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Animal Models |
From the Departments of Ophthalmology andNeuroscience*
and Molecular Biology andGenetics,
The Johns Hopkins University Schoolof Medicine, Baltimore, Maryland; and the Jules Stein Eye Institute andthe Department of Neurobiology and Brain ResearchInstitute,
University of California at LosAngeles School of Medicine, Los Angeles, California
Abstract
Transgenic mice with vascular endothelial growth factor (VEGF) driven by the rhodopsin promoter (rho/VEGF mice) develop neovascularization that originates from the deep capillary bed of the retina and grows into the subretinal space. In rho/VEGF mice, VEGF expression in photoreceptors begins between postnatal days 5 and 7, the period when the deep capillary bed is developing. An important question is whether or not the developmental stage of the deep capillary bed is critical for occurrence of neovascularization. Also, although rho/VEGF mice are extremely useful for the study of ocular neovascularization, there are some applications for which the early onset of VEGF expression is a disadvantage. In this study, we used the reverse tetracycline transactivator (rtTA) inducible promoter system coupled to either the rhodopsin or interphotoreceptor retinoid-binding protein (IRBP) promoter to control the time of onset of VEGF transgene expression in photoreceptors. In the absence of doxycycline, adult double-transgenic rho/rtTA-TRE/VEGF or IRBP/rtTA-TRE/VEGF mice showed little VEGF transgene expression and no phenotype. The addition of doxycycline to the drinking water resulted in prominent transgene expression and evidence of neovascularization within 3 to 4 days. Like rho/VEGF mice, the neovascularization originated from the deep capillary bed of the retina, but it was more extensive and caused outer retinal folds followed by total retinal detachment. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay demonstrated that the mice with inducible expression of VEGF that developed retinal detachment had much higher ocular levels of VEGF mRNA and protein compared to rho/VEGF mice that manifest a much milder phenotype. These data demonstrate that regardless of developmental stage of the vascular bed, increased expression of VEGF in the retina is sufficient to cause neovascularization, and high levels of expression cause severe neovascularization and traction retinal detachment. Mice with inducible expression of VEGF in the retina provide a valuable new model of ocular neovascularization.
Although these studies clearly implicated VEGF as a stimulatory factor for ocular neovascularization, other studies showed that in some situations expression of VEGF by itself was insufficient to induce retinal neovascularization. The retinas of patients with nonproliferative diabetic retinopathy or other retinal diseases may show elevated levels of VEGF without retinal neovascularization.15,16 In primates, multiple intravitreous injections of VEGF result in iris neovascularization17 and retinal vascular abnormalities including endothelial cell proliferation,18 but do not cause definite retinal neovascularization. Similarly, intraocular implantation of pellets that provide sustained release of VEGF in primates fails to stimulate retinal neovascularization.19 However, intraocular implantation of similar pellets that release VEGF in rabbits results in transient retinal neovascularization.19
To resolve the issue of whether or not increased levels of VEGF are capable of stimulating retinal neovascularization, a full-length cDNA for VEGF165 was coupled to the bovine rhodopsin promoter and transgenic mice were generated that selectively express VEGF in photoreceptors.20 In these rho/VEGF mice, VEGF expression was turned on in photoreceptors at postnatal day 5 to postnatal day 6 and throughout the next week, neovascularization sprouted from the deep capillary bed of the retina and grew into the subretinal space. This indicates that increased expression of VEGF at a time when retinal vascular development is occurring is sufficient to cause neovascularization from the deep capillary bed, but it also raises several questions. Why does the neovascularization only originate from the deep capillary bed and not from superficial capillaries or choroidal vessels? Is the time that the transgene is turned on, which is during retinal vascular development, a critical determinant of the phenotype? Would alteration of the level of VEGF expression result in alteration of the location and/or amount of neovascularization? In this study, we have addressed these questions by using retina-specific promoters combined with an inducible promoter system, the reverse tetracycline transactivator system.21
Materials and Methods
Generation of Double-Transgenic Mice with Doxycycline-Inducible Expression of VEGF in the Retina
Transgenic mice that carry the reverse tetracycline transactivator under control of the photoreceptor-specific promoters for rhodopsin (rho/rtTA mice, line D) or interphotoreceptor retinoid-binding protein (IRBP/rtTA mice, line K) have been generated and characterized.22 A plasmid containing the tetracycline response element (TRE) was digested with BamHI, and the 598-bp BamHI fragment of VEGF that was previously used to generate rho/VEGF transgenic mice20 was inserted. After transformation, a clone with correct orientation of the VEGF fragment was identified by sequencing. DNA was purified by the CsCl gradient technique and cut with XhoI and HindIII yielding a 1529-bp TRE/VEGF/SV40 poly A fusion gene. The fusion gene was purified and transgenic mice were generated as previously described.20 Forty-eight mice were obtained and screened by polymerase chain reaction (PCR) of tail DNA using transgene-specific primers (primer 1: 5'-TCGAGTAGGCGTGTACGG-3', primer 2: 5'-GCAGCAGCCCCCGCATCG-3') that result in a 429-bp specific product. Tail DNA was obtained by overnight digestion of a 1- to 2-mm tail segment in 0.5 mg/ml proteinase K, 10 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 20 mmol/L ethylenediaminetetraacetic acid, and 2% Triton X-100 at 55°C. Four founder mice were identified and mated with C57BL/6 mice. One founder failed to reproduce and therefore three TRE/VEGF transgenic lines were obtained. Mice from each of these lines were mated with rho/rtTA and IRBP/rtTA mice and double transgenics were identified by PCR of tail DNA. The TRE/VEGF transgene was identified using the primers listed above. The rho/rtTA or IRBP/rtTA transgenes were identified as previously described22 using rtTA primer (5'-GTTTACCGATGCCCTTGGAATTGACGAGT-3'), IC40 primer (5'-GATGTGGCGAGATGCCCTTGGAATTGACGAGT-3'), and IC41 primer (5'-CAAGCAACTCCTGATGCCAAAGCCCTGCCC-3').
Retinal Reverse Transcriptase (RT)-PCR
Adult double-transgenic mice were given drinking water containing
10 mg/ml of doxycycline and 5% sucrose. After various times, mice were
sacrificed, eyes were removed, and retinas were dissected. Retinal RNA
was isolated using the guanidine isothiocyanate method as described by
Chomczynski and Sacchi.23
Reverse transcription was
performed with 
0.5 µg of total RNA, reverse transcriptase
(SuperScript II; Life Technologies, Inc., Gaithersburg, MD), and 5.0
µmol/L of oligo dT primer. Aliquots of the cDNAs were used for PCR
amplification using primers that specifically amplify human VEGF
(5'-CACCCATGGCAGAAGGAGGAG-3' and 5'-CAAATGCTTTCTCCGCTCTGA-3').
Titrations were performed to ensure that PCR reactions were performed
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.
Real-Time RT-PCR
Real time PCR allows for precise quantitation of mRNA using total RNA from a single retina and obviates the need for Northern blots using pooled retinas. Retinal RNA was isolated using Trizol solution (Life Technologies, Inc.) as directed by the manufacturer. Residual DNA was removed by DNase-treatment with DNA-free (Ambion, TX). One µg of RNA was reverse-transcribed using Superscript II (Life Technologies, Inc.). Real-time quantitative PCR was performed and analyzed using the SYBR Green I format on the LightCycler rapid thermal cycler system (Roche Molecular Biochemicals, Indianapolis, IN) using a previously published procedure and primer pairs specific for human VEGF.24 Hot Start Taq DNA polymerase and the FastStart Master SYBR Green I reaction mix (Roche Molecular Biochemicals) were used. During amplification, SYBR Green fluorescence was automatically acquired during the elongation step of each cycle by the LightCycler software. After the amplification protocol, melting curve analysis was performed. Quantification was done with the LightCycler Software by linear regression analysis using a series of specimen dilution curves and a series of standard curves generated with known amounts of cDNA (linearized plasmid containing the requisite VEGF cDNA diluted in 10-fold increments up to 1:10,000). Linear regression (log concentration versus cycle number) for each dilution set of standard and each sample had similar slopes (-1/log efficiency), comparable y intercepts, an r correlation value of -1.00, and a mean squared error lower than 0.25. Multiple runs did not differ in amplification efficiency by more than ±0.05. Through the use of appropriate external standards for quantification, suitable internal controls for assuring the integrity of the reverse transcriptase reaction, and accurate determinants for analysis it was possible to determine very precise mRNA levels.
Histopathological Evaluation of Retinas
Untreated double-transgenic mice and those treated with various doses of doxycycline for various time periods were sacrificed and eyes were rapidly removed and frozen in optimum cutting temperature embedding compound (OCT; Miles Diagnostics, Elkhart, IN). Frozen sections (10 µm) of eyes were histochemically stained with biotinylated Griffonia simplicifolia lectin B4 (GSA) (Vector Laboratories, Burlingame, CA) that selectively binds to vascular cells. Slides were incubated in methanol/H2O2 for 10 minutes at 4°C, washed with 0.05 mol/L Tris-buffered saline (TBS), pH 7.6, and incubated for 30 minutes in 10% normal porcine serum. Slides were incubated 2 hours at room temperature with biotinylated lectin and after rinsing with 0.05 mol/L TBS, they 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 TBS, slides were incubated with diaminobenzidine to give a brown reaction product. Some slides were counterstained with hematoxylin and all were mounted with Cytoseal.
Quantitation of Ocular VEGF Levels
At the time mice were sacrificed, eyes were removed, a cut was made at the limbus, the lens was carefully removed, and the remainder of each eye was snap-frozen in liquid nitrogen. Eye homogenates were prepared by Dounce homogenization followed by three freeze/thaw cycles in phosphate-buffered saline with 100 µmol/L of phenylmethyl sulfonyl fluoride. Homogenates were microfuged and the protein concentration of supernatants was measured using a Bio-Rad Protein Assay Kit (BioRad, Hercules, CA). Enzyme-linked immunosorbent assay of the samples was performed using the Quantikine VEGF assay kit (R&D Systems, Minneapolis, MN) using the manufacturer’s instructions. Serial dilutions of recombinant VEGF165 were assayed to generate a standard curve with the limit of detection at 16 pg/ml.
Results
Double-Transgenic Mice with Doxycycline-Inducible Expression of VEGF in the Retina
Three TRE/VEGF transgenic lines were obtained and mice from each
of these lines were mated with rho/rtTA or IRBP/rtTA transgenic mice
and double-hemizygous rho/rtTA-TRE/VEGF and IRBP/rtTA-TRE/VEGF
transgenics were identified by PCR of tail DNA. At 3 weeks of age, mice
were treated with 10 mg/ml of doxycycline in their drinking water.
After 2 weeks, mice were sacrificed and retinal RNA was isolated.
RT-PCR for human VEGF showed that both IRBP/rtTA-TRE/VEGF and
rho/rtTA-TRE/VEGF double-hemizygous transgenics generated from one of
the TRE/VEGF lines had minimal expression of human VEGF in the absence
of doxycycline, but strong expression when the mice had been given
doxycycline in their drinking water (Figure 1)
. Double-hemizygous transgenics
generated from the other two TRE/VEGF lines failed to show good
inducible expression of VEGF; one showed low-level transgene expression
and the other showed little difference in expression in the presence or
absence of doxycycline (not shown).
|
Untreated rho/rtTA-TRE/VEGF or IRBP/rtTA-TRE/VEGF mice had normal
retinas with no evidence of neovascularization (Figure 2A
, and Figure 3, A and B
). Mice at several ages were examined and through 5 weeks of
age (Figure 3B)
. GSA-stained retinas showed completely normal
superficial, intermediate, and deep capillary beds and no
ectopic vessels. Both
types of double-transgenic mice were treated with 10 mg/ml of
doxycycline in their drinking water and sacrificed
between 4 and 10 days
later. Four days after starting doxycycline, both rho/rtTA-TRE/VEGF and
IRBP/rtTA-TRE/VEGF mice showed neovascularization originating from the
deep capillary bed and extending into the photoreceptor layer. In some
mice, the appearance was very similar to that seen in V6 rho/VEGF mice
(Figure 2, I and J)
.20
At 5 or 6 days after initiation of
doxycycline, several different phenotypes were seen including mild
neovascularization (Figure 3D)
such as that in V6 rho/VEGF mice, severe
neovascularization with thick cords of vascular cells extending into
and disrupting the photoreceptor layer (Figure 3, E and F
, arrows)
resulting in outer retinal folds and focal retinal detachments
(asterisks), partial retinal detachments (Figure 2C)
, or extensive
traction retinal detachments (Figure 2; E to H
, and Figure 3; G to J
).
Four eyes examined at 8 to 10 days after initiation of doxycycline all
had retinal detachments (Figure 2, G and H
; Figure 3, I and J
).
|
|
To determine whether the severity of the phenotype in
IRBP/rtTA-TRE/VEGF double transgenics could be altered by decreasing
the dose of doxycycline, adult mice were divided into four groups and
given a daily subcutaneous injection of 0.01, 0.05, 0.1, or 0.5 mg/g
body weight. The doxycycline was delivered by injection rather than
oral administration to better control the dose provided. Mice were
sacrificed at 8 days after initiation of the injections. Gross
pathological examination of eyes and microscopic evaluation of serial
sections stained with GSA showed that the incidence of retinal
detachment increased from 0 of 10 mice treated with 0.01 mg/g body
weight/day to 17 of 18 mice treated with 0.5 mg/g body weight/day
(Figure 4)
. The remaining mouse in the
0.5 mg/g group had retinal neovascularization without retinal
detachment and therefore 100% of the mice in the high-dose group had
an abnormal phenotype. The percentage of mice with neovascularization
and no detachment was greatest in the 0.05 mg/g group and decreased in
the 0.1 mg/g and 0.5 mg/g groups in which the vast majority of mice
that had neovascularization also had retinal detachment. Therefore,
both the incidence of an abnormal phenotype and its severity increased
as the dose of doxycycline was increased.
|
Double-transgenic rho/rtTA-TRE/VEGF or IRBP/rtTA-TRE/VEGF mice
were treated with daily subcutaneous injections of one of
several doses of doxycycline and after 8 days the mice were sacrificed
and eyes were removed. Retinal RNA was isolated from one eye of each
mouse and histopathological evaluation was performed on the opposite
eye. The level of retinal human VEGF mRNA assessed by real
time RT-PCR in wild-type mice was undetectable. Double-transgenic mice
that did not receive doxycycline or those that received 0.01 mg/g body
weight/day had relative copy numbers of VEGF mRNA in the
range of 1000 to 10,000 (Figure 5)
. In
general, mice treated with higher doses of doxycycline (0.1 and 0.5
mg/g body weight/day) had higher levels of VEGF mRNA in the
retina than those treated with lower doses (0.01 or 0.05 mg/g body
weight/day).
|
Fewer rho/rtTA-TRE/VEGF mice were available and therefore none were treated with 0.01 mg/g body weight/day of doxycycline and only three doses starting with 0.05 mg/g body weight/day were examined. All of the mice showed a phenotype and although the numbers were small, as the dose of doxycycline increased, there was an increase in both average VEGF mRNA level and percentage of mice with retinal detachment. Therefore, in both types of double-transgenic mice, there was a positive correlation between dose of doxycycline, level of retinal VEGF mRNA, and severity of the phenotype. The level of retinal VEGF mRNA was assessed in six rho/VEGF mice and the mean (±SEM) relative copy number was 23,860 ± 1633.
Ocular VEGF Protein Levels in rho/VEGF Mice and Mice with Inducible Expression of VEGF
Hemizygous rho/VEGF mice (n = 5 mice,
10 eyes) were sacrificed and eye homogenates were prepared at postnatal
day 21, a time when VEGF mRNA levels are at steady-state and there is
extensive retinal neovascularization.20,25
Measurement of
ocular VEGF protein levels in the retina in all 10 rho/VEGF mice was
below the level of detection of the assay (16 pg/ml, Figure 6
). At postnatal day 21,
double-transgenic rho/rtTA-TRE/VEGF (n = 10) and
IRBP/rtTA-TRE/VEGF mice (n = 10) were given
daily subcutaneous injections of 0.5 mg/g body weight of doxycycline
and after 3 days the mice were sacrificed and eye homogenates were
prepared. The mean ocular VEGF level was 189.3 pg/µg of protein in
rho/rtTA-TRE/VEGF mice and 823.5 pg/µg protein in IRBP/rtTA-TRE/VEGF,
both significantly greater than levels in V6 rho/VEGF transgenic mice.
|
Increased expression of VEGF in photoreceptors beginning at postnatal day 5 to postnatal day 6 in rho/VEGF mice results in neovascularization that originates from the deep capillary bed of the retina and grows into the subretinal space mimicking many aspects of choroidal neovascularization.20 This demonstrates that in neonatal mice, increased expression of VEGF in the retina is sufficient to cause neovascularization. In this study, we have extended those findings by using an inducible promoter system to demonstrate that increased expression of VEGF is also sufficient to cause neovascularization in the retina of adult mice. We have recently initiated doxycycline in adult mice ranging from 1 to 6 months of age and like rho/VEGF mice, the origin of the neovascularization at all ages was from the deep capillary bed of the retina. This suggests that the differential sensitivity of the deep capillary bed to increased expression of VEGF is not related to developmental stage, because that sensitivity is also present in adult mice.
Although the origin of neovascularization in mice with inducible expression of VEGF is the same as that in rho/VEGF mice, the resulting phenotype is more severe. Three to 4 days after initiation of doxycycline, particularly in mice treated with 0.05 to 0.1 mg/g body weight, the neovascularization is similar to that seen in rho/VEGF mice. By 5 to 6 days after onset of doxycycline, many mice treated with low doses and almost all mice treated with high doses showed cords of vascular cells invading the outer nuclear layer that became very thick and exerted traction that caused outer retinal folds, focal detachments, and then total detachments. During the advanced stages, neovascularization spreads throughout the subretinal space and all parts of the retina resulting in disorganized retina and large masses of vascular cells. One difference between rho/VEGF and rho/rtTA-TRE/VEGF and IRBP/rtTA-TRE/VEGF mice that is likely to contribute to the difference in phenotype is that the double transgenics, when treated with appropriate doses of doxycycline, express much higher levels of VEGF.
In addition to demonstrating that increased expression of VEGF is sufficient to cause retinal neovascularization in adult mice, rho/rtTA-TRE/VEGF and IRBP/rtTA-TRE/VEGF mice provide a new animal model that is complementary to rho/VEGF mice. The severe phenotype of the inducible transgenics provides a more stringent test of efficacy for anti-angiogenic agents. We have identified treatments that completely inhibit neovascularization in rho/VEGF transgenics.13,26 By extending the range of disease severity for testing, the inducible transgenics provide a possible means of distinguishing between treatments that have maximum efficacy in the models with a less severe phenotype. Also, the inducible transgenics provide another outcome variable that may simplify testing. If treatments are effective, they should prevent retinal detachments, which can be rapidly detected. Alteration of the incidence of retinal detachment at a particular dose of doxycycline may provide a rapid assay for anti-angiogenic efficacy that obviates the need for time consuming image analysis. Finally, the inducible transgenics offer more flexibility in experimental design because the time of onset and the severity of disease can be regulated via the time of initiation of doxycycline treatment and the dose administered. Some experiments that are not possible with rho/VEGF mice can be done with the inducible transgenics. For instance, the long latency of recombinant adenoassociated virus (AAV) vectors makes it impossible to use AAV to express genes in the eye before the onset of neovascularization in rho/VEGF mice to determine whether expression of those genes prevents neovascularization. In the inducible transgenics, it is possible to inject expression constructs packaged in AAV and wait as long as necessary before turning on expression of VEGF.
Therefore, mice with inducible expression of VEGF in photoreceptors provide a valuable new animal model that will help to explore the pathogenesis of ocular neovascularization and identify new ways to treat this blinding complication of ocular diseases.
Footnotes
Address reprint requests to Peter A. Campochiaro, M.D., Maumenee 719, The Johns Hopkins University School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287-9277. E-mail: pcampo{at}jhmi.edu
Supported by the National Eye Institute (grants EY05951, EY12609, EY00444, EY0331, and K08 EY13420 to P. G.); Research to Prevent Blindness (core grant P30EY1765), a Lew R. Wasserman Merit Award (to P. A. C.), a Career Development Award (to D. J. Z.), and an unrestricted grant; the Foundation Fighting Blindness, the Ruth and Milton Steinbach Foundation, the Juvenile Diabetes Research Foundation (to P. G.), Michael Panitch, and Dr. and Mrs. William Lake. P. A. C. is the George S. and Dolores Dore Eccles Professor of Ophthalmology and Neuroscience. D. B. is the Dolly Green Professor of Ophthalmology at UCLA.
Accepted for publication October 26, 2001.
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