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Animal Models |
From the Albert Ludwigs University,*
Freiburg, Germany;
the Laboratory for Surgical Research,
Children’s Hospital, Boston, Massachusetts; the Department of
Ophthalmology,§
Harvard Medical School,
Massachusetts Eye and Ear Infirmary, Boston, Massachusetts; Brigham and
Women’s Hospital and Harvard Medical School,
Boston, Massachusetts; and the Laboratory of Retinal Cell and Molecular
Biology,¶
National Eye Institute, National
Institutes of Health, Bethesda, Maryland
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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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The pathogenesis of exudative ARMD is primarily unknown. Histopathological studies of choroidal neovascular membranes from patients with ARMD have demonstrated the presence of various growth factors that include basic fibroblast growth factor,3,4 vascular endothelial growth factor (VEGF),5-7 and transforming growth factor-ß.3,8 The hypoxia-regulated protein VEGF is one of the major stimulators of angiogenesis. VEGF was first reported in 1983 in highly vascularized tumors.9 VEGF is secreted as a homodimeric protein that specifically stimulates proliferation of endothelial cells in blood vessels.10 Five known isoforms with 121 to 206 amino acids are generated from a single gene by alternative mRNA splicing.11-14 The 165- and 121-kd proteins are commonly expressed in the ischemic retina.15 Two high-affinity tyrosine kinase transmembrane VEGF receptors, flt-1 and flk-1, are expressed on vascular endothelial cells.16,17
Recent evidence suggests a central role for VEGF in the development of choroidal neovascularization secondary to ARMD. RPE cells produce VEGF in vivo under physiological conditions,18 and in vitro after experimental ischemia/reperfusion.19 In patients with ARMD, high concentrations of VEGF and VEGF receptors were detected in the subfoveal fibrovascular membrane, the surrounding tissue and the RPE.5,7 In vitro, VEGF mRNA as well as the VEGF protein concentration are increased in RPE cells that were exposed to hypoxia, reactive oxygen species, or cytokines.19,20
To date, there is no generally accepted experimental in vivo model for choroidal neovascularization that occurs in ARMD. Reproducibility of existing models is limited, partly because of technical artifacts and because the induction of choroidal neovascularization was accompanied by a nonspecific, local inflammatory reaction.21-27 Recent work reported a transgenic mouse model in which overexpression of VEGF in the photoreceptors was under the control of a constitutively active rhodopsin promoter. These mice developed retinal neovascularization but failed to develop choroidal neovascularization that is characteristic of ARMD.28 It was hypothesized that the RPE may serve as a barrier that blocks VEGF diffusion from the photoreceptors to the choroid. We sought to determine whether VEGF overexpressed directly in the RPE would overcome this barrier and be sufficient to induce choroidal neovascularization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of Transgenic Mice
A full-length cDNA for murine VEGF164 (a gift from Dr. Yin-Shan Ng and Dr. P. D’Amore) was cloned into the SmaI site of pBluescript II KS. A rabbit ß-globin-3' UTR sequence (a gift from Dr. H. Bujard) was directionally cloned at the 3' end of the VEGF-coding sequence to add a polyadenylation tail to the transcript to increase mRNA stability. A fragment (-655 to +52) of the cloned 2.8-kb murine RPE65 promoter was directionally cloned into the 5' end of the VEGF-coding sequence.29 The sequence of the final construct was confirmed (2,517 bp). The construct sequence was excised and used to generate three founder mice at the National Institute of Child Health and Human Development Transgenic Mouse Development Facility, University of Alabama at Birmingham. To expand the transgenic lines the founder mice were crossed into a C57BL/6J-TyrC-2J background (Jackson Laboratories, Bar Harbor, ME). Founder no. 1 died without giving offspring, the transgene incorporated in founder no. 2 had a deletion and point mutation. All investigated animals were offspring from founder no. 3 and heterozygous, as they were generated by mating a transgenic parent with a C57BL/6J-TyrC-2J mouse.
Polymerase Chain Reaction
Mice were screened for the presence of the transgene by polymerase
chain reaction (PCR) of tail DNA. Tail pieces were digested overnight
at 56°C in 0.2% sodium dodecyl sulfate, 100 mmol/L Tris-HCl, pH 8.5,
5 mmol/L ethylenediaminetetraacetic acid, 200 mmol/L NaCl, and 30 µl
proteinase K at 20 mg/ml. For PCR at 63°C, a 5' primer
(f-RPE65-1, ACC TCG AGG CAA TGG TGA AGA CAG TGA
TG), and a 3' primer (r-exon-1, TGG TGG AGG TAC AGC AGT AA) (Figure 1)
were used to amplify 800 bp of the
transgene-specific sequence.
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Offspring were screened for the complete presence of the
transgene by Southern blot and sequencing. EcoRI-digested
tail DNA (10 µg) was used for Southern blot analysis. Hybridization
was performed with 
-32-phosphate dCTP (New
England Nuclear Life Science Products Inc., Boston, MA)-labeled DNA
probe to exon 3 of the VEGF gene for 24 hours at 42°C. After washes
(5x SSPE and 0.5% sodium dodecyl sulfate at room temperature,
1x SSPE and 0.5% sodium dodecyl sulfate at room temperature, and
0.1x SSPE and 1% sodium dodecyl sulfate at 64°C) the Hybond-N+
nucleic acid transfer membrane (Amersham Life Science, Arlington
Heights, IL) was autoradiographed (Kodak Scientific Imaging Film Ready
Pack, X-OMAT AR; Eastman-Kodak, Rochester, NY).
Sequencing of tail DNA was performed with three sets of primer pairs to
cover most of the construct (Figure 1)
: f-RPE65-2
(CTC TAA TCT TCA CTG GAA GCT) with r-exon-7 (CAC ACT TGC AAG TAC GTT
CGT), f-exon-1 (CGT CAG AGA GCA ACA TCA TCA CC) with r-ß-globin-1
(GGA GAC AAT GGT TGT CAA CA), f-exon 1 with r-ß-globin-2 (CTT CCG AGT
GAG AGA CAC AA). The PCR product was subcloned into a pCRII-TOPO vector
(Invitrogen, Carlsbad, CA) and sequenced in a biopolymer facility
(Department of Cardiology, Children’s Hospital, Boston, MA).
Veterinary Care and Euthanasia
Mice were housed in a barrier-care facility and fed a diet of animal chow and water ad libitum. Euthanasia was achieved with application of 75 mg/kg of pentobarbital intraperitoneally followed by cervical dislocation. All procedures were performed according to the ARVO guidelines and Children’s Hospital recommendations.
Reverse Transcriptase (RT)-PCR
Whole eyes from postnatal day 15, 1 month-, 2 month-, 4 month-, 5
month-, and 7-month-old mice were analyzed. Total RNA was purified by
homogenizing the eyes in RNazol-B (Tel Test Inc., Friendswood, Texas)
according to the manufacturer’s instructions. Reverse transcription
was performed with 2 µg of total RNA, oligo (dT) primers (Ambion
Inc., Austin, Texas), and Moloney murine leukemia virus reverse
transcriptase (New England Biolabs, Beverly, MA). The resulting cDNAs
were diluted 1:10 and used for subsequent PCR amplification of
endogenous VEGF isoforms at 52°C with the primers f-exon-4 (5' ATC
ATG CGG ATC AAA CCT CAC CA) and r-exon-8 (3' TAC GGA TCC TCC GGA CCC
AAA GTG CTC) (Figure 1)
. Amplification of a transgene-specific 600-bp
sequence was achieved at 62°C with 5' f-RPE65-2
and 3' r-exon-7 primers. Housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA was amplified with 5' primer TTA GCA CCC CTG
GCC AAG G and 3' primer CTT ACT CCT TGG AGG CCA TG at 62°C.
In Situ Hybridization
Briefly, a 380-bp transgene sequence spanning exon 4 of VEGF164 and part of the ß-globin was cloned into a pCR II-TOPO vector. Antisense and sense probes labeled with digoxigenin-dUTP were generated with the digoxigenin-labeling kit (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions. The antisense probe was not specific for transgenic VEGF mRNA but also recognized endogenous VEGF mRNA. Transgenic and control mice eyes fixed in 4% paraformaldehyde and paraffin-embedded were hybridized at 55°C overnight in 50% formamide, 0.3 mol/L NaCl, 20 mmol/L Tris-HCl, pH 8, 5 mmol/L ethylenediaminetetraacetic acid, 10 mmol/L Na2HPO4, 10% dextran sulfate, 1x Denhardt’s, and 0.5 mg/ml yeast RNA. Washes [5x standard saline citrate (SSC), 2x SSC plus 50% formamide, 2x SSC, and 0.2x SSC] preceded immunohistochemistry with a 1:200 anti-digoxigenin Fab conjugated to alkaline phosphatase and color development with BM purple (all Boehringer Mannheim) for 3 to 5 days at 4°C. Eyes were evaluated under light microscopy.
Screening for Morphological Differences
Routine Harris hematoxylin and eosin staining was performed to screen for morphological differences between control and transgenic mice aged 15 days to 11 months. Briefly, deparaffinized and rehydrated 4- to 5-µm paraffin sections were placed in Harris hematoxylin for 10 minutes at room temperature and rinsed in water. Quick dips in acid alcohol and rinses in tap water preceded quick dips in ammonia water. After rinsing in tap water, the sections were stained for 1 minute at room temperature with eosin followed by dehydration to 100% ethanol and xylene. The sections were mounted in permount.
Furthermore, control and transgenic eye sections were stained with routine periodic acid-Schiff and Gill’s hematoxylin to visualize Bruch’s membrane. Briefly, deparaffinized and rehydrated 4- to 5-µm paraffin sections were incubated with Schiff’s reagent for 6 minutes at room temperature, rinsed 3 x 5 minutes with tap water and incubated for 15 minutes at room temperature in periodic acid solution. After rinses in tap water the sections were stained with Gill’s hematoxylin for 2 minutes at room temperature and developed in ammonia water. The sections were mounted in aquamount.
Immunohistochemistry
Transgenic and littermate control mice were sacrificed and their eyes enucleated. Eyes were embedded in Tissue Tek (Sakura Finetechnical Co, Tokyo, Japan) for frozen sections, or fixed in 4% paraformaldehyde. Four-µm-thick paraffin sections were cut.
For VEGF immunohistochemistry, frozen sections were fixed in acetone and blocked with 0.1 mol/L phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), 2% rabbit serum, 0.3% Triton X-100, and 0.01% sodium azide. Incubation for 1 hour at room temperature with polyclonal goat anti-mouse VEGF (dilution 1:50 in blocking buffer; R&D Systems, Minneapolis, MN) was followed by incubation with an affinity-purified polyclonal rabbit anti-goat IgG (diluted 1:200 in blocking solution; Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature. Amplification with 1:100 streptavidin conjugated to horseradish peroxidase for 30 minutes at room temperature preceded color development with diaminobenzidine (DAKO Corp., Carpinteria, CA). Counterstain was performed with Gill’s hematoxylin. Corresponding cross-sections in proximity of the optic nerve entry were evaluated under light microscopy.
CD31 immunohistochemistry was performed on deparaffinized sections. Endogenous peroxidase activity was quenched with 1% H2O2 and antigen retrieval was performed with 0.0036 mg/ml proteinase K in 0.2 mol/L Tris-HCl, pH 7.5. The tissue was preincubated with TNB-rabbit blocking solution (10% rabbit serum in 0.15 mol/L NaCl, 0.1 mol/L Tris-HCl, pH 7.5, and 0.5% blocking reagent from a TSA indirect amplification kit (New England Nuclear Life Sciences Technology) for 1 hour at room temperature. The sections were then incubated overnight at 4°C with 1:200 purified anti-mouse CD31 (PECAM-1, MEC13.3; PharMingen, San Diego, CA). A 1:400 dilution of affinity-purified, biotinylated rabbit anti-rat IgG (Vector Laboratories) was applied in TNB-rabbit for 1 hour at room temperature. Signal amplification was obtained with the TSA indirect kit followed by a streptavidin-biotin-alkaline phosphatase complex (ABC Kit; Vector Laboratories). Color development was performed with Fast Red (BioGenex, San Ramon, CA) and counterstain with Gill‘s hematoxylin. Specificity of staining was assessed by omitting the primary antibody. Corresponding choroidal regions with constant retinal thickness were evaluated with a reticule grid at magnification x1,000 under light microscopy. Choroidal thickness of control and transgenic choroids at the optic nerve entry and 10 µm from the ora serrata was measured in µm in 5 sections each. Statistical analysis of the means was performed with a two-tailed Student’s t-test with unequal variance.
ADPase Staining
Choroidal flatmounts were incubated in 5 mmol/L ethylenediaminetetraacetic acid for 30 minutes at room temperature and fixed in 10% buffered formalin overnight at 4°C. ADPase staining was performed as previously described.30 Briefly, incubation with a 1:100 dilution of 1 mg/ml ADP in 0.2 mol/L of Tris-maleate, pH 7.2, 3 mmol/L Pb(NO3)2, and 6 mmol/L MgCl2 for 30 minutes at 37°C preceded color development with 2% ammonium sulfide. Flatmounts were inspected under light microscopy. Corresponding areas at equal magnifications of the vortex vein-, the optic nerve-, and the long ciliary artery region were photographed. To determine vessel density, the area of stained vessels relative to the total area was analyzed with NIH image software. Statistical analysis of the values was performed with a two-tailed Student’s t-test with unequal variance.
Lectin Perfusion
Mice were anesthetized with 0.5 ml of avertin intraperitoneally. A thoracotomy exposed the heart and a 20-gauge blunt-ended feeding needle (Fine Science Tools, Foster City, CA) was inserted into the left ventricle and fixed with a Dieffenbach Serrefine clamp (Arista). The right atrium was perforated to drain 10 ml of PBS, 10 ml of 4% paraformaldehyde, 5 ml of 1% BSA, and 10 ml of 40 mg/ml fluorescein isothiocyanate-labeled Lycopersicon esculentum lectin solution. The eyes were enucleated, prepared as a flatmount, and inspected by fluorescent microscopy.
5-Bromo-2'-Deoxy-Uridine (BrdU) Staining
Mice of the indicated age were injected with 0.25 ml to 0.5 ml of
undiluted BrdU (Boehringer Mannheim) intraperitoneally and sacrificed
after 1.15 hours. The eyes were enucleated, fixed in 70% ethanol, 0.2
mol/L glycine, pH 2, and processed for 4-µm paraffin sections.
Sections were stained according to the BrdU labeling and detection kit
II (Boehringer Mannheim) instructions. CD31 and BrdU double stainings
were performed before the evaluation of choroidal cell proliferation to
establish which cells in the choroid were BrdU-positive. Corresponding
cross-sections (n 
7) with equal optic nerve
diameter were analyzed by light microscopy with magnification x800 and
the number of stained choroidal cell nuclei was counted. Statistical
analysis of the means was performed with a two-tailed Student’s
t-test with unequal variance.
Measurement of Blood Vessel Leakage
Choroidal and retinal blood vessel leakage was quantitated in transgenic and control choroids and retinae (n = 6) of 3-month-old mice using Evans blue dye. Evans blue noncovalently binds to plasma albumin in the blood stream (Xu, Quaum, IOVS in print) and in situations of increased vessel leakage is extravasated into the interstitial space. After clearance of Evans blue from the vessel lumina, the amount of extravasated dye is extracted from the interstitial space and quantitated.
Evans blue dye was dissolved in normal saline sonicated for 5 minutes, and filtered through a 5-µm filter. Under deep anesthesia, 30 mg/kg of Evans blue was injected into the tail vein and circulated for 1 hour. The chest cavity was opened and the left ventricle of the heart cannulated. Each mouse was then perfused with citrate-buffered 1% paraformaldehyde, pH 4.2, 37°C for 2 minutes at a physiological pressure of 100 mmHg to clear the dye out of the vessel lumina. Immediately after perfusion, the eyes were enucleated and the retinae and sclera-choroid complex were carefully dissected and collected in separate tubes. After thorough drying (Speed-Vac) of the tissue, the dry weight was measured. Evans blue was extracted by subsequent incubation of the tissue in 75 µl of formamide for 18 hours at 70°C. The extract was ultracentrifuged at a speed of 70,000 rpm for 45 minutes. The absorbance of the supernatant was measured at the absorption maximum for Evans blue in formamide (620 nm) with a spectrophotometer. The dye concentration in the extracts was calculated from a standard curve of Evans blue in formamide and normalized to the dry tissue weight.
CD18 Staining
Choroidal flatmounts were fixed in acetone, permeabilized for 24 hours at room temperature in 1% Triton X-100 in PBS, and blocked with PBS, 3% BSA, and 1% Triton X-100. Biotinylated rat anti-mouse CD18 antibody (Chemicon, Temecula, CA) was incubated at a dilution of 1:100 in PBS, 1% BSA, and 1% Triton X-100 for 24 hours at room temperature, followed by a 1:250 dilution of fluorescein isothiocyanate-conjugated streptavidin (Vector Laboratories) in 0.5% Triton X-100 in PBS at room temperature for 24 hours. Staining of leukocytes was evaluated under a fluorescent microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Offspring from line no. 3 mice were generated in a C57BL/SJL
background, mated with C57BL/6J-TyrC-2J and
expanded by five back-crosses to maintain an albino phenotype. As
determined by Southern blot analysis, genomic incorporation of a full
copy of the transgene was complete, as both hybridization products for
the endogenous VEGF gene (an 
9-kb band) and for the transgenic
VEGF164 gene (a 1,256-bp band) were detected
(Figure 2)
. The transgene copy number was
2 to 3, as the intensity of the transgene hybridization product was
2–3 stronger than the endogenous hybridization product. Control
littermates showed only the endogenous VEGF gene hybridization product.
Sequencing of the transgene showed the correct sequence of the complete
RPE65/VEGF164/ß-globin-3'
UTR construct (data not shown).
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Time course and expression level of transgenic
VEGF164 was assessed by RT-PCR and in
situ hybridization. In RT-PCR, transgene-specific primers
amplified a VEGF164 cDNA-specific 600-bp sequence
throughout the life of transgenic mice but not of control mice (Figure 3A)
. Normalized transgenic
VEGF164 mRNA increased toward 4 months of age and
decreased afterward. GAPDH bands of similar intensity demonstrated
equal loading (Figure 3B)
. In situ hybridization with an
anti-sense exon 4 to ß-globin probe detected endogenous as well as
transgenic VEGF164 mRNA. The anti-sense probe
showed minimal hybridization product in the RPE cell nuclei of control
eye sections (Figure 4A)
, which was
markedly increased in the RPE cell nuclei of transgenic eye sections
(Figure 4C)
. No difference was seen between the hybridization observed
in choroidal cells of transgenic and control eyes. As well, no
difference in VEGF mRNA was detected in the inner nuclear or ganglion
cell layer of the retina between both groups (data not shown). A sense
exon 4 -ß-globin probe showed no hybridization (Figure 4, B and D)
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Immunohistochemical staining for VEGF (Figure 5, E and F)
in transgenic mice showed
specific and intense staining of RPE cells and choroidal vessels. VEGF
immunoreactivity was observed uniformly on the basal side of the RPE
cells (Figure 5, E and F
; inserts). No apical RPE cell and
photoreceptor staining could be observed. Choroidal staining was not
uniform throughout the eye. Areas of very intense and cluster-like
staining alternated with areas of less intense staining. The lamina
choriocapillaris showed intense staining, as well as some areas of the
lamina vasculosa. In contrast, control choroids were almost free of
VEGF staining in the lamina choriocapillaris and vasculosa, and also
lacked the cluster-like staining pattern observed in transgenic
choroids. The RPE cells of control mice stained uniformly and less
intense throughout the eye. In transgenic and control animals
comparable retinal staining was observed in ganglion cells and cells of
the inner nuclear layer (data not shown). An age-dependent increase of
RPE cell and choroidal staining was observed in transgenic animals.
This increase was most pronounced at 3 to 4 months of age (data not
shown).
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Control and transgenic eye sections of mice aged 15 days to 11
months screened by hematoxylin and eosin and by PAS and hematoxylin
staining showed differences in the choroid of transgenic mice, which
was thickened (Figure 5, A and B)
. In transgenic as well as control
eyes the RPE cells and Bruch’s membrane were intact and showed no
areas of disruption or damage (Figure 5, C and D)
. In both groups the
retina and sclera did not differ.
Immunohistochemistry for the endothelial cell marker CD31 was performed
to evaluate the differences in vascularization between transgenic and
control choroids of mice aged 15 days to 11 months (Figure 5
; G, H, I,
and J). Transgenic mice demonstrated a thickened and irregular
choroidal vasculature. In some areas the vessels appeared in clusters
that bulged toward the sclera. The lamina choriocapillaris stained most
intensely. Choriocapillary density was increased and the distance
between capillary vessel lumina was reduced. The lamina vasculosa also
appeared more dense with dilated vessels and wide lumina. Measurements
of total choroidal thickness in standardized areas of the choroid
(n = 4 per group) showed a significant increase
(P < 0.05) of 35 ± 4% in the optic nerve
region and of 40 ± 6% in the proximity of the ora serrata in
transgenic animals relative to control mice. Choroidal vessels did not
penetrate Bruch’s membrane or appear in the subretinal space at any
age. No differences in the retinal vasculature were seen by CD31
immunohistochemistry or by ADPase staining of retinal whole mounts.
For further evaluation of the choroidal changes observed with CD31
staining, we performed adenosine diphosphatase stains of transgenic and
control choroidal flatmounts at 1 to 11 months of age. Choroidal
flatmounts of 1- to 2-month-old mice stained poorly (data not shown),
whereas choroids of transgenic mice older than 2 months revealed a more
intense staining than controls (Figure 6, A and D)
. As already described by Lutty and colleagues,30
ADPase activity in the retina was most intense in new vessels, which
also seems to be the case for new vessels in the choroid of mice.
Reaction product was confined almost exclusively to the vasculature,
with arteries staining more intense than veins or capillaries as shown
before.30
In transgenic choroids, the regular architecture
was perturbed. The vessels appeared elongated and tortuous. Abnormal
sprouts were observed and derived particularly from the stem of the
long and short ciliary arteries (Figure 6E
relative to 6B). The area of
the vortex vein showed an increase in staining intensity and the
vessels appeared engorged (data not shown). Choriocapillary vessels
around the optic nerve entry also stained intensely, looped, and
appeared tortuous (Figure 6F
relative to 6C). Control flatmounts showed
very little choriocapillary staining in the area of the optic nerve
entry. Vascular density of corresponding flatmount areas was
significantly increased in transgenic choroids in respect to control
choroids (Figure 7)
.
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. Although control choroids
showed a normal vascular pattern, the vascular architecture of
transgenic choroids was irregular (Figure 8, A and B
versus
C and D). Choroidal vessels were denser and engorged with nests of high
vascular density with dilated, tortuous, and abnormal vessels. The
nests were elevated out of the two dimensional plane of the normal
choriocapillaris (Figure 8D)
. Other regions in transgenic choroids were
characterized by tortuous vessels that branched off the stem of larger
vessels (data not shown), mainly the long ciliary artery. Vessels of
the choriocapillaris were denser with only small spaces between the
capillaries (Figure 8C
versus 8A). The staining was not as
uniform as in control choroids.
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Lectin perfusion revealed cells adherent to the inside of
capillaries that were abundant and appeared in clusters of two to four
cells in transgenic choroids (Figure 8E)
. The choroidal capillaries
were not occluded because of their wide caliber. Only a few adherent
cells and no clusters were seen in control choroids. CD18 staining of
3-month-old control and transgenic mice (data not shown) demonstrated
that the adherent cells were neutrophils and/or monocytes. These cells
were more abundant in transgenic choroids and formed rolls and
clusters, whereas control choroids contained only single adherent
cells. Flatmounts that were processed without primary CD18 antibody
showed no staining.
Evans Blue Leakage in Transgenic Mice
Evans blue vascular leakage was measured in both retina and
choroid of transgenic and control eyes. Choroidal leakage was
significantly higher (P < 0.001) in the
transgenic eyes (24.6 ± 0.7 mg/ml per mg choroidal-scleral
tissue) compared to the control eyes (6.9 ± 2.7 mg/ml per mg
choroidal-sclera tissue) (Figure 9)
. In
the retina, however, transgenic and control eyes did not show
differences in Evans blue vessel leakage.
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BrdU immunohistochemistry was performed to detect abnormal
proliferation of choroidal cells in transgenic mice at 1, 3, and 7
months of age. To establish that BrdU was labeling endothelial cells,
we first performed CD31 and BrdU double stains and demonstrated that
>90% of the BrdU-labeled cells were intraluminal and therefore most
likely represented endothelial cells (data not shown). These
intraluminal choroidal BrdU-stained nuclei were counted and were found
to be increased in transgenic mice at all ages inspected (Figure 10)
. Proliferation peaked at 3 months
of age and then subsequently decreased. In contrast, proliferation of
choroidal cells in control choroids decreased from birth to nearly
undetectable levels at 7 months of age. Transgenic choroids also
displayed clusters of more than four proliferating cells that were
abundant in central parts of the eye. Toward the ora serrata, control
and transgenic choroids showed single cell proliferations (data not
shown). BrdU staining was comparable in retina, ciliary body, and
cornea of control and transgenic mice as well as in heart, liver, and
skin (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The transgene expression in RPE cells was demonstrated by RT-PCR and in situ hybridization. In situ hybridization showed strong staining against VEGF164 mRNA in the transgenic RPE cell nuclei that was higher than the basal VEGF164 mRNA expression in control RPE cells. Basal VEGF164 mRNA expression was also detected in choroidal cells although the expression was similar in transgenic and control choroids. Because RPE cells and choroidal fibroblasts have been reported to express VEGF mRNA,18,35 basal VEGF164 mRNA expression may relate to the maintenance of vascular integrity and permeability of the choroid.
The transgenic overexpression of VEGF by the RPE cells resulted in strong staining against VEGF protein in the RPE cells, Bruch‘s membrane, and choroid. VEGF was not detected in the photoreceptor layer in either animal group. This in vivo finding supports that the secretion of VEGF mostly occurs on the basal side of the RPE cell. The polarized VEGF secretion by RPE cells is thought to direct VEGF toward the choroidal vasculature where it may regulate choroidal integrity by binding to the VEGF receptors flt-1 and flk-1/KDR on the adjacent choriocapillaris.18 In vitro, this polarized secretion of VEGF was reported in human RPE cells in culture.36
VEGF was originally described as vascular permeability factor (VPF)37,38 because of its ability to increase microvascular permeability.38 In normal human eyes, VEGF secreted from the RPE is thought to maintain a fenestrated epithelium in the choriocapillaris.36 VEGF is increased in diseases associated with increased leakage such as diabetic retinopathy39,40 and ARMD where it can be detected in the subfoveal fibrovascular membranes.5 Animal models41,42 have demonstrated that VEGF is associated with leakage from abnormal vessels. In our model the quantitative measurement of Evans blue leakage from choroidal vessels demonstrated a significant increase in vessel permeability in transgenic choroids in respect to control choroids. In retinal vessels this difference in Evans blue leakage was not observed. We propose that the increased choroidal vessel permeability in our model was a direct effect of increased transgenic VEGF164 secretion that affected choroidal vessel permeability in transgenic choroids.
In lectin perfused flatmounts and CD18 staining of transgenic choroids, an increase of adherent cells was noted in respect to control choroids. Lectins have been described to bind to the surface of adherent intravascular leukocytes43,44 and CD18 staining identifies neutrophils and monocytes. Histopathology of human specimens of choroidal neovascularization has demonstrated inflammatory cells.6,45,46 Most animal models of choroidal and subretinal neovascularization induce a significant extravascular inflammatory response that is regarded as secondary to the mechanical disruption of the retina or choroid. However, VEGF in our model induces an intravascular adherence of leukocytes without preceding mechanical disruption. This finding is in accordance with another transgenic mouse model in which VEGF was overexpressed in epidermal keratinocytes. Overexpression in keratinocytes resulted in an increase in intravascular leukocyte adhesion in postcapillary skin venules.41 VEGF has also been shown to increase leukocyte adherence in early angiogenesis by up-regulation of intercellular adhesion molecule-1 (ICAM-1).47-50 ICAM-1 mediated the invasion of leukocytes in corneal angiogenesis in vivo47 and in the retinal vasculature.49 We suspect that the observed leukostasis in our transgenic mice is induced by a VEGF-mediated up-regulation of ICAM-1.
The paracrine secretion of VEGF into the choriocapillaris with subsequent choroidal neovascularization did not result in a disruption of the Bruch’s membrane/RPE cell complex and subsequent subretinal neovascularization, as seen in ARMD. Subretinal neovascularization can be induced in animals by introducing growth factors including basic fibroblast growth factor and VEGF into the subretinal space, or by laser disruption of the Bruch’s membrane/RPE complex.21,22,27,31,51,52 However these models are associated with significant extravascular inflammation and/or tissue disruption that may be permissive to subretinal extension of the choroidal neovascularization. Clinical disorders with breaks in Bruch‘s membrane such as myopia, trauma, pseudoxanthoma elasticum, and histoplasmosis have an increased incidence of choroidal neovascularization. Therefore our model reinforces the idea that an intact Bruch’s membrane/RPE barrier prevents choroidal neovascularization from penetrating into the subretinal space.
In summary, the present work establishes a transgenic mouse model in which overexpression of VEGF in RPE cells is sufficient for the development of intrachoroidal neovascularization. Our model is not complicated by technical artifacts and demonstrates that transgenic overexpression of VEGF from the intact RPE is not sufficient to induce subretinal neovascularization. Because degeneration of RPE cells and Bruch’s membrane is a common morphological feature in human ARMD, we suggest that breaks in this barrier are necessary in the progression of disease in humans. Finally we propose that this new model can be used to study agents that may inhibit choroidal neovascularization.
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Acknowledgements |
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Footnotes |
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Supported by the American Health Assistance Foundation No. M2000020, and Gottlieb Daimler and Karl Benz Foundation, project number 02-18/99.
Accepted for publication December 8, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. Janssen, J. Hoellenriegel, M. Fogarasi, H. Schrewe, M. Seeliger, E. Tamm, A. Ohlmann, C. A. May, B. H. F. Weber, and H. Stohr Abnormal Vessel Formation in the Choroid of Mice Lacking Tissue Inhibitor of Metalloprotease-3 Invest. Ophthalmol. Vis. Sci., July 1, 2008; 49(7): 2812 - 2822. [Abstract] [Full Text] [PDF]
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K. Yamada, E. Sakurai, M. Itaya, S. Yamasaki, and Y. Ogura Inhibition of Laser-Induced Choroidal Neovascularization by Atorvastatin by Downregulation of Monocyte Chemotactic Protein-1 Synthesis in Mice Invest. Ophthalmol. Vis. Sci., April 1, 2007; 48(4): 1839 - 1843. [Abstract] [Full Text] [PDF]
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R. A. Costa, R. Jorge, D. Calucci, J. A. Cardillo, L. A. S. Melo Jr, and I. U. Scott Intravitreal Bevacizumab for Choroidal Neovascularization Caused by AMD (IBeNA Study): Results of a Phase 1 Dose-Escalation Study. Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4569 - 4578. [Abstract] [Full Text] [PDF]
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I. Semkova, S. Fauser, A. Lappas, N. Smyth, N. Kociok, B. Kirchhof, M. Paulsson, V. Poulaki, and A. M. Joussen Overexpression of FasL in retinal pigment epithelial cells reduces choroidal neovascularization FASEB J, August 1, 2006; 20(10): 1689 - 1691. [Abstract] [Full Text] [PDF]
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S. S. Dandekar, S. A. Jenkins, T. Peto, H. P. N. Scholl, K. S. Sehmi, F. W. Fitzke, A. C. Bird, and A. R. Webster Autofluorescence Imaging of Choroidal Neovascularization Due to Age-Related Macular Degeneration Arch Ophthalmol, November 1, 2005; 123(11): 1507 - 1513. [Abstract] [Full Text] [PDF]
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G. Malek, L. V. Johnson, B. E. Mace, P. Saloupis, D. E. Schmechel, D. W. Rickman, C. A. Toth, P. M. Sullivan, and C. Bowes Rickman Apolipoprotein E allele-dependent pathogenesis: A model for age-related retinal degeneration PNAS, August 16, 2005; 102(33): 11900 - 11905. [Abstract] [Full Text] [PDF]
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C-M Lai, S A Dunlop, L A May, M Gorbatov, M Brankov, W-Y Shen, N Binz, Y K. Lai, C E Graham, C J Barry, et al. Generation of transgenic mice with mild and severe retinal neovascularisation Br J Ophthalmol, July 1, 2005; 89(7): 911 - 916. [Abstract] [Full Text] [PDF]
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N.H. V. Chong, J. Keonin, P. J. Luthert, C. I. Frennesson, D. M. Weingeist, R. L. Wolf, R. F. Mullins, and G. S. Hageman Decreased Thickness and Integrity of the Macular Elastic Layer of Bruch's Membrane Correspond to the Distribution of Lesions Associated with Age-Related Macular Degeneration Am. J. Pathol., January 1, 2005; 166(1): 241 - 251. [Abstract] [Full Text] [PDF]
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M. G. Slomiany and S. A. Rosenzweig IGF-1-Induced VEGF and IGFBP-3 Secretion Correlates with Increased HIF-1{alpha} Expression and Activity in Retinal Pigment Epithelial Cell Line D407 Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2838 - 2847. [Abstract] [Full Text] [PDF]
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M. A. Zarbin Current Concepts in the Pathogenesis of Age-Related Macular Degeneration Arch Ophthalmol, April 1, 2004; 122(4): 598 - 614. [Abstract] [Full Text] [PDF]
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H. Ida, T. Tobe, H. Nambu, M. Matsumura, M. Uyama, and P. A. Campochiaro RPE Cells Modulate Subretinal Neovascularization, but Do Not Cause Regression in Mice with Sustained Expression of VEGF Invest. Ophthalmol. Vis. Sci., December 1, 2003; 44(12): 5430 - 5437. [Abstract] [Full Text] [PDF]
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E. Sakurai, A. Anand, B. K. Ambati, N. van Rooijen, and J. Ambati Macrophage Depletion Inhibits Experimental Choroidal Neovascularization Invest. Ophthalmol. Vis. Sci., August 1, 2003; 44(8): 3578 - 3585. [Abstract] [Full Text] [PDF]
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J.-M. Rakic, V. Lambert, L. Devy, A. Luttun, P. Carmeliet, C. Claes, L. Nguyen, J.-M. Foidart, A. Noel, and C. Munaut Placental Growth Factor, a Member of the VEGF Family, Contributes to the Development of Choroidal Neovascularization Invest. Ophthalmol. Vis. Sci., July 1, 2003; 44(7): 3186 - 3193. [Abstract] [Full Text] [PDF]
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M. V. Kalayoglu, C. Galvan, O. S. Mahdi, G. I. Byrne, and S. Mansour Serological Association Between Chlamydia pneumoniae Infection and Age-Related Macular Degeneration Arch Ophthalmol, April 1, 2003; 121(4): 478 - 482. [Abstract] [Full Text] [PDF]
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F. Wang, K. G. Rendahl, W. C. Manning, D. Quiroz, M. Coyne, and S. S. Miller AAV-Mediated Expression of Vascular Endothelial Growth Factor Induces Choroidal Neovascularization in Rat Invest. Ophthalmol. Vis. Sci., February 1, 2003; 44(2): 781 - 790. [Abstract] [Full Text] [PDF]
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K. G. Carrasquillo, J. A. Ricker, I. K. Rigas, J. W. Miller, E. S. Gragoudas, and A. P. Adamis Controlled Delivery of the Anti-VEGF Aptamer EYE001 with Poly(lactic-co-glycolic)Acid Microspheres Invest. Ophthalmol. Vis. Sci., January 1, 2003; 44(1): 290 - 299. [Abstract] [Full Text] [PDF]
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V. Lambert, C. Munaut, M. Jost, A. Noel, Z. Werb, J.-M. Foidart, and J.-M. Rakic Matrix Metalloproteinase-9 Contributes to Choroidal Neovascularization Am. J. Pathol., October 1, 2002; 161(4): 1247 - 1253. [Abstract] [Full Text] [PDF]
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9-kb upper band) in
all samples. The probe also hybridized to the exon 3 sequence of
transgenic VEGF164 in transgenic mice
(T)
(1,259-bp band).
EcoRI digestion of the plasmid
(P) that contained the
transgenic construct also generated a 1,259 bp band.
