively. The samples were then boiled for 3 min followed by
centrifugation at 10,000 × g for 10 min at 4°C.
Samples of the supernatants were applied on a one-dimensional 5-17%
gradient SDS-PAGE gel, together with the high molecular weight standard
proteins (3 µl, Amersham) 220, 97.4, 66, 46, 30, 21.5, and 14.3 kDa.
The samples were electrophoresed according to Laemmli (1970). After the
electrophoresis, the proteins were transferred to Hybond-C extra
nitrocellulose membrane (Amersham) by semidry blotting. The membrane
was blocked in 5% BSA in PBS for 1 hr in room temperature and then
incubated with the primary antibody against VEGF, 1:500 in 2% BSA in
PBS overnight at 4°C. After rinsing in PBS, the membrane was
incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG
(Bio-Rad, Hercules, CA) 1:1000 in PBS, for 1 hr at room temperature.
After rinsing in PBS, the blots were developed for 5 hr in a substrate
solution composed of 0.34 mM
5-bromo-4-chloroindolyl-phosphate, 0.36 mM nitro blue
tetrazodium chloride in 0.1 M
NaHCO3, and 1.0 mM
MgCl2, pH 9.8.
BrdU labeling. BrdU labeling was used to visualize the
presence of proliferating cells in cultured SCG and DRG. BrdU is a thymidine analog that is incorporated into DNA of cells in the S-phase
(Moran et al., 1985). The ganglia were cultured in Eppendorf tubes as
described above. SCG were cultured in the presence of 50 ng/ml of VEGF
and DRG in 100 ng/ml of the growth factor.
After 24 hr of culturing BrdU (Sigma, St. Louis, MO) was added to the
medium to a final concentration of 55 µM. After an
additional 24 hr culture period, the ganglia were washed in PBS three
times for 5 min each, fixed in 4% formalin in PBS for 3 hr, and washed in PBS. The ganglia were then cryoprotected and sectioned as described above.
The slides were processed for BrdU and S-100 double labeling as
previously described (Sondell et al., 1997). S-100 is a glial cell
protein (Hydén and McEwen, 1966; Li et al., 1997). The
preparations were studied in an Olympus (Tokyo, Japan) fluorescence
microscope, and for each section the number of BrdU-labeled
nuclei surrounded by S-100 immunoreactivity was counted.
Statistics. Student's t test was used to
calculate significant differences between VEGF-treated preparations and
their control with respect to axonal outgrowth, apoptosis, BrdU
labeling, section area, and signal transduction. Scheffe's
F test was used to compare the mean values of axonal
outgrowth from ganglia exposed to different concentrations of VEGF.
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RESULTS |
Axonal outgrowth
Axons from the SCG grew out from three principal sites, the
severed internal and external carotid nerves and the inlet of the
sympathetic trunk. SCG cultured in the presence of 10, 50, 100, or 200 ng/ml VEGF for 48 hr exhibited a significantly better axonal outgrowth
than their corresponding contralateral controls (Fig.
1). Maximal effect was observed in a
concentration range of 10-50 ng/ml. At higher concentrations, the
stimulatory effect of VEGF diminished. VEGF at 1, 10, 50, and 100 ng/ml
also increased axonal density i.e., the number of axons which grew out
from the explanted SCG (Table 1). Maximal
effect was observed at 50-100 ng/ml (Fig.
2a-e). The effects
of VEGF were distinctly different from that of NGF (50 ng/ml), which
induced a massive but shorter axonal outgrowth (Fig.
2c).
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Figure 1.
Axonal outgrowth from SCG at 48 hr. Values are
mean + SEM and obtained from three separate experiments. Six animals
were used per concentration tested, and nine measurements were
performed on each ganglia (n = 9 × 6). Five
animals were used at the concentration of 10 ng/ml
(n = 9 × 5). Student's t test
was used to calculate significant differences. *p < 0.05 compared with untreated contralateral ganglia.
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Figure 2.
SCG cultured 72 hr. a, SCG control.
b, SCG exposed to VEGF (50 ng/ml). Note the increase in
the number and density of long axons (arrows).
c, SCG cultured in the presence of NGF (50 ng/ml). The
high density of axons is obvious, but there are only few long axons.
d, SCG control, 6 d in culture, axons with growth
cones emerging from the Matrigel. e, SCG exposed to VEGF
(50 ng/ml), axons at the same distance from the ganglia as in the
control. Note the density of axons. To be able to illustrate both
ganglia and axons, the area surrounding the ganglia was subjected to
enhancement of contrast in Adobe Photoshop 3.0. In
a-c, the ganglia were photographed in an
inverted phase-contrast microscope using the 10× phase-contrast ring
to the 4× objective, giving rise to a dark-field effect. In
d and e, conventional phase-contrast
microscopy was used. Scale bars: a, 300 µm;
d, 70 µm.
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The axonal outgrowth from DRG occurred mainly from the cut ends of the
dorsal root and the sciatic nerve. Cultures exposed to 30, 50, and 100 ng/ml showed significantly longer axons than their contralateral
untreated counterparts (Fig. 3). Maximal
effect was observed at 100 ng/ml VEGF (Fig.
4a,b). As in the
case of SCG, higher concentrations of VEGF had a less stimulatory
effect. Thus, at 200 ng/ml the length of axons was significantly
shorter than at 100 ng/ml.
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Figure 3.
Axonal outgrowth from DRG at 48 hr. Values are
mean + SEM. For this study six animals were used per concentration
tested, and six measurements were performed on each ganglia. The
experiment was repeated three times (n = 6 × 6), except for the concentrations 5 and 200 ng/ml, which were repeated
twice with ganglia from five animals (n = 6 × 5). Student's t test was used to calculate significant
differences. *p < 0.05 compared with untreated
contralateral ganglia.
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Figure 4.
DRG cultured for 72 hr. a, DRG
control. b, DRG exposed to VEGF (100 ng/ml). Note the
increase in the number of long axons. c, DRG cultured in
the presence of NGF (50 ng/ml). The high density of axons is obvious.
Scale bar, 150 µm.
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In general, axonal outgrowth from the DRG appeared scattered and was
less profound than that from the SCG. VEGF did not affect the number of
axons growing out from the DRG (Table 1) at any of the concentrations
tested. In the DRG, NGF (50 ng/ml) induced a denser axonal outgrowth
than did VEGF (Fig. 4c).
To test the effect of VEGF in combination with NGF, a lower
concentration of NGF (1 ng/ml) was used together with 50 and 100 ng of
VEGF per milliliter for SCG and DRG, respectively. Cultures exposed to
both VEGF and NGF showed a significantly longer axonal outgrowth than
cultures exposed to either NGF or VEGF alone (Fig. 5).
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Figure 5.
The effect of 1 ng/ml -NGF on axonal outgrowth
from SCG and DRG cultured 48 hr in the presence or absence of VEGF.
Values are mean + SEM. The experiment was repeated twice. Ganglia from
five animals were used in each group, and nine measurements were
performed on each SCG (n = 9 × 5) and six on
each DRG (n = 6 × 5). Student's
t test was used to calculate significant differences.
*p < 0.05. The SEM for the control and the VEGF
bars is too small to be visible in the figure.
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Non-neuronal cells could be noticed in the close vicinity of the cut
ends of the ganglia first after 72 hr. No interference with axonal
outgrowth and density evaluation occurred because the evaluation was
performed at 48 hr.
Cell death
TUNEL staining for dead cells in cultured SCG (Fig.
6) and DRG reveals a significant
difference between VEGF-treated ganglia and their corresponding
controls for both nerve cells and satellite/Schwann cells (Fig.
7a,b). In SCG
exposed to 50 ng/ml and DRG to 100 ng/ml VEGF, the number of
TUNEL-positive nerve cells was reduced to 57 and 60% of the control
values. The corresponding values for the number of satellite/Schwann
cells was a reduction to 69 and 80% of the control values in the
respective ganglia.
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Figure 6.
TUNEL-positive nuclei in SCG cultured 48 hr,
control. Arrows point to nuclei of nerve cells, and
arrowheads point to satellite cells. Scale bar, 15 µm.
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Figure 7.
a, Number of TUNEL-positive nerve
cells and satellite/Schwann cells in SCG cultured in the presence of 50 ng/ml VEGF. Values are mean + SEM obtained from two separate
experiments. Ganglia from five animals were used, and counting was
performed on 22 sections. *p < 0.05 compared with
untreated contralateral ganglia. b, Number of
TUNEL-positive nerve cells and satellite/Schwann cells in DRG cultured
in the presence of 100 ng/ml VEGF. Values are mean + SEM from two
independent experiments. Four animals were used, and counting was
performed on 18 sections. Student's t test was used to
calculate significant differences. *p < 0.05 compared with untreated contralateral ganglia. No significant
difference of area was found between experiment and control for either
SCG or DRG. The SEM for the nerve cells is too small to be visible in
the figures.
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The analysis of section areas revealed no significant difference
between experiment and control for either SCG or DRG.
VEGF staining
In freshly dissected SCG, virtually all nerve cells were
immunoreactive to VEGF (Fig.
8a,b). In contrast,
in DRG only small neurons, here defined as neuronal profiles containing
a nucleus and with a diameter <35 µm, exhibited VEGF
immunoreactivity (Fig. 8c). The number of
VEGF-immunoreactive cells constituted 32.2 ± 3.7% (mean ± SD, n = 15) of DRG neurons. In both types of ganglia the blood vessels were VEGF-immunoreactive. Ceramide staining of the
Golgi apparatus (Fig. 8d) exhibited a fluorescent staining pattern similar to the VEGF staining.
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Figure 8.
a, VEGF-positive cells in SCG. Note
the Golgi-like distribution of VEGF (arrow).
Arrowheads are pointing to blood vessels.
b, SCG control, using preabsorbed primary antibody. No
immunoreactive cells are visible. c, VEGF-positive cells
in DRG. Counting VEGF-positive cells and total number of neuronal
profiles on DRG was performed on 15 sections from three animals. The
arrows indicate neurons containing VEGF
immunoreactivity, and arrowheads are pointing to blood
vessels. Scale bar, 30 µm. d, Ceramide staining of the
Golgi apparatus (arrow) in neurons in SCG. Scale
bar, 30 µm.
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Receptor and signal transduction
On sections of freshly dissected SCG and DRG, flk-1 receptor
immunoreactivity was localized to the cell membranes of neurons, although to a different extent. Although a few SCG neurons possessed the receptor (data not shown), virtually all neurons in the DRG exhibited flk-1 immunoreactivity (Fig.
9a). Faint staining was also
found in cell nuclei. No immunoreactivity was observed in the satellite
cells surrounding the neurons. In whole-mount preparation on glass
coverslips, most axons and growth cones of both SCG and DRG showed
flk-1 immunoreactivity (Fig. 9b). Teased nerve fibers of the
sciatic nerve revealed the presence of flk-1 immunoreactivity on the
membranes of Schwann cell and around the Schwann cell nuclei (Fig.
9c). It was also found on blood vessels.
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Figure 9.
a, The VEGF receptor flk-1
(arrowheads) in DRG. b, Flk-1
immunoreactivity localized to the growth cones (arrows)
of regenerating axons of SCG and DRG 6 d in culture.
c, Flk-1 staining of Schwann cells in teased
preparation. The arrows indicate flk-1 immunoreactivity
around the cell nuclei of Schwann cells, and arrowheads
point to flk-1 positive staining at the nodes of Ranvier. Scale bar, 35 µm.
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The protein kinase inhibitor K252a at a concentration (50 nM) that inhibited trk tyrosine kinases (Ohmichi et al.,
1992) and effectively suppressed NGF induced axonal outgrowth (data not shown) did not affect the VEGF-induced axonal outgrowth (Fig. 10). K252a alone had no significant
effect on axonal outgrowth.
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Figure 10.
The effect of 50 nM K252a on axonal
outgrowth from SCG and DRG cultured 48 hr in the presence or absence of
VEGF. Control and all groups contained DMSO in corresponding amounts as
used to dissolve K252a. Values are mean + SEM of two independent
experiments. Ganglia from five animals were used in each group, and
nine measurements were performed on each SCG (n = 9 × 5) and six on each DRG (n = 6 × 5).
Student's t test was used to calculate significant
differences. No significant difference in outgrowth length was found
between the ganglia treated with VEGF and those treated with a
combination of VEGF and K252a.
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Addition of PD 98059 (50 µM), an MAPK kinase inhibitor,
to cultures of SCG and DRG exposed to 50 and 100 ng/ml VEGF,
respectively, blocked the VEGF-induced outgrowth of axons (Fig.
11). PD 98059 alone had no significant
effect on axonal outgrowth.
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Figure 11.
The effect of 50 µM PD 98059 on
axonal outgrowth from SCG and DRG cultured 48 hr in the presence or
absence of VEGF. Control and all groups contained DMSO in corresponding
amounts as used to dissolve PD 98059. Values are mean + SEM. The
experiment was repeated at two different occasions. Ganglia from four
animals were used in each group, and nine measurements were performed
on each SCG (n = 9 × 4) and six on each DRG
(n = 6 × 4). Student's t test
was used to calculate significant differences. *Significantly different
from the VEGF bar, *p < 0.05.
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Electrophoresis and Western blotting
Figure 12 shows the
VEGF-immunoreactive proteins bands of homogenates of SCG, DRG, and pure
recombinant VEGF165 after electrophoresis and blotting.
Human recombinant VEGF165 appeared at molecular weights of
37 and 18 kDa. The lanes containing proteins from mouse SCG and DRG
revealed specific staining at 36, 33, and a barely visible band at 31 kDa.
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Figure 12.
VEGF immunoreactivity on Western blots of
electrophoretically separated recombinant VEGF165 and
homogenates of SCG and DRG. Molecular weights to the left in
kilodaltons. Lane 1, 80 ng of recombinant
VEGF165. Lane 2, 160 ng of recombinant
VEGF165. Lanes 3, 4, DRG.
Lanes 5, 6, SCG. Similar results were
obtained in two additional experiments.
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BrdU labeling
In SCG BrdU-positive nuclei were observed only in axon-containing
areas except for occasionally labeled cells in the perineurium (data
not shown). The number of BrdU-positive nuclei in ganglia exposed to 50 ng/ml VEGF was significantly larger than in the contralateral control
ganglia (Table 2). Immunostaining for
S-100 protein showed that these nuclei belonged to Schwann cells. The same results were obtained for DRG exposed to VEGF (100 ng/ml). Analysis of section areas showed no significant difference between experiment and control for either SCG or DRG.
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DISCUSSION |
The major findings in this study were that VEGF, assumed to
specifically affect endothelial cells, is present in the neurons of SCG
and DRG and that addition of VEGF to explanted ganglia stimulates
axonal outgrowth, nerve and satellite/Schwann cell survival, and
proliferation of Schwann cells. We also found the flk-1 receptor on
neurons and Schwann cells. We therefore conclude that VEGF has both
neurotrophic and mitogenic activity on cells in the peripheral nervous system.
The immunocytochemistry revealed a Golgi-like distribution of VEGF,
i.e., an association of the protein with the synthetic machinery of the
cell body, implying that VEGF is synthesized by the nerve cells and
that the immunoreactivity does not represent VEGF that has reached the
cell bodies by retrograde transport before explantation of the ganglia.
Interestingly, not all the neurons of the DRG exhibited VEGF
immunoreactivity. The positive cells were small and constituted ~30%
of the neurons. Such a cellular distribution is characteristic also for
neurons that express the high-affinity NGF (TrkA) receptor and
calcitonin gene-related peptide (Averill et al., 1995; Fagan et
al., 1996). It is therefore tempting to speculate that VEGF is
expressed by the TrkA-positive neurons. This speculation is further
supported by our finding that low concentration of NGF had an additive
effect together with VEGF on axonal outgrowth from both SCG and DRG.
At least four different isoforms of VEGF have been found in murines,
and in rat brain VEGF164 dominates (Bacic et al., 1995). The immunoblotting experiments showed two VEGF-immunoreactive bands
with slightly lower molecular weights than those of the recombinant
VEGF165. The resolution of this experiment, aimed at
characterization of the antibody rather than separation of isoforms,
was not sufficient to allow conclusions with respect to the most
prevalent form of VEGF in the mouse DRG and SCG. However, murine VEGF
has lower molecular weight than its human counterparts, which is
consistent with the present results. Furthermore, VEGF can be
glycosylated at several sites, a factor known to affect the
electrophoretic mobility in SDS-PAGE.
VEGF exerts its action via high-affinity binding to phosphotyrosine
kinase receptors; fms-like tyrosine kinase (flt-1) and fetal liver
kinase receptor (flk-1) (De Vries et al., 1992; Quinn et al., 1993).
However, neither neurons nor glial cells appear to possess the flt-1
receptor (Peters et al., 1993). In the present experiments, effects on
axonal outgrowth and cell survival were observed at physiological
concentrations i.e., in the range of the Kd for the flk-1
receptor (Terman et al., 1992; Waltenberger et al., 1994).
Immunocytochemistry revealed the presence of flk-1 on both nerve cell
bodies, axons, and growth cones. Flk-1 immunoreactivity was present on
more neurons in the DRG than in SCG. However, in cultures grown on
glass coverslips, there was no obvious difference in axonal staining of
flk-1 between the ganglia. Taken together our results suggest that the
effects of VEGF are mediated through this receptor. Still, we cannot
rule out the possibility that VEGF interacts with other receptors or
acts indirectly through non-neuronal cells that might release
neurotrophic factors. Such reciprocal cell-cell interaction has been
reported for NT-3/neuregulin, acting on sympathetic neuroblasts and
non-neuronal cells (Verdi et al., 1996). If indirect mechanisms of
actions are considered, we can at least exclude the involvement of trk
receptors because K252a failed to affect the VEGF response. It has also
been reported that VEGF165 binds to neurophilin-1 (Soker et
al., 1998), a neuronal cell surface molecule that is important for
chemorepulsive signaling during peripheral axonal outgrowth in the
developing mice (Kitsukawa et al., 1997). Still, our findings suggest
that VEGF acts via autocrine and/or paracrine mechanisms on neurons
because the receptor and the protein may be present on the same cell.
For axonal outgrowth, we observed a bell shaped dose- response curve
for both SCG and DRG. Such a dose-response has also been noted for
other growth factors, including bFGF and NGF, acting on PC12 cells or
DRG neurons (Rydel and Greene, 1987; Conti et al., 1997; Rutishauser
and Edelman, 1980). The explanation in the case of NGF is that at high
concentrations, the axons come close enough to interact and the
outgrowth is inhibited in favor of fasciculation. However, in our
experiment where no drastic differences in axon density was found with
raised concentrations of VEGF, such an effect appears unlikely. Another
explanation could be that external addition of higher concentrations of
VEGF to the cultured ganglia lead to a downregulation of the flk-1 receptor.
The effects of VEGF were distinctly different from those of NGF. NGF
also appeared far more efficient than VEGF when the number of
regenerating axons was considered. On the other hand, VEGF treatment
promoted survival of both neurons and satellite/Schwann cells in the
adult ganglia, which NGF does not (Edström et al., 1996; our
unpublished observations). However, one must proceed with caution when
these types of comparisons are made because we do not know the
intraganglionic concentrations of neither NGF nor VEGF. Whereas the
former is mainly target-derived, the present findings suggest that VEGF
is produced within ganglia. Furthermore, the Matrigel used in our
experiments contains a variety of growth factors, including NGF, but
also heparin which binds VEGF, hampering exact estimations and
comparisons of potency.
In experiments using endothelial cells overexpressing the human
counterpart to mouse flk-1, VEGF acts via stimulation of the MAPK
pathway (Kroll and Waltenberger, 1997). In the present experiment, we
could block VEGF-induced axonal outgrowth by adding PD 98059, suggesting that in the ganglia VEGF acts through the MAPK pathway.
The present findings raise several questions with respect to the role
of VEGF in the peripheral nervous system. For instance, what functions
does VEGF have for the normal metabolism of the neurons within the
ganglia? We found VEGF in the neurons of both SCG and DRG, and we found
that both ganglia possess receptors for VEGF. One possibility is that
VEGF has a role similar to that observed for brain-derived neurotrophic
factor, which in DRG has been suggested to be an autocrine
survival factor (Acheson et al., 1995). It could also be anticipated
that VEGF by virtue of its stimulatory action on axonal outgrowth,
Schwann cell proliferation, and angiogenesis plays an important role in
the response to nerve injury.
| |
FOOTNOTES |
Received Dec. 14, 1998; revised April 19, 1999; accepted April 27, 1999.
This work was supported by grants from the Swedish Medical and Natural
Science Research Councils and the foundations of Åke Wiberg,
Hierta-Retzius, and Crafoord. We thank Marie Adler-Maihofer and Inger
Antonsson for expert technical assistance.
Correspondence should be addressed to Mariann Sondell, Department of
Animal Physiology, University of Lund, Helgonav. 3B, S-223 62 Lund, Sweden.
| |
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J. M. Rosenstein, N. Mani, A. Khaibullina, and J. M. Krum
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S. Ishida, T. Usui, K. Yamashiro, Y. Kaji, E. Ahmed, K. G. Carrasquillo, S. Amano, T. Hida, Y. Oguchi, and A. P. Adamis
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H. Brown
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A. Wick, W. Wick, J. Waltenberger, M. Weller, J. Dichgans, and J. B. Schulz
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TOP
ABSTRACT
INTRODUCTION
