The Role of VEGF and VEGFR2/Flk1 in Proliferation of
Retinal Progenitor Cells in Murine Retinal Degeneration
Koji M. Nishiguchi, Makoto Nakamura, Hiroki Kaneko, Shu Kachi, and Hiroko Terasaki
PURPOSE. To analyze the role of VEGF and its receptors,
VEGFR2/Flk1 and VEGFR1/Flt1, on retinal progenitor cells
(RPCs) in a murine model of inherited retinal degeneration
(rd1 mice).
METHODS. After proliferating RPCs in the retina of rd1 mice
were labeled with bromodeoxyuridine (BrdU), expressions of
VEGFR2/Flk1 and VEGFR1/Flt1 were immunohistochemically
analyzed. To examine its effect on the proliferation of BrdUpositive RPCs in rd1 mice, VEGF was administered into retinal
culture medium with or without blocking agents against
VEGFR2/Flk1 or VEGFR1/Flt1 in vitro or injected into vitreous
cavity in vivo.
RESULTS. BrdU-labeled RPCs in rd1 mice expressed VEGFR2/
Flk1 but not VEGFR1/Flt1. These cells later expressed retinal
neuronal markers such as Pax6 and rhodopsin. Exposure of the
retinas from postnatal day (P) 9 rd1 mice to VEGF increased
the number of proliferating RPCs by 61% in vitro. This effect
was blocked by concomitant administration of VEGFR2/Flk1
kinase inhibitor. In vivo, a single intravitreal injection of VEGF
in rd1 mice at P9 increased by 138% the number of RPCs and
cells that developed from RPCs in the peripheral retina at P18.
CONCLUSIONS. VEGF stimulates the proliferation of RPCs
through VEGFR2/Flk1 in rd1 mice. The observed proliferation
of RPCs that have the potential to differentiate into retinal
neurons may enhance the regeneration of the degenerating
retina. (Invest Ophthalmol Vis Sci. 2007;48:4315– 4320) DOI:
10.1167/iovs.07-0354
V
ascular endothelial growth factor (VEGF) is a potent
growth factor known to play a major role in the formation
and maintenance of vascular structures.1,2 These functions are
mediated through two of its tyrosine kinase receptors,
VEGFR1/Flt1 and VEGFR2/Flk1, both expressed on vascular
endothelial cells.3 However, VEGF has recently been reported
to be an important signaling molecule for neuroprotection and
neurogenesis.4 –10 For example, reduced expression of VEGF in
mice by a targeted deletion in the promoter region of the VEGF
gene resulted in neurodegeneration resembling amyotrophic
lateral sclerosis (ALS), a neurodegenerative disease affecting
the motoneurons in the brain and spinal cord.7,8 Similarly, in
From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan
(Grant C16591746 [MN]; Grants B16390497 and B18390466 [HT]) and
a Grant-in-Aid from the Ministry of Health, Labor, and Welfare of Japan,
Tokyo, Japan (HT).
Submitted for publication March 24, 2007; revised April 23, 2007;
accepted June 25, 2007.
Disclosure: K.M. Nishiguchi, None; M. Nakamura, None; H.
Kaneko, None; S. Kachi, None; H. Terasaki, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Koji M. Nishiguchi, Department of Ophthalmology, Nagoya University Graduate School of Medicine, 65 Tsuruma,
Showa-ku, Nagoya 466-8550, Japan; kojinish@med.nagoya-u.ac.jp.
Investigative Ophthalmology & Visual Science, September 2007, Vol. 48, No. 9
Copyright © Association for Research in Vision and Ophthalmology
humans, reduction in VEGF expression increases the risk for
ALS.7,8 On the other hand, VEGF injected into the central
nervous system through the ventricles in rodent models of ALS
proved one of the most effective treatments for this disease.9,10
In addition, in vitro and in vivo analyses have suggested that
VEGF can stimulate the proliferation of neural stem/progenitor
cells in the brain, including those from the cerebral cortex and
the hippocampus. This proliferation was mediated through
VEGFR2/Flk1 signaling.11–14
In the eye, VEGF plays a significant role in the normal
development of the retinal and choroidal vascular systems and
in pathologic changes15,16 such as diabetic retinopathy,17,18
choroidal revascularization in age-related macular degeneration,19 and retinopathy of prematurity.3,20,21 Treatments
aimed at blocking VEGF signaling to overcome these diseases are under evaluation in humans and have already led to
promising results in several forms of retinal and choroidal
vascular diseases.22–26
Although the importance of VEGF in neurogenesis of developing retinas in wild-type rats27 and chickens28 has been reported, the role of VEGF in eyes with retinal diseases other
than the vasculopathy remains unclear. In particular, it is unclear whether VEGF has the potential to prevent retinal degeneration by protecting retinal neurons against apoptosis or by
stimulating the proliferation of retinal progenitor cells (RPCs).
To analyze the role of RPCs and the effect of VEGF on RPCs,
we studied the rd1 mouse, which has an inherited retinal
degeneration, as a model of human hereditary retinal degeneration. During the early period after birth, the normal mouse
retina is morphologically immature, and RPCs form a neuroblast layer. By postnatal day (P) 9, the retina differentiates to
form a highly complex structure with three distinct neural
layers. In rd1 mice, however, rod photoreceptors degenerate
rapidly between P9 and P21; this degeneration is followed by
a slower loss of cone photoreceptors.29 Here we show that
proliferating RPCs are capable of differentiating into cells that
express retinal neural markers in postnatal rd1 mouse. VEGF
stimulates the proliferation of these RPCs through VEGFR2/
Flk1 in vitro. Moreover, a single intravitreal injection of recombinant VEGF in young rd1 mice promotes the proliferation of
RPCs and results in an increased number of these cells or cells
that developed from RPCs in the peripheral retina. These
suggest that VEGF may have the potential to stimulate regeneration of the retinal neurons in the degenerating retina.
METHODS
Animals
We used the C3H/HeJ strain of mice, homozygous for the rd1 mutation, as a murine model of an inherited retinal degeneration.30,31 The
C57BL/6J strain of mice was used as the wild-type control. Mice were
kept on a 12-hour light/12-hour dark cycle. All experimental procedures adhered to the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research and the guidelines for the use of
animals at Nagoya University School of Medicine.
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Nishiguchi et al.
Immunohistochemical Analyses
To label cells that are in the S-phase of the cell cycle, mice were
injected once intraperitoneally with BrdU (150 mg/kg) and were killed
after periods ranging from 2 hours to 21 days. Corneas were removed
from the enucleated eyes, and the eyecups were fixed in 4% paraformaldehyde (PFA) for 2 hours at room temperature followed by cryoprotection in 30% sucrose overnight at 4°C. We embedded the eyecups
in OCT compound (Tissue-Tek; Sakura Finetek, Tokyo, Japan) and cut
12-␮m frozen sections through the dorsal to ventral meridian.
Sections were stained with primary antibodies for rhodopsin (1:
1500; Chemicon, Temecula, CA), VEGFR1/Flt1 (1:30; R&D Systems,
Minneapolis, MN), VEGFR2/Flk1 (1:30; R&D Systems), Pax 6 (1:1000;
Developmental Studies Hybridoma Bank, Iowa City, IA), or BrdU (1:
1000 [Developmental Studies Hybridoma Bank] and 1:1000 [Oxford
Biotechnology, Raleigh, NC]), followed by Alexa 488-, 568-, or 647conjugated secondary antibodies (all at 1:1500; Molecular Probes,
Eugene, OR). A TUNEL assay kit (Roche, Basel, Switzerland) was used,
in accordance with to the manufacturer’s instructions, to detect apoptotic cells.
Next the flat mount specimens were subjected to BrdU-staining, as
follows: after the PFA-fixed eyecups were flattened with radial incision,
they were permeabilized in 0.5% phosphate-buffered saline Triton
X-100 (PBST) for 2 hours, incubated in 2 N HCl for 45 minutes, and
neutralized with 0.1 M Na2B4O7 for 15 minutes. After blocking with 5%
goat serum in PBS for 1 hour, the eyecups were incubated with
anti–BrdU antibodies for 12 hours and with secondary antibody for 9
hours.
Retinal Explant Culture
Retinal explants were cultured as described in detail32 with slight
modification. Briefly, the retina was peeled from the RPE of P9 eyes
under an ophthalmic surgical microscope. Four radial incisions were
made to flatten the retina, and the retina was placed on a chamber filter
(Millicell; Millipore, Bedford, MA) with the photoreceptor side up. The
chamber was placed in a culture plate with 1 mL culture medium that
contained 50% minimal essential medium with HEPES (Gibco, Grand
Island, NY), 25% Hanks balanced salt solution (Gibco), 25% heatinactivated horse serum (Gibco), antibiotics mixture (25 U/mL penicillin, 25 ␮g/mL streptomycin, 62.5 ng/mL amphotericin B; Gibco),
and 200 ␮M L-glutamine (Sigma). This medium was supplemented with
0 to 20 ng/mL VEGF (PeproTech, Rocky Hill, NJ), 0 to 100 ng/mL
anti–VEGFR1/Flt1-neutralizing antibody (R&D Systems), or 0 to 100
nmol/mL SU1498, a potent inhibitor of VEGFR2/Flk1 kinase (Calbiochem, La Jolla, CA). Cultures were maintained at 34°C in 5% CO2. The
medium was changed 24 and 72 hours after the culture was begun, and
BrdU (50 ␮g/mL) was added at 96 hours. Culturing of the retinal
explant was stopped at 120 hours, and the retinas were fixed, cryoprotected, frozen, and sectioned as described. We analyzed five retinas
from five animals for the different combinations of VEGF and SU1498
concentrations. BrdU- or TUNEL-positive cells in 512 ␮m of the outer
nuclear layer (ONL) were counted with the counter masked to the
composition of the culture media. Counts were performed from three
different areas of each retina and were averaged. One-way ANOVA
followed by the Dunnett post hoc test for multiple comparisons was
performed to determine the significance of any differences. All experiments were conducted in duplicate.
Measurement of Retinal VEGF
Wild-type controls and rd1 mice were killed at P6, P9, P12, P15, and
P18. Their eyes were enucleated and immediately placed in PBS on ice.
Retinas were peeled from the retinal pigment epithelium (RPE), placed
in 150 ␮L SDS lysis buffer, sonicated, and centrifuged at 14,000 rpm for
10 minutes at 4°C. Then the level of VEGF in the supernatant of each
retina was determined using a mouse ELISA kit (R&D Systems) according to the manufacturer’s instructions. At least four retinas from four
animals of each age group were studied. Student’s t-test was used for
statistical analysis.
IOVS, September 2007, Vol. 48, No. 9
Intravitreal Injection and Counting of
BrdU-Positive Cells
In P9 rd1 mice, 50 ng human recombinant VEGF (PeproTech) in 0.5
␮L PBS was injected into the right eye, and 0.5 ␮L PBS was injected
into the left. Injections were performed with a 33-gauge needle
through the corneal limbus into the vitreous cavity under an ophthalmic surgical microscope. At P12, the mice were injected intraperitoneally with BrdU (150 mg/kg). At P30, the mice were killed immediately after fundus examination under a microscope to exclude the eyes
with retinal detachment, cataract, or opaque vitreous. After the eyes
were histologically processed into cilioretinal flat mounts, the specimens were stained for BrdU as described. Digital images of the dorsal
and ventral cilioretinal margins containing the peripheral retina and
the entire ciliary body were taken from each flat mount specimen.
Numbers of BrdU-positive cells were determined by averaging the
counts from dorsal and ventral images, obtained with the operator
masked to the previous treatment. Student’s paired t-test was used for
statistical analysis. All experiments were conducted in duplicate.
RESULTS
RPCs Differentiate into Cells Committed to Rod
Photoreceptor Lineages in rd1 Mice
In wild-type rodents, RPCs are capable of differentiating into
rhodopsin-positive cells27,33,34 presumably destined to become
rod photoreceptors. To determine whether postnatal RPCs in
mice that experience retinal degeneration are similarly capable
of differentiating into a photoreceptor lineage, we analyzed the
fate of the cells labeled with BrdU at P6 by histologic evaluation at P18.
At P6, the retina is still developing, and BrdU-positive cells
that are in the S-phase of the cell cycle form a layer of neuroblasts at the peripheral retina. Later at P18, when the retina is
degenerating, the cells that incorporated BrdU also express
Pax6 (a marker for multipotent RPCs35) or rhodopsin (a marker
for rod photoreceptors; Fig. 1). Even if these dividing cells
were labeled with BrdU at P12, when the neuroblast layer
disappeared and the retina developed into three neuronal layers, a small number of BrdU-positive cells still expressed these
markers at P18 in the peripheral retina (data not shown). We
failed to find BrdU-positive cells that developed into cells of
cone photoreceptor lineage in the retina. However, rare BrdUpositive cells that expressed a marker for a subset of cone
photoreceptors, M-cone opsin, were identified in the adjacent
ciliary body (data not shown). These findings suggest that
BrdU-positive cells in rd1 mouse retina are RPCs capable of
differentiating into the rod photoreceptor lineage.
RPCs Express VEGFR2/Flk1
In the mammalian brain, VEGF participates in neurogenesis by
stimulating the proliferation of neural stem/progenitor cells
through VEGFR2/Flk1.11–14 An earlier study on dissociated
retinal cells from wild-type mice retinas showed that VEGFR2/
Flk1 is expressed on BrdU-positive RPCs.36 To determine
whether this was also true of RPCs in rd1 mice, we examined
the in situ expression of two ligands of VEGF, VEGFR1/Flt1 and
VEGFR2/Flk1, in P6 rd1 mice. RPCs were labeled with BrdU 2
hours before the eyes were removed. As expected, BrdUpositive RPCs showed staining for VEGFR2/Flk1. BrdU incorporation and VEGFR2/Flk1 expression were most pronounced
in RPCs at the peripheral retina (Fig. 2); remaining cells in the
neuroblast layer expressed these markers to a lesser degree.
We also identified cells that were positive for BrdU but negative for VEGFR2/Flk1 or vice versa. On the other hand,
VEGFR1/Flt1 showed a staining pattern not related to the
IOVS, September 2007, Vol. 48, No. 9
RPC Proliferation by VEGF in rd1 Mouse
4317
FIGURE 1. BrdU-positive RPCs differentiate into cells that express photoreceptor markers in rd1 mouse.
rd1 mice were injected with BrdU at
P6 and were humanely killed for histologic analyses at P18. P6-labeled
BrdU-positive RPCs expressed Pax6
(a marker for multipotent RPCs; upper row) or rhodopsin (a marker for
rod photoreceptors; lower row) by
P18. ONL, outer nuclear layer; INL,
inner nuclear layer.
BrdU-positive cells; the rare BrdU-positive cells with VEGFR1/
Flt1 expression were vascular endothelial cells (Fig. 2).
These BrdU-positive cells were negative for TUNEL staining
(data not shown), indicating that BrdU incorporation does not
represent DNA synthesis associated with apoptosis.37
VEGFR2/Flk1 Mediates Retinal Stem Cell
Proliferation In Vitro
To determine whether the level of VEGF expression in rd1
mouse retinas has any impact on retinal neurogenesis (proliferation of RPCs) or on neuroprotection (decrease in photoreceptors apoptosis), we analyzed the effect of different concentrations of VEGF in the culture media on BrdU incorporation
and apoptosis of the photoreceptors in retinal explants.
The number of BrdU-positive cells in the ONL increased
with the addition of VEGF for 6 days, and we observed a
F IGURE 2. VEGFR2/Flk1, but not
VEGFR1/Flt1, is expressed by BrdUpositive RPCs in the peripheral retina
of P6 rd1 mouse. rd1 mice were injected with BrdU at P6 and were killed
for histologic analyses 2 hours later.
Although VEGFR1/Flt1 showed an expression pattern not associated with
BrdU-positive cells (upper row; few
BrdU-positive cells with VEGFR1/Flt1
expression represent vascular endothelial cells), VEGFR2/Flk1 was localized with BrdU-positive RPCs (lower
row). NBL, neuroblast layer.
maximal increase of these cells by 61.2% at a concentration of
10 ng/mL (P ⬍ 0.05; Fig. 3A). On the other hand, the number
of TUNEL-positive cells in the ONL was not altered by the
VEGF (Fig. 3A). This experiment was repeated once, with
similar results (data not shown). These findings indicated that
VEGF can stimulate neurogenesis in vitro, but it did not protect
neurons from apoptosis under our experimental conditions.
We then studied the role of the two VEGF receptors using
this in vitro retinal explant by adding different concentrations
of SU1498, an agent that blocks the kinase activity of VEGFR2/
Flk1, or by adding anti–VEGFR1/Flt1-neutralizing antibodies to
the culture medium containing 10 ng/mL VEGF. The number
of BrdU-positive RPCs in the ONL was decreased with exposure to SU1498 at a concentration of 10 to 20 nmol/mL (P ⬍
0.01; Fig. 3B), whereas exposure to anti–VEGFR1-neutralizing
antibodies under similar experimental conditions did not alter
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IOVS, September 2007, Vol. 48, No. 9
FIGURE 4. VEGF expression is reduced after P12 in the rd1 mouse
retina. The amount of VEGF expressed per retina was measured from
rd1 mice (n ⫽ 4) and wild-type mice (n ⫽ 4 or 6) with ELISA. A
significant reduction of VEGF expression is observed only after P12
(P ⬍ 0.001– 0.01). Bars represent the mean (⫾ SEM).
FIGURE 3. VEGF stimulates VEGFR2/Flk1 to enhance the proliferation
of BrdU-positive RPCs in retinal explants from rd1 mice. (A) The
addition of VEGF to the culture medium of 10 ng/mL for 6 days
resulted in an increase in the number of BrdU-positive RPCs in the ONL
(left; P ⬍ 0.05), with no detectable difference in the number of
TUNEL-positive apoptotic photoreceptors (right). (B) The addition of
SU1498 (10 –20 nmol/mL), a VEGFR2/Flk1 inhibitor, to the culture
medium containing VEGF (10 ng/mL) resulted in a decrease of BrdUpositive RPCs in the ONL (left; P ⬍ 0.01), with no effect in the number
of TUNEL-positive apoptotic photoreceptors (right). Bars represent
the mean (⫾ SEM).
the number (data not shown). Blocking either VEGFR1/Flt1 or
VEGFR2/Flk1 activity had no effect on photoreceptor apoptosis (data not shown and Fig. 3B, respectively).
These results, together with the finding that only VEGFR2/
Flk1 is expressed in RPCs, indicate that the neurogenic potential of VEGF is mediated by VEGFR2/Flk1 but not VEGFR1/Flt1
in the rd1 mouse retina. Our findings are similar to the reports
for neural stem/progenitor cells in the brain.11–14
or would protect retinal neurons from degenerating, similar to
its observed effect on the neurodegenerative disease in the
brain in vivo. A single 50-ng intravitreal injection of VEGF in 0.5
␮L PBS (right eye) or 0.5 ␮L PBS (left eye) was given at P9.
Proliferating RPCs were then labeled with BrdU at P12, after
the initiation of photoreceptor degeneration. The eyes from
these mice were later collected at P30 and studied histologically. Only minor portions of the eyes were included in the
study, especially those treated with VEGF, because of the high
rate of the induced retinal detachments.
Because few BrdU-positive proliferating RPCs were found
exclusively at the peripheral retina in the P12 histologic sections, which continued to decline in number (Fig. 5), we
analyzed flat mount specimens and focused on the RPCs at the
peripheral retina. As expected, the P12-labeled BrdU-positive
cells identified at P30, most likely either RPCs or cells that
developed from RPCs, were almost exclusively found in the
peripheral retina. The number of BrdU-positive cells in the peripheral retina was significantly increased by 138% in the VEGFtreated eyes compared with the contralateral eyes injected with
PBS (P ⫽ 3.9 ⫻ 10–3; Fig. 6). We also observed a significantly
increased number of BrdU-positive cells in the adjacent ciliary
body of eyes injected with VEGF (data not shown).
However, at P6 or P9, intravitreal injection of VEGF into rd1
mice did not show any evidence of neuroprotection of the
degenerating retina. In other words, no significant difference
VEGF Expression Is Reduced in Developing rd1
Mouse Retina
Reduced expression of VEGF is associated with neurodegeneration in the brains and spinal cords of mice and humans.7,8
To determine whether the level of VEGF is reduced in the
retina of rd1 mouse, we measured its expression in mice
between P6 and P18. From P6 to P9, when the morphology of
the retina was still normal, the VEGF level expressed in the
retinas of rd1 mice was comparable to that expressed in the
retinas of wild-type controls. However, at P12, after the rapid
photoreceptor degeneration had begun, the VEGF expression
level was 47.2% lower (P ⬍ 0.001) in rd1 retinas than in
wild-type retinas and remained lower thereafter (Fig. 4).
VEGF Stimulates Proliferation of RPCs In Vivo
Because VEGF has the potential to stimulate the proliferation of
RPCs in vitro and its expression was reduced in the rd1 mouse,
we sought to determine whether increasing the VEGF level in
the retina by an intravitreal injection of VEGF in rd1 mice
would also increase the number of RPCs, as in retinal cultures,
FIGURE 5. BrdU-positive RPCs are found exclusively in the peripheral
retina at P12 and continue to decrease in number at P30. (A) At P12,
a few BrdU-positive cells (filled arrowheads) were found exclusively at
the peripheral retina. (B) Number of BrdU-positive (mean ⫾ SEM) cells
in the peripheral retina at P12 (n ⫽ 8) and P30 (n ⫽ 4). Cor, cornea;
Scl, sclera; RPE, retinal pigment epithelium; CB, ciliary body.
IOVS, September 2007, Vol. 48, No. 9
FIGURE 6. A single intravitreal injection of VEGF at P9 increases the
number of RPCs labeled with BrdU at P12 in the peripheral retina of
rd1 mice at P18 in cilioretinal flatmounts. (A) Increased number of
BrdU-positive RPCs is observed in the peripheral retina of rd1 mouse
treated with VEGF compared with PBS at P18. (B) Number of BrdUpositive (mean ⫾ SEM) cells in the peripheral retina treated with VEGF
(n ⫽ 5) and PBS (n ⫽ 5). CB, ciliary body.
was found in the number of rhodopsin-positive cells in the rd1
retinas treated with VEGF and those treated with PBS. The
experiment was repeated once with similar results.
DISCUSSION
In mammals, previous studies have shown the significance of
VEGF to stimulate the proliferation of neural stem/progenitor
cells in the brain. Our results show that VEGF stimulates the
proliferation of RPCs through VEGFR2/Flk1 in vitro and further
provide in vivo evidence that VEGF promotes the proliferation
of RPCs in rd1 mice.
To determine the role of RPCs in rd1 mice, we studied the
expression of several neural markers on BrdU-positive cells to
track their fate after differentiation. In particular, few RPCs
labeled with BrdU at P6 developed into cells that expressed
rhodopsin, a marker specific for rod photoreceptors, at P18.
However, given that the rd1 mice carry genetic defects in rod
photoreceptor–specific Pde6b,30,31 the RPC-derived rhodopsin-positive cells may not ultimately contribute to functional
vision. Nonetheless, loss of rod photoreceptors induces secondary loss of cone photoreceptors in this mouse model29 and
in many forms of hereditary retinal degeneration in humans.38
Therefore, an effort to promote the regeneration of the rod
photoreceptors from these RPCs as a possible treatment of
these diseases is reasonable. Earlier studies have shown that
VEGFR2/Flk1 is expressed on BrdU-positive RPCs of dissociated retinal cells from wild-type mouse retinas and that VEGF
binds to these cells.36 In this study, we confirmed in in situ
RPC Proliferation by VEGF in rd1 Mouse
4319
preparations that VEGFR2/Flk1 is expressed on RPCs in rd1
mouse retinas. This expression was most pronounced in the
peripheral retina, where RPCs persist exclusively after retinal
development.
The ability of VEGF to stimulate the proliferation of RPCs
was confirmed in the in vitro analyses using retinal explants
from rd1 mice. The number of BrdU-positive RPCs increased
with the addition of VEGF to the culture medium. We observed
a maximal increase of 61.2% in the number of RPCs after the
addition of 10 ng/mL VEGF. A further increase in the VEGF
concentration did not increase the number of RPCs. These
results are in close agreement with the results of an in vitro
study that assessed the potential of VEGF to stimulate the
proliferation of neural stem/progenitor cells from the brain.11
In addition, the in vitro analyses indicated that VEGF acts
through VEGFR2/Flk1 expressed by RPCs to stimulate neurogenesis. This observation is in agreement with results from
neural stem/progenitor cells in the brain.11 However, we were
unable to detect any reduction in the rate of photoreceptor
apoptosis by adding VEGF to the culture medium. Therefore, it
appears that VEGF does not protect retinal neurons from apoptosis, at least with the in vitro culture and in vivo intravitreal
injection conditions and the rd1 mice used in the present
study.
In the murine brain and spinal cord, reduced expression of
VEGF is associated with ALS-like neurodegeneration.7,8 To determine whether VEGF is also reduced in the retina of rd1
mice, we measured its level of expression between P6 and P18.
From P6 to P9, when the rd1 retina is morphologically intact,
the level of VEGF expression in the retinas from rd1 mice was
comparable to that of wild-type controls. However, at P12,
when the photoreceptors are rapidly degenerating,29 VEGF
expression was significantly lower in the rd1 retinas and remained reduced thereafter. A simple explanation for this observation is that the reduction in VEGF expression resulted
from the decreased number of VEGF-expressing photoreceptors. However, given that VEGF is reported to be mainly expressed in the ganglion cell and inner nuclear layers in the
retina,39 the decrease in cell number caused by photoreceptor
degeneration may be insufficient to explain the observed degree of reduction in VEGF expression. In addition, because a
significant reduction in the expression of VEGF, a potent angiogenic factor, in the retina is likely to result in an antiangiogenic state, our results are also in agreement with the previous
report that in retinal degeneration, vessels respond poorly to
angiogenic stimulation.40
The importance of VEGF as a potential target for the treatment of neurodegeneration was recently highlighted by the
observation that an intraventricular injection of VEGF in a
rodent model of neurodegenerative disease was one of the
most effective treatments.9,10 These reports, together with the
results of our in vitro analyses, the coincidence of the reduced
VEGF expression and the progression of retinal degeneration,
prompted us to administer VEGF as a potential treatment for
retinal degeneration. Our results indicated that intravitreal administration of VEGF had no detectable therapeutic effect on
the progression of photoreceptor degeneration. However, we
found that a single intravitreal injection of VEGF in P9 rd1 mice
resulted in an increased number of RPCs or cells that developed from RPCs at P18, further validating that VEGF can
stimulate the proliferation of RPCs and may have the potential
to enhance retinal regeneration. However, the effect of VEGF
was modest considering the limited number of RPCs observed
exclusively in the peripheral retina and the larger degree of
photoreceptors lost throughout the retina. Nonetheless, these
results provide us with a rationale to continue our study of
VEGF and to investigate the mechanism for neural regeneration
as a treatment of retinal degeneration.
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Nishiguchi et al.
Based on the results of our experiments, we believe there is
only a minute concern on the use of anti–VEGF therapy in adult
patients, in whom presumably few, if any, retinal stem cells
exist at the peripheral retina. However, caution must be exercised regarding its application in children, especially before
their retinas are fully developed, because VEGF appears to
regulate retinal neurogenesis in various ways.27,28
Acknowledgments
The anti–Pax6 and anti–BrdU monoclonal antibodies were obtained
from the Developmental Studies Hybridoma Bank, developed under
the auspices of the National Institute of Child Health and Human
Development and maintained by the Department of Biological Sciences
at the University of Iowa (Iowa City, Iowa).
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