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1 From the Department of Ophthalmology, Kansai Medical University, Osaka, Japan; and the 2 Department of Pharmaceutical Sciences, University of Missouri, Kansas City, Missouri.
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Abstract |
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METHODS. Choroidal neovascularization was induced by laser photocoagulation in rat eyes. At intervals of up to 2 weeks after photocoagulation, the eyes were removed and prepared for in situ hybridization and immunohistochemical study. In situ hybridization was performed with digoxigenin-labeled PEDF riboprobes. Protein expression of PEDF and VEGF was studied immunohistochemically.
RESULTS. In normal adult rat eyes, PEDF mRNA was observed mainly in the corneal epithelial and endothelial cells, lens epithelial cells, ciliary epithelial cells, retinal ganglion cells, and the RPE cells. During the development of choroidal neovascularization, PEDF mRNA, PEDF protein, and VEGF protein were strongly detected in many cells within the laser lesions at 3 days after photocoagulation, after which levels gradually declined. However, PEDF was still expressed in the RPE cells that proliferated and covered the neovascular tissues at 2 weeks, whereas VEGF protein was weakly expressed in endothelial cells in choroidal neovascularization.
CONCLUSIONS. PEDF is expressed in different cell types of normal rat eyes. The expression of PEDF was detected in the choroidal neovascular tissues induced by photocoagulation, and these findings suggest that PEDF may modulate the process of choroidal neovascularization.
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Introduction |
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Recent studies have implicated PEDF in activities that are inhibitory to angiogenesis.15 16 PEDF has been shown to inhibit the migration of endothelial cells in vitro in a dose-dependent manner and is more effective than the angiogenesis inhibitors, angiostatin, thrombospondin-1, and endostatin.15 The results of these studies placed PEDF among the most potent natural inhibitors of angiogenesis.
Choroidal neovascularization (CNV) is a devastating complication of macular diseases, especially age-related macular degeneration.17 18 CNV involves the formation of neovascular tissues from the choriocapillaris that extend into the subretinal space. Various growth factors, including basic fibroblast growth factor (bFGF),19 20 21 transforming growth factor (TGF)-ß,22 and vascular endothelial growth factor (VEGF),23 24 25 26 27 have been reported to mediate the development of CNV.
In this study, we investigated the mRNA of PEDF in the eyes of normal adult rats and in eyes during the process of CNV induced by laser photocoagulation. We also studied PEDF protein expression and compared it with the expression of VEGF in the choroidal neovascular tissues.
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Materials and Methods |
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Tissue Processing
Animals were killed with an overdose of intraperitoneal pentobarbital sodium at 3 and 7 days and 2 weeks after photocoagulation (11 rats at each time point). The eyes (8 of 11 rats at each time point) were enucleated and fixed for 2 hours in 4% paraformaldehyde in 0.1% diethylpyrocarbonate (DEPC)-treated phosphate-buffered saline (PBS) at 4°C. The tissue was then prepared for in situ hybridization, according to the manufacturer’s recommendations (Roche Molecular Biochemicals, Mannheim, Germany). Briefly, the fixed eyes were placed in 30% sucrose in PBS for 20 minutes at 4°C, dehydrated through a graded ethanol series, and embedded in paraffin. Sections, 4 µm thick, were deparaffinized, stained with hematoxylin-eosin (HE), and observed under a light microscope.
The eyes (three rats at each time point) were enucleated and fixed for 2 hours in 4% paraformaldehyde in PBS at 4°C, dehydrated through a graded ethanol series, and embedded in paraffin. The sections were then prepared for immunohistochemical study.
In Situ Hybridization
Serial or very closely adjacent 4-µm-thick sections were cut and stored at 4°C before use for in situ hybridization. Mouse PEDF cDNA (1084 bp, corresponding region of mouse PEDF cDNA, nucleotides 10-1188, GenBank accession number NM_011340; GenBank is provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD, and is available at http://www.ncbi.nlm.nih.gov/genbank) was subcloned into a vector (pBluescript II KS; Stratagene, La Jolla, CA). The resultant plasmid was linearized with HindIII and used as the template for the synthesis of an antisense riboprobe, using T7 polymerase, or linearized with XbaI for synthesis of a sense riboprobe, using T3 polymerase.
In vitro transcription was performed in the presence of digoxigenin-11-uridine-triphosphate (DIG-UTP) to produce the DIG-UTP-labeled single-stranded antisense or sense RNA probe using a kit (DIG RNA Labeling Kit; Roche Molecular Biochemicals), according to the manufacturer’s instructions. The amount of labeled RNA was determined by agarose gel electrophoresis and ethidium bromide staining. The RNA transcripts were then subjected to alkaline hydrolysis. The efficiency of transcription was checked by transferring the probe onto a nitrocellulose membrane (Hybond-N, Amersham, Amersham, UK).
Localization of PEDF mRNA was determined on 4-µm sections. The sections were deparaffinized in xylene and placed in PBS after a series of graded ethanol baths, refixed with 4% paraformaldehyde in PBS-0.1% DEPC for 15 minutes, treated with proteinase K (20 µg/mL) in TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) for 20 minutes, and fixed in the same fixative for 10 minutes.
After a rinse in PBS, the slides were incubated in 0.1 M HCl for 10 minutes to inhibit endogenous alkaline phosphatase activity. The sections were then rinsed with 0.1 M triethanolamine-HCl (pH 8.0) for 1 minute and with 0.25% acetic anhydride for 10 minutes, dehydrated through a graded ethanol series, and dried.
In situ hybridization was performed according to the manufacturer’s recommendations (Roche Molecular Biochemicals). An appropriate amount of DIG-labeled RNA-antisense probe (or DIG labeled RNA-sense probe as a control) was diluted in hybridization buffer (50% formamide, 10 mM Tris-HCl [pH 7.6], 200 µg/mL transfer [t]RNA, and 1x Denhardt’s solution: 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 1 mM EDTA [pH 8.0]), denatured, and hybridized to pretreated sections overnight at 50°C in a 50% formamide-saturated chamber.
After hybridization, the sections were washed with 50% formamide and 2x SSC at 50°C for 30 minutes, rinsed in TNE (10 mM Tris-HCl [pH 8.0], 500 mM NaCl, and EDTA 1 mM) at 37°C, digested for 10 minutes at 37°C with 20 µg/mL RNase A in TNE, and rinsed once in 2x SSC at 50°C for 20 minutes and twice in 0.2x SSC at 50°C for 20 minutes.
The probe was detected according to the instructions supplied with the kit (DIG Nucleic Acid Detection; Roche Molecular Biochemicals). Briefly, the slides were washed in DIG buffer 1 (100 mM Tris-HCl [pH 7.5], 150 mM NaCl) for 5 minutes at room temperature, incubated for 1 hour in DIG buffer 1 containing 1.5% blocking reagent to block nonspecific binding, and incubated with anti-DIG alkaline phosphatase-labeled antibody complex in DIG buffer 1 for 30 minutes. Slides were then washed with DIG buffer 1 for 15 minutes and DIG buffer 3 (100 mM Tris-HCl [pH 9.5], 100 mM NaCl, 50 mM MgCl2) for 5 minutes, followed by incubation in coloring reagent containing nitroblue tetrazolium salt (NBT, 0.34 mg/mL) and 5-bromo-4-chloro-3-indolylphosphate (BCIP, 0.18 mg/mL) in DIG buffer 3 for 12 hours at room temperature. Alkaline phosphatase activity appeared as a dark blue-to-purple precipitate. Color development was stopped by placing the slides in TE (pH 8.0). The sections were counterstained with methyl green that stained the nuclei a light blue-green. Sections were observed under a light microscope to detect PEDF mRNA. As a negative control, hybridization was performed with a sense strand riboprobe under identical conditions.
Immunohistochemical Analysis for PEDF and VEGF
To detect PEDF or VEGF protein, serial or very closely adjacent 4-µm-thick sections were used for immunohistochemical study. Immunoperoxidase analyses were performed with a kit (LSAB; Dako, Glostrup, Denmark), according to the manufacturer’s protocol. All steps were performed at room temperature, unless otherwise stated. Briefly, sections were deparaffinized, fixed in cold acetone (4°C) for 10 minutes and then treated with 3% hydrogen peroxide to remove endogenous peroxidase activity. After blocking, primary antibody, affinity-purified rabbit polyclonal antibody against bovine PEDF (prepared by JT-T; 1:500) or affinity-purified rabbit polyclonal antibody against human VEGF (Catalog no. VEGF [A-20rsqb]: sc-152; dilution 1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), was applied to sections for 60 minutes, and the sections were incubated with biotinylated goat anti-rabbit IgG. The slides were incubated with horseradish peroxidase (HRP)-conjugated avidin. For a chromogen, 3-amino-9-ethyl-carbazole (AEC; Dako) was used, and the slides were counterstained with methyl green. Between each step, the sections were washed three times with PBS. For control staining, preimmune rabbit IgG or mouse IgG was used instead of the primary antibody. Sections were observed under a light microscope to detect the localization of immunoreactivity for PEDF or VEGF.
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Results |
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Expression of PEDF mRNA in the CNV
Three days after photocoagulation, strong expression of PEDF mRNA was observed in the cells within the laser lesions. PEDF mRNA signals were observed in the proliferating RPE cells, pigment-laden macrophages, and fibroblasts that appeared in the photocoagulated lesions (Figs. 5A
5B)
. Seven days after photocoagulation, the PEDF mRNA signals in the laser lesions were less than those at 3 days after photocoagulation; however, a strong expression of PEDF mRNA was detected in the spindle-shaped RPE cells that were observed sealing over the region of CNV (Figs. 5C
5D)
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Sections hybridized with the sense PEDF control probes showed no precipitates (Fig. 5) .
Immunoreactivity for PEDF and VEGF in the CNV
Three days after photocoagulation, immunoreactivity for PEDF and VEGF was observed in many cells within the laser lesions (Figs. 7A
7B
8A)
. Immunoreactivity for PEDF was observed in the proliferating RPE cells, pigment-laden macrophages, and fibroblasts that appeared in the photocoagulated lesions. VEGF expression was also strongly observed in the proliferating RPE cells, pigment-laden macrophages, and fibroblasts. Seven days after photocoagulation, immunoreactivity for PEDF was still observed in many cells within the laser lesions, but it was especially detected in the spindle-shaped RPE cells that sealed the region of the CNV (Figs. 7C
7D)
. In contrast, immunoreactivity for VEGF was prominently present in endothelial cells of the choroidal neovascular tissues (Figs. 8B)
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Discussion |
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We found a strong expression of PEDF mRNA in the corneal epithelial and endothelial cells and also in the lens epithelial cells. Thus, the cornea and the lens are also sites for the synthesis of ocular PEDF and probably the sites of the PEDF in the aqueous humor and the vitreous. It is interesting that PEDF is expressed in the avascular cornea and lens, because PEDF has been shown to have antiangiogenic activities and to inhibit neovascularization in rat corneas.15
Indirect immunofluorescence and immunohistochemical studies have shown that PEDF antibodies are expressed on both the pigmented and nonpigmented cells that comprise the ciliary epithelium, and more specifically, the plasma membrane domain of the nonpigmented cells in the pars plicata region.32 However, we found expression of PEDF mRNA and immunoreactivity for PEDF predominantly in the nonpigmented epithelium. One reason for this discrepancy may be a species difference (i.e., rat versus cow). However, it should be noted that the PEDF expression in the pigmented epithelial cells may have been masked by the pigments.
An interesting finding was the strong expression of PEDF mRNA in the ganglion cells and PEDF protein in the ganglion cell layer and inner and outer plexiform layers. This indicates that ganglion cells are also a site of PEDF synthesis in the rat eye, and synthesized PEDF may be distributed in the retina and contribute to the high levels in the vitreous (1.6 mg/mL in cows and 1.7 mg/mL in humans).14 33
CNV and Activity of PEDF
PEDF has been shown to inhibit neovascularization in rat corneas, to inhibit the proliferation of capillary endothelial cells, and to inhibit the migration of endothelial cells toward angiogenic inducers such as platelet-derived factor and VEGF. In addition, an underlying stimulation of angiogenesis was observed in PEDF-negative vitreous samples.15
These observations suggest that PEDF is a strong antiangiogenic factor and should act as an inhibitor of ocular angiogenesis.
Recently, we found that PEDF is produced at high levels by regenerating RPE cells after laser photocoagulation.34 This suggests that upregulation of PEDF in RPE cells explains the inhibition and the regression of neovascularization after panphotocoagulation.35 36
We have also observed that the vitreous levels of PEDF in eyes of patients with diabetic retinopathy were lower than in eyes with nondiabetic retinopathy—that is, those with rhegmatogenous retinal detachments and idiopathic macular holes.33 In addition, the level of PEDF in eyes with inactive diabetic retinopathy was higher than in eyes with active diabetic retinopathy. These observations also support the idea that PEDF is a strong inhibitor of ocular angiogenesis.
It is believed that homeostasis of angiogenesis is regulated by two counterbalancing systems: angiogenic stimulators and angiogenic inhibitors. The balance is critical for the regulation of angiogenesis. It has been reported that the angiogenic factors (i.e., VEGF and bFGF) play a major role in mediating intraocular neovascularization,37 38 39 40 and we previously reported that bFGF and VEGF were expressed in the cells in an experimental CNV. We suggested that both factors play a major role in promoting angiogenesis.20 21 22 26 27 However, we have also demonstrated a strong expression of TGF-ß2 in the cells composing the experimental CNV and, because TGF-ß2 inhibits the proliferation of endothelial cells,41 42 it may play a role in controlling the experimentally induced CNV.22 Thus, it has been supposed that a balance between inhibitors and inducers of angiogenesis would be very important in the development of CNV. Gao et al.43 recently reported that there was an unbalanced expression of VEGF and PEDF in ischemia-induced retinal neovascularization. They demonstrated that the retinal PEDF levels, in contrast to that of VEGF, were negatively correlated with pathologic retinal neovascularization. Their results provided evidence supporting the hypothesis that an upset balance between angiogenic stimulators and inhibitors is a cause of pathologic neovascularization.
We have found that the PEDF expression was widely distributed in the laser lesions at 3 days after photocoagulation. Many cells expressing PEDF in these lesions were considered to be proliferating RPE cells, macrophages, and fibroblasts, as described in our previous studies.20 21 22 26 27 31 One and 2 weeks after photocoagulation, CNV had developed, and expression of PEDF was decreased in the cells within the choroidal neovascular tissues, whereas strong expression of PEDF was present in the proliferating RPE cells covering the CNV. The expression of VEGF protein, in contrast, was weakly present in the endothelial cells of the CNV.
Despite the observations indicating that PEDF expression was detected in the laser lesions at 3 days after photocoagulation, CNV had developed. It is more likely that the angiogenic factors (i.e., VEGF and bFGF) would have prevailed over the antiangiogenic activity of PEDF and resulted in the neovascularization, because we found that VEGF protein expression was strongly detected in many cells in the laser lesions. One and 2 weeks after photocoagulation, PEDF was still strongly detected in RPE cells sealing the CNV. Two weeks after photocoagulation, VEGF expression had declined as previously reported26 27 and was detected only weakly in the endothelial cells in choroidal neovascular tissue. Therefore, PEDF, mainly secreted from RPE cells, may have led to the regression of CNV.
More recently, it has been demonstrated that PEDF inhibits aberrant blood vessel growth in a murine model of ischemia-induced retinopathy and that PEDF appears to inhibit angiogenesis by causing apoptosis of activated endothelial cells.16 The investigators suggested that the PEDF action on endothelial cells is likely to be receptor mediated, but by way of a different receptor than that of the survival signal to neural cells.44 Therefore, we suggest that the prominent expression of PEDF in RPE cells may mediate apoptosis of endothelial cells and lead to the regression of CNV.
A recent study showed that a subretinal injection of an adenoviral vector encoding PEDF results in strong inhibition of CNV and retinal neovascularization.45 These results suggest that PEDF is a strong antiangiogenic factor and can be a therapeutic agent for ocular angiogenesis.
In conclusion, our results suggest that because PEDF is an inhibitor of vascular endothelial cell proliferation15 16 and an inhibitor of ocular angiogenesis,33 34 it may play a significant role in the normal rat eye and also in experimentally induced CNV.
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Footnotes |
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Submitted for publication May 15, 2001; revised December 7, 2001; accepted December 18, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Nahoko Ogata, Department of Ophthalmology, Kansai Medical University, Fumizono-cho 10-15, Moriguchi, Osaka 570-8507, Japan; ogata{at}takii.kmu.ac.jp
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