Intrachoroidal Neovascularization in Transgenic Mice Overexpressing Vascular Endothelial Growth Factor in the Retinal Pigment Epithelium
2001; Elsevier BV; Volume: 158; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64063-1
ISSN1525-2191
AutoresCatherine Schwesinger, Charles Yee, Richard M. Rohan, Antonia M. Joussen, António Fernández, Tobias Meyer, Vassiliki Poulaki, Joseph J.K., T. Michael Redmond, Suyan Liu, Anthony P. Adamis, Robert J. D’Amato,
Tópico(s)Retinal Imaging and Analysis
ResumoChoroidal neovascularization in age-related macular degeneration is a frequent and poorly treatable cause of vision loss in elderly Caucasians. This choroidal neovascularization has been associated with the expression of vascular endothelial growth factor (VEGF). In current animal models choroidal neovascularization is induced by subretinal injection of growth factors or vectors encoding growth factors such as VEGF, or by disruption of the Bruch's membrane/retinal pigment epithelium complex with laser treatment. We wished to establish a transgenic murine model of age-related macular degeneration, in which the overexpression of VEGF by the retinal pigment epithelium induces choroidal neovascularization. A construct consisting of a tissue-specific murine retinal pigment epithelium promoter (RPE65 promoter) coupled to murine VEGF164 cDNA with a rabbit β-globin-3′ UTR was introduced into the genome of albino mice. Transgene mRNA was expressed in the retinal pigment epithelium at all ages peaking at 4 months. The expression of VEGF protein was increased in both the retinal pigment epithelium and choroid. An increase of intravascular adherent leukocytes and vessel leakage was observed. Histopathology revealed intrachoroidal neovascularization that did not penetrate through an intact Bruch's membrane. These results support the hypothesis that additional insults to the integrity of Bruch's membrane are required to induce growth of choroidal vessels into the subretinal space as seen in age-related macular degeneration. This model may be useful to screen for inhibitors of choroidal vessel growth. Choroidal neovascularization in age-related macular degeneration is a frequent and poorly treatable cause of vision loss in elderly Caucasians. This choroidal neovascularization has been associated with the expression of vascular endothelial growth factor (VEGF). In current animal models choroidal neovascularization is induced by subretinal injection of growth factors or vectors encoding growth factors such as VEGF, or by disruption of the Bruch's membrane/retinal pigment epithelium complex with laser treatment. We wished to establish a transgenic murine model of age-related macular degeneration, in which the overexpression of VEGF by the retinal pigment epithelium induces choroidal neovascularization. A construct consisting of a tissue-specific murine retinal pigment epithelium promoter (RPE65 promoter) coupled to murine VEGF164 cDNA with a rabbit β-globin-3′ UTR was introduced into the genome of albino mice. Transgene mRNA was expressed in the retinal pigment epithelium at all ages peaking at 4 months. The expression of VEGF protein was increased in both the retinal pigment epithelium and choroid. An increase of intravascular adherent leukocytes and vessel leakage was observed. Histopathology revealed intrachoroidal neovascularization that did not penetrate through an intact Bruch's membrane. These results support the hypothesis that additional insults to the integrity of Bruch's membrane are required to induce growth of choroidal vessels into the subretinal space as seen in age-related macular degeneration. This model may be useful to screen for inhibitors of choroidal vessel growth. Choroidal neovascularization remains the leading cause of severe vision loss in patients with the exudative form of age-related macular degeneration (ARMD). Choroidal vessels grow through breaks in Bruch's membrane and proliferate under the retinal pigment epithelium (RPE) and the sensory retina. The RPE as well as the choriocapillaris are morphologically altered before neovascularization occurs.1Adamis AP Shima DT Yeo KT Yeo TK Brown LF Berse B D'Amore PA Folkman J Synthesis and secretion of vascular permeability factor/vascular endothelial growth factor by human retinal pigment epithelial cells.Biochem Biophys Res Commun. 1993; 193: 631-638Crossref PubMed Scopus (369) Google Scholar, 2Lutty G Grunwald J Majji AB Uyama M Yoneya S Changes in choriocapillaris and retinal pigment epithelium in age-related macular degeneration.Mol Vis. 1999; 5: 35PubMed Google Scholar These immature vessels leak serum and blood that can induce a fibrotic reaction known as a disciform scar. Despite recent advances in medical and surgical treatment of choroidal neovascularization the long-term prognosis of ARMD is still poor. The pathogenesis of exudative ARMD is primarily unknown. 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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.21Soubrane G Cohen SY Delayre T Tassin J Hartmann MP Coscas GJ Courtois Y Jeanny JC Basic fibroblast growth factor experimentally induced choroidal angiogenesis in the minipig.Curr Eye Res. 1994; 13: 183-195Crossref PubMed Scopus (53) Google Scholar, 22Kimura H Sakamoto T Hinton DR Spee C Ogura Y Tabata Y Ikada Y Ryan SJ A new model of subretinal neovascularization in the rabbit.Invest Ophthalmol Vis Sci. 1995; 36: 2110-2119PubMed Google Scholar, 23Orzalesi N Migliavacca L Miglior S Subretinal neovascularization after naphthalene damage to the rabbit retina.Invest Ophthalmol Vis Sci. 1994; 35: 696-705PubMed Google Scholar, 24Pollack A Korte GE Weitzner AL Henkind P Ultrastructure of Bruch's membrane after krypton laser photocoagulation. I. 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Repair of Bruch's membrane and the role of macrophages.Arch Ophthalmol. 1986; 104: 1377-1382Crossref PubMed Scopus (34) Google Scholar, 26Lutty GA McLeod DS Pachnis A Costantini F Fabry ME Nagel RL Retinal and choroidal neovascularization in a transgenic mouse model of sickle cell disease.Am J Pathol. 1994; 145: 490-497PubMed Google Scholar, 27Spilsbury K Garrett KL Shen WY Constable IJ Rakoczy PE Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization.Am J Pathol. 2000; 157: 135-144Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar 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.28Okamoto N Tobe T Hackett SF Ozaki H Vinores MA LaRochelle W Zack DJ Campochiaro PA Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization [see comments].Am J Pathol. 1997; 151: 281-291PubMed Google Scholar 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. Unless indicated, reagents were purchased from Sigma Chemical Co., St. Louis, MO. A full-length cDNA for murine VEGF164 (a gift from Dr. Yin-Shan Ng and Dr. P. D'Amore) was cloned into the Sma I site of pBluescript II KS. A rabbit β-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.29Boulanger A Liu S Henningsgaard AA Yu S Redmond TM The upstream region of the RPE65 gene confers retinal pigment epithelium-specific expression in vivo and in vitro and contains critical octamer and E-box sites.J Biol Chem. 2000; 275: 31274-31282Crossref PubMed Scopus (44) Google Scholar 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. 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. Offspring were screened for the complete presence of the transgene by Southern blot and sequencing. Eco RI-digested tail DNA (10 μg) was used for Southern blot analysis. Hybridization was performed with α-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 (5× SSPE and 0.5% sodium dodecyl sulfate at room temperature, 1× SSPE and 0.5% sodium dodecyl sulfate at room temperature, and 0.1× 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-β-globin-1 (GGA GAC AAT GGT TGT CAA CA), f-exon 1 with r-β-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). 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. 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. Briefly, a 380-bp transgene sequence spanning exon 4 of VEGF164 and part of the β-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, 1× Denhardt's, and 0.5 mg/ml yeast RNA. Washes [5× standard saline citrate (SSC), 2× SSC plus 50% formamide, 2× SSC, and 0.2× 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. 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 × 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. 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 ×1,000 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. 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.30Lutty GA McLeod DS A new technique for visualization of the human retinal vasculature.Arch Ophthalmol. 1992; 110: 267-276Crossref PubMed Scopus (117) Google Scholar 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. 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. 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 ×800 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. 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. 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. 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/β-globin-3′ UTR construct (data not shown). 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 β-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 -β-globin probe showed no hybridization (Figure 4, B and D).Figure 4In situ hybridization. Original magnification, ×1,600. A: In control eyes, hybridization with an antisense probe demonstrates basa
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