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Regular Articles |
§
From the Departments of Ophthalmology,*
Neuroscience,
and Molecular Biology and
Genetics,§
The Johns Hopkins University School
of Medicine, Baltimore, Maryland, and Department of Microbiology and
Kaplan Cancer Center,
New York University
School of Medicine, New York, New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, attention has focused on vascular endothelial cell growth factor (VEGF). It is up-regulated by hypoxia,4,5 and its levels are increased in the retina and vitreous of patients6-9 or laboratory animals10,11 with ischemic retinopathies. Increased expression of VEGF in retinal photoreceptors of transgenic mice stimulates neovascularization within the retina,12,13 and VEGF antagonists partially inhibit retinal or iris neovascularization in animal models.14-16
Despite the strong evidence implicating VEGF in retinal neovascularization, however, it is likely that VEGF does not act by itself. Rather, it appears to collaborate with other angiogenic factors in the stimulation of retinal neovascularization.17 This issue is important in terms of understanding the mechanism of new blood vessel growth in a particular vascular bed and also has relevance to the design of treatments. As noted above, VEGF antagonists can only partially inhibit experimental retinal neovascularization, and it is not known whether this is because of difficulties with delivery or potency of the antagonists or whether the residual activity is due to other angiogenic factors.
One factor that may work in combination with VEGF in stimulating retinal neovascularization in ischemic retinopathies is insulin-like growth factor-I (IGF-I). Mice with ischemic retinopathy that have low IGF-I levels due to antagonism of growth hormone have less retinal neovascularization than mice with normal IGF-I levels.17 Members of the fibroblast growth factor (FGF) family, particularly basic FGF or FGF2, have also been implicated. In fact, FGF2 has been a candidate retinal angiogenesis factor longer than VEGF, and there have been many studies investigating its possible role in retinal neovascularization.18 As with VEGF, FGF2 levels are increased in the vitreous19 and neovascular tissue20 removed during surgery from patients with retinal neovascularization due to diabetic retinopathy. However, other studies have argued against a role for FGF2.21,22 Whether or not it participates in the stimulation of retinal neovascularization remains unanswered and controversial.
FGF2 also has neurotrophic effects in the retina, and exogenous administration of FGF223-25 or increased expression of endogenous FGF226 decreases retinal cell death after a variety of insults. However, alteration of FGF2 levels in the retina for therapeutic benefit may be hazardous if it stimulates retinal neovascularization. Therefore, it is important to clarify the role of FGF2 in the retina both to better understand the pathogenesis of retinal neovascularization and to determine whether undesired effects on the retinal vasculature preclude therapeutic uses for FGF2.
In this study, we have used mice with targeted disruption of the Fgf2 gene and transgenic mice that overexpress FGF2 in the retina to investigate the role of FGF2 in retinal neovascularization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice with targeted disruption of the Fgf2 gene were produced by homologous recombination and will be described elsewhere.27 Gene disruption was confirmed by Southern blotting of tail DNA and by Western blot analysis of eyes and other tissues.
Eyes from four wild-type and four Fgf2 knockout mice were dissected, frozen in liquid nitrogen, and stored at -70°C. Protein extracts were prepared by homogenizing the frozen samples in 2 ml of 20 mmol/L Tris-HCl buffer (pH 7.4) containing 5 mmol/L ethylenediaminetetraacetic acid, 1% Nonidet P-40, 0.5% deoxycholate, 2 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride using a Tissumizer (Tekmar Co.). Extracts were clarified by centrifugation in a Sorvall GS 34 rotor at 12,000 rpm for 30 minutes. Protein concentration was determined in the supernatant using the Bio-Rad protein assay reagent (Richmond, CA). Equal amounts of total protein from the wild-type and knockout extracts (20 mg) were incubated for 2 hours at 4°C with 200 µl of heparin-sepharose CL-6B beads preswollen in the extraction buffer. Beads were collected by spinning 1 minute at 14,000 rpm in a microfuge. After three washes with 1 ml of 20 mmol/L Tris-HCl buffer (pH 7.4), the beads were boiled 5 minutes in 100 µl of loading buffer containing 100 mmol/L dithiothreitol and 0.1% sodium dodecyl sulfate and loaded on a 15% polyacrylamide-sodium dodecyl sulfate gel. Proteins were transferred to a nitrocellulose filter and analyzed by Western blot using a polyclonal antibody against human FGF2 (Santa Cruz Biotechnology, Santa Cruz, CA) and the Amersham enhanced chemiluminescense detection system (Arlington Heights, IL). Human recombinant FGF2 used as a control was a gift from Dr. David Moscatelli.
Generation of Transgenic Mice That Overexpress FGF2 in the Retina
A full-length cDNA for human FGF228
was generously
provided by Dr. Judith Abraham. It was digested with
EcoRI, filled in, and blunt end ligated into the
BamHI site of gBR200 containing the bovine rhodopsin
promoter, an intron and a polyadenylate addition site derived
from the mouse protamine gene (mP1), and a eukaryotic
consensus ribosomal binding site.29
After transformation, a
clone with correct orientation was selected. DNA was double CsCl
purified and cut with KpnI and SphI to provide a
3777-bp fusion gene (Figure 1)
. The
fusion gene was purified, and transgenic mice were generated using
established techniques as previously described.29
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. Reverse Transcription (RT)-PCR
At appropriate time points, mice were sacrificed, eyes were
removed, and retinas were dissected. Retinal RNA was isolated using the
guanidinium isothiocyanate method as described by Chomczynski
and Sacchi.30
RT was carried out with ~0.5 µg of total
RNA, reverse transcriptase (SuperScript II, Life Technologies,
Gaithersburg, MD), and 5.0 µmol/L oligodeoxythymidylate
primer. Aliquots of the cDNAs were used for PCR amplification at 58°C
with primers for the hFgf2/mP1
fusion gene that amplify across an intron-exon border, P3
(5'-TGGTATGTGGCACTGAAACG-3') and P2 (Figure 1)
. The expected PCR
products for the hFgf2/mP1 fusion
gene fragment from genomic DNA and mRNA are 572 and 478 bp,
respectively. Titrations were performed to ensure that PCR reactions
were carried out in the linear range of amplification. Mouse S16
ribosomal protein primers (5'-CACTGCAAACGGGGAAATGG-3' and
5'-TGAGATGGACTGTCGGATGG-3') were used to provide an internal control
for the amount of template in the PCR reactions.
Immunohistochemistry
Transgenic and littermate control mice were sacrificed, and their eyes were removed, fixed in 4% paraformaldehyde, and embedded in paraffin. Ten-µm sections were cut and immunohistochemically stained as previously described31,32 with a 1:50 dilution of a monoclonal anti-hFGF2 antibody33 generously provided by Dr. Thomas Reilly (Wilmington, DE). Specificity of staining was assessed by substitution of nonimmune serum for primary antibody.
Evaluation of Transgenic Mice for Retinal Neovascularization
Ten-µm serial sections were cut through the eyes of at least five mice 1 month old or older from each FGF2 transgenic line. The sections were stained with hematoxylin and eosin or histochemically stained with biotinylated griffonia simplicifolia isolectin B4 as described below and examined with an Axioskop microscope (Zeiss, Thornwood, NY). Eyes from transgenic mice that overexpress VEGF in the retina12 were used for comparison.
Murine Model of Oxygen-Induced Ischemic Retinopathy
Ischemic retinopathy was produced in FGF2-deficient mice, transgenic mice with high expression of FGF2 in the retina, and wild-type mice by a method described by Smith et al.34 Seven-day-old mice and their mothers were placed in an airtight incubator and exposed to an atmosphere of 75 ± 3% oxygen for 5 days. Incubator temperature was maintained at 23 ± 2°C, and oxygen was measured every 8 hours with an oxygen analyzer. After 5 days, the mice were removed from the incubator and placed in room air for 5 days. After 5 days, the mice were killed, and their eyes were rapidly removed and frozen in optimal cutting temperature embedding compound or fixed in 10% formalin and embedded in paraffin.
Quantitation of Retinal Neovascularization
Frozen sections (10 µm) of eyes from FGF2-deficient and wild-type mice were histochemically stained with biotinylated griffonia simplicifolia isolectin B4 (Vector Laboratories, Burlingame, CA), which selectively binds to endothelial cells. Slides were incubated in methanol/H2O2 for 30 minutes at 4°C, washed with 0.05 mol/L Tris-buffered saline, pH 7.4, and incubated for 30 minutes in 10% normal porcine serum. Slides were rinsed with 0.05 mol/L Tris-buffered saline and incubated 1 hour at 37°C with biotinylated lectin. After being rinsed with 0.05 mol/L Tris-buffered saline, slides were incubated with avidin coupled to peroxidase (Vector Laboratories) for 45 minutes at room temperature. After being washed for 10 minutes with 0.05 mol/L Tris buffer, pH 7.6, slides were incubated with diaminobenzidine (Research Genetics, Huntsville, AL) to give a brown reaction product and mounted with Cytoseal (Stephens Scientific, Riverdale, NJ).
To perform quantitative assessments, 10-µm serial sections were cut through the entire extent of each eye. The entire eye was sampled by staining sections roughly 50 to 60 µm apart, which provided 13 sections per eye for analysis. Lectin-stained sections were examined with an Axioskop microscope, and images were digitized using a 3 charge-coupled device color video camera (IK-TU40A, Toshiba, Tokyo, Japan) and a frame grabber. Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) was used to delineate lectin-stained cells on the surface of the retina, and their area was measured. The mean of the 13 measurements from each eye was used as a single experimental value.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice with targeted disruption of the
Fgf2 gene show no detectable
expression of FGF2 in the retina by Western blot, whereas wild-type
mice show a strong signal (Figure 2)
.
Retinas histochemically stained with griffonia simplicifolia isolectin
show a reaction product localized to vascular endothelial cells
providing excellent visualization of normal or abnormal retinal
vessels. In wild-type postnatal day 17 (P17) mice, normal retinal blood
vessels are seen as circumferential and radial tubes in the inner
retina (Figure 3, A and B)
. At high
magnification in Figure 3B
, no endothelial cells are seen on the
surface of the retina, although in some sections from normal mice a few
endothelial cells from large retinal vessels are seen on the surface
(not shown). In P17, FGF2-deficient mice, the retinal vessels are
normal in appearance (Figure 3, C and D)
. The total area of endothelial
cell staining in the retinas of FGF2-deficient mice was not
statistically different from that of wild-type mice (Figure 4A
, columns 1 and 3).
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Wild-type P17 mice with oxygen-induced ischemic retinopathy show
clumps of endothelial cells on the surface of the retina (Figure 3E
,
arrows) that are not seen in nonischemic retinas (Figure 3A)
. At higher
magnification, retinal vessels are seen connecting to endothelial cell
aggregates on the surface of the retina (Figure 3F
, arrow). The total
area of endothelial cell staining is significantly greater in the
retinas of mice with ischemic retinopathy compared with those of normal
mice (Figure 4A
, columns 1 and 2). The area of endothelial cell
staining on the surface of the retina, which has previously been
demonstrated to directly correlate with the number of endothelial
cells,35
is dramatically greater for ischemic compared with
normal retinas (Figure 4B
, columns 1 and 2).
FGF2-deficient mice with oxygen-induced ischemic retinopathy show
clumps of endothelial cells on the surface of the retina (Figure 3, G and H
, arrows), similar to those seen in wild-type mice with ischemic
retinopathy (Figure 3, E and F)
. The total area of endothelial cell
staining was significantly greater in ischemic compared with
nonischemic FGF2-deficient retinas (Figure 4A
, columns 3 and 4) but was
not different from that in wild-type ischemic retinas (Figure 4A
,
columns 2 and 4). Likewise, endothelial cell staining on the surface of
the retina was dramatically greater in ischemic compared with
nonischemic FGF2-deficient retinas (Figure 4B
, columns 3 and 4) but was
not different from that in wild-type ischemic retinas (Figure 4B
,
columns 2 and 4).
Generation of Rhodopsin Promoter/FGF2 Transgenic Mice
Our laboratory has previously demonstrated that overexpression of
VEGF in photoreceptors is sufficient to stimulate retinal
neovascularization.12
We used a similar strategy, coupling
the rhodopsin promoter to the Fgf2
gene (Figure 1)
and making transgenic mice, to investigate whether
overexpression of FGF2 in photoreceptors has a similar effect. Five
independent transgenic founders that incorporated the rhodopsin
promoter/Fgf2 fusion gene were
obtained (designated bF8, bF14, bF16, bF19, and bF28). The founders
were backcrossed with C57BL/6J mice to establish transgenic lines. Mice
that were heterozygous at the transgene locus were used in all
analyses.
Expression of FGF2 in the Retinas of Transgenic Mice
Fgf2 transgene mRNA expression
was assessed by semiquantitative RT-PCR. Mice from four of the five
transgenic lines showed good transgene expression (Figure 5)
, whereas, as expected, there was no
detectable signal in transgene-negative mice (Figure 5
, tg(-) lane).
Immunohistochemical staining for FGF2 showed reaction product within
photoreceptors in the retinas of transgene-positive mice, whereas
retinas from transgene-negative mice or transgene-positive mice for
which nonimmune serum was substituted for primary antibody showed only
background staining in the retina (Figure 6)
.
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The retinas of five transgene-positive mice from each of the four
lines with overexpression of FGF2 in photoreceptors were systematically
examined for retinal neovascularization. Examination at high power of
10 µm serial sections showed normal-appearing retinas with no
evidence of retinal neovascularization in any of the eyes from the four
different lines. Histochemical staining with griffonia simplicifolia
isolectin in retinas with overexpression of FGF2 in photoreceptors
showed only normal retinal blood vessels (Figure 6, D and E)
. This
contrasts with mice that overexpress VEGF in photoreceptors, which show
lectin-stained retinal neovascularization in the outer nuclear layer
(Figure 6F
, arrows).
There Is No Difference in Retinal Neovascularization in Mice with Ischemic Retinopathy That Overexpress FGF2 in the Retina Compared with Wild-Type Mice
Oxygen-induced ischemic retinopathy was induced in the offspring
of transgene-positive bF14 and wild-type mice, and after the amount of
retinal neovascularization was quantitated, the mice were genotyped.
Transgene-positive mice with ischemic retinopathy showed no significant
increase in the area of endothelial cell staining in the retina
compared with wild-type mice with ischemic retinopathy (Figure 7)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we have investigated this hypothesis by using genetically engineered mice to examine the effect of altered expression of FGF2 on the retinal vasculature. Mice deficient in FGF2 show no difference in morphology or total area of retinal vessels compared with wild-type mice, and when their retinas are made ischemic, they develop the same amount of retinal neovascularization. This indicates that FGF2 is not necessary for the normal development of retinal vessels, nor is it required for the occurrence of retinal neovascularization. Also, in contrast to mice with increased expression of VEGF in retinal photoreceptors, which develop intraretinal and subretinal neovascularization,12 mice that overexpress FGF2 in photoreceptors do not develop any neovascularization. Furthermore, mice that overexpress FGF2 and have oxygen-induced ischemic retinopathy show no significant difference in the amount of retinal neovascularization compared with wild-type mice with ischemic retinopathy. Therefore, FGF2 is neither necessary nor sufficient for retinal neovascularization to occur in ischemic retinopathy. In contrast to these data, a recent study used a similar strategy with genetically engineered mice that have altered expression of growth hormone and IGF-I to demonstrate that IGF-I contributes to retinal neovascularization.17
Is it possible that FGF2 stimulates vascular endothelial cell proliferation, migration, and proteolytic enzyme production in vitro37-39 and its exogenous administration stimulates neovascularization in some vascular beds, but it does not play a substantial role in retinal neovascularization? It is reasonable to consider this possibility, because the surrounding microenvironment modulates the effects of angiogenic stimuli,40 and therefore the effects of FGF2 in vitro or in other vascular beds in vivo may not be predictive of its effects in the retina. For instance, glial cells provide an important influence on retinal vessels that is not present for vessels not associated with the nervous system. Glial cells induce barrier characteristics in microvessels41 and may also act to inhibit retinal neovascularization.42 There are also differences between retinal and brain microvessels, with the former having a greater amount of pericyte coverage,43 and pericytes have been demonstrated to inhibit endothelial cell proliferation.44 Therefore, inhibitory influences in the retina may prevent retinal vascular endothelial cells from responding to FGF2 in the same way they do in other tissues or when isolated and placed in culture.
These same inhibitory influences also modulate the effects of VEGF and IGF-I, and yet it has been possible to demonstrate that VEGF and IGF-I are important stimulators of retinal neovascularization. This suggests that there may be other control mechanisms that are unique or stronger for FGF2. The Fgf2 gene does not have a signal sequence,45 and although FGF2 gets out of cells by a mechanism that has not been clearly defined, the amount that gets out may be substantially less than the amount of VEGF that is secreted or the amount of circulating IGF-I. Comparison of the amounts of VEGF and FGF2 externalized from retinal cells under baseline and hypoxic conditions demonstrate much higher levels of VEGF.46 Also, binding to proteoglycans in extracellular matrix47-49 and to soluble FGF receptors, which are present in the retina and vitreous in substantial quantities,50,51 may sequester extracellular FGF2 and make it inaccessible to retinal vessels.
Recently, Zhou et al52 demonstrated that Fgf2 knockout mice have decreased vascular smooth muscle contractility, low blood pressure, and thrombocytosis. However, they could not identify any abnormalities in angiogenic processes occurring during development, and the mice had normal female reproductive activity, which is dependent on angiogenesis. In addition, although it was previously thought that FGF2 plays an important role in the repair of intravascular injury, they found that FGF2-deficient mice have a normal hyperplastic response after mechanical intra-arterial injury. These results, and those of the present study, suggest that it may be necessary to change our thinking with regard to the role of FGF2 in angiogenesis and wound repair.
Disruption of FGF signaling in photoreceptors by expression of a
dominant-negative FGF receptor results in photoreceptor
degeneration.53
This indicates that at least one FGF is a
survival factor for photoreceptors. FGF2 is likely to play such a role
in photoreceptors and other retinal cells. It is constitutively
expressed in photoreceptors and other retinal cells,54-58
and its expression is increased by several types of insults, including
retinal trauma, light exposure, inherited retinal degenerations, or
optic nerve transsection.59-62
This may represent an
adaptation to limit cell damage, because these same insults decrease
cell death from subsequent insults.63,64
Also, injection of
FGF2 into the vitreous cavity of rats decreases cell death from an
inherited retinal degeneration, constant light exposure, or
ischemia.23-25
Increased expression of endogenous FGF2 by
administering an 
-adrenergic agent also decreases cell death from
constant light exposure.26
Therefore, there is reason to
believe that increasing FGF2 expression in the retina is a potentially
useful treatment for several types of pathology. Our data suggest that
this approach is unlikely to be limited by FGF2-induced retinal
neovascularization.
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Footnotes |
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Supported by US Public Health Service grants EY05951, EY10017, and EY09769; core grant P30EY1765 from the National Eye Institute; a Juvenile Diabetes Foundation fellowship grant (to NO); a Lew R. Wasserman Merit Award (to PAC); a career development award (to DJZ); an unrestricted grant from Research to Prevent Blindness, Inc.; a grant from the Rebecca P. Moon, Charles M. Moon, Jr., and Dr. P. Thomas Manchester Research Fund; a grant from the Association for Retinopathy of Prematurity and Related Diseases; a grant from Lois Duffey; and National Cancer Institute grant CA42568.
Accepted for publication May 20, 1998.
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-v ß-3 inhibit retinal neovascularization in a murine model. Lab Invest 1996, 75:563-573[Medline]
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