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Biochemistry and Molecular Biology  |   March 2010
Effects of Human Recombinant PEDF Protein and PEDF-Derived Peptide 34-mer on Choroidal Neovascularization
Author Affiliations & Notes
  • Juan Amaral
    From the Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
  • S. Patricia Becerra
    From the Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
  • Corresponding author: S. Patricia Becerra, NEI-NIH, Building 6, Room 134, 6 Center Drive, MSC 0608, Bethesda, MD 20892-0608; becerrap@nei.nih.gov
Investigative Ophthalmology & Visual Science March 2010, Vol.51, 1318-1326. doi:10.1167/iovs.09-4455
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      Juan Amaral, S. Patricia Becerra; Effects of Human Recombinant PEDF Protein and PEDF-Derived Peptide 34-mer on Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2010;51(3):1318-1326. doi: 10.1167/iovs.09-4455.

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      © 2015 Association for Research in Vision and Ophthalmology.

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Purpose. Pigment epithelium-derived factor (PEDF) is a serpin with antiangiogenic properties. Previously, the authors showed that PEDF injected into the subconjunctiva reaches the choroid. Here, they examined the effects of PEDF polypeptide fragments on vessel sprouting and on choroidal neovascularization (CNV) after subconjunctival administration.

Methods. Recombinant human PEDF (rhuPEDF) was cleaved at its serpin-exposed loop by limited chymotrypsin proteolysis. Synthetic PEDF peptides 34-mer (Asp44-Asn77) and 44-mer (Val78-Thr121) were used. Ex vivo chick aortic vessel sprouting assays were performed. CNV was induced in rats by laser injury of Bruch's membrane. Daily subconjunctival injections (0.01–10 pmol/d protein) were performed for 5 days starting at day of injury or at the seventh day after injury. New vessel volumes were quantified using optical sections of choroid/RPE flat-mounts labeled with isolectin-Ib4. PEDF distribution was evaluated by immunofluorescence of choroid/RPE/retina cross-sections.

Results. Full-length rhuPEDF, cleaved rhuPEDF, or peptide 34-mer exhibited ex vivo antiangiogenic activity, but peptide 44-mer was inefficient. PEDF immunostaining around CNV lesions diminished after laser injury. Subconjunctival administration of rhuPEDF or 34-mer at 0.1 pmol/d decreased CNV lesion volumes by 52% and 47%, respectively, whereas those of 44-mer were similar to vehicle injections. Doses of 0.1 and 1 pmol/d rhuPEDF decreased fully developed CNV complex volumes by 45% and 50%, respectively, compared with vehicle injections.

Conclusions. A functional region for the inhibition of vessel sprouting and CNV resides within the 34-mer region of PEDF. Furthermore, subconjunctival administration of optimal range dosages of rhuPEDF or 34-mer can suppress and regress rat CNV lesions, demonstrating that these agents reach the choroid/RPE complex as functionally active molecules.

Pigment epithelium-derived factor (PEDF) is a multifunctional serpin protein present in extracellular compartments of the eye. 1,2 It inhibits angiogenesis both in vitro and in vivo 3,4 and also exhibits neurotrophic activities. 5,6 Several studies have shown a decrease in PEDF protein levels in patients with age-related macular degeneration 7,8 and diabetic retinopathy. 9 Intravitreal injections of PEDF protein can delay the death of retinal cells in animal models of retinal degeneration, 10,11 and intraocular PEDF gene transfer can rescue photoreceptors from light-induced cell damage in an animal model of retinitis pigmentosa. 12,13 Intravitreal, subretinal, and periocular administration of viral vectors encoding PEDF are also effective in suppressing neovascularization in animal models of oxygen-induced retinopathy and choroidal neovascularization (CNV) and in regression of ocular neovascularization. 1418 Moreover, results of a phase I clinical trial have suggested that intravitreous injections of PEDF adenoviral vectors can be a viable approach to inhibit CNV as the treatment of neovascular age-related macular degeneration. 19 These results imply that the transfected tissues express the heterologous PEDF from viral vectors and, subsequently, translate and secrete polypeptide products extracellularly to yield bioactive antiangiogenic PEDF protein. In this regard, subcutaneous administration of “low” dosages of PEDF protein in mice suppresses laser-induced CNV, suggesting that the functional antiangiogenic protein reached the choroid. 20 However, the effects of local administration of PEDF protein on CNV inhibition have not yet been investigated. 
The serpin superfamily of proteins are related through their highly conserved folded conformation. 21 Most serpin members are inhibitors of serine proteases, such as antithrombin III, α-antitrypsin, and α-antichymotrypsin. However, PEDF belongs to the subgroup of serpins that lack antiprotease activity, such as ovalbumin, maspin and angiotensinogen. 5 Serpins with demonstrable antiangiogenic activities include PEDF, 22 maspin, 23 and angiotensinogen. 24 Antithrombin III is also included, but only if its antiprotease activity is abolished by cleavage at the serpin-exposed peptide loop near its carboxy-end. 25 Whereas the serpin-exposed loop is essential for antiprotease activity of serpins, it is dispensable for the antiangiogenic activity of maspin, angiotensinogen, and antithrombin III. 2325 It is not known whether the antiangiogenic activity of PEDF is independent of its serpin-exposed loop. 
The extracellular PEDF is a glycoprotein composed of amino acid residues Asp21-Pro418 (human amino acid sequence numbering) with an apparent molecular weight of 50 kDa. 26 It is highly resistant to proteolysis with the exception of the serpin-exposed loop, which contains the homologous serpin-reactive site. 27 After cleavage of this loop, the remaining core PEDF protein of 46 kDa retains the neurotrophic activity and binding affinities for extracellular matrix components (e.g., glycosaminoglycans and collagens) of the intact PEDF. 2730 More interestingly, the primary and tertiary structures of PEDF contain distinct regions for each of the neurotrophic and antiangiogenic activities and the collagen-, heparin-, and hyaluronan-binding affinities, all which are away from the serpin-exposed loop. 27 A synthetic peptide 44-mer, derived from amino acid positions Val78-Thr121 of human PEDF, is neurotrophically active and binds receptors on the surfaces of target cells. 28,31,32 It can induce neuronal differentiation on retinoblastoma cells, 28 protect the retina from ischemic damage in an ischemia reperfusion animal model, 33 protect spinal cord motor neurons from chronic glutamate toxicity, 32 and induce neuroendocrine differentiation in prostate cancer cells 34 like the full-length PEDF. Another synthetic peptide 34-mer, derived from positions Asp44-Asn77, does not share these neurotrophic activities but, rather, has antiangiogenic properties. 34 It can inhibit human umbilical vein endothelial cell (HUVEC) growth and migration, induce the apoptosis of HUVECs, and suppress corneal angiogenesis 34 like the full-length PEDF. The effects of 34-mer and 44-mer on CNV have not yet been reported. 
Here, we evaluate the antiangiogenic effects of PEDF core (PEDF cleaved at its serpin-exposed loop) and peptides 34-mer and 44-mer using an ex vivo chick aortic ring assay. Given that recombinant human PEDF (rhuPEDF) protein can traverse the sclera from the subconjunctiva and reach the choroid/RPE complex, 35,36 we further investigated the effects of subconjunctival injections of full-length rhuPEDF and PEDF-derived peptides on laser-induced CNV. Our results show that the serpin-exposed loop was dispensable for PEDF antiangiogenic activity and that the 34-mer region contained the CNV inhibitory activity of the full-length PEDF protein. The data imply that after subconjunctival injections, rhuPEDF protein or 34-mer peptide traverse the sclera and reach the choroid/RPE complex as functionally active molecules. 
Materials and Methods
Reagents
Recombinant human PEDF protein was purified from the conditioned media of BHK cells containing the expression vector pMA-PEDF with a full-length human PEDF cDNA under the control of the SV40 promoter. 37 Human angiostatin K1–5 was purchased from Calbiochem (San Diego, CA). Synthetic PEDF peptides 34-mer and 44-mer (Biosynthesis, Inc.) were as described 28 from the human PEDF sequence 44–77 and 78–121 amino acid positions, respectively. 
Cleaved PEDF
Recombinant rhuPEDF was cleaved at its homologous reactive serpin loop by control proteolysis using a method previously described. 27 Briefly, chymotrypsin (Sigma-Aldrich) and rhuPEDF at a protease/substrate ratio of 1:100 (wt/wt) were mixed in buffer A (20 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA [pH 7.4]). After incubation at room temperature for 30 minutes, the reaction was stopped with AEBSF at 0.5 mM final concentration. The cleaved protein product was confirmed by SDS-PAGE using 10% to 20% gradient polyacrylamide gels in SDS-tricine running buffer (Invitrogen, Carlsbad, CA) followed by staining with Coomassie Blue. 
Chick Aortic Ring Assay
The ex vivo chick embryo aortic ring assay was performed as previously described. 38 Briefly, aortic rings were prepared from the aortic arches of 13-day-old chicken embryos (CBT Farms, Chestertown, MD) and were placed in a 48-well plate previously covered with 10 μL synthetic matrix (Matrigel; BD Biosciences, Franklin Lakes, NJ). A total of 300 μL growth-factor–free human endothelial serum-free basal growth medium (Invitrogen) containing the proper concentration of the test substances was added to each well. Endothelial cell growth supplement (ECGS; Biomedical Collaborative Products, Bedford, MA) at a concentration of 400 μg/mL was used as an angiogenesis promoter. 
Four independent rings per treatment were measured. The plates were kept in a humid incubator at 37°C in 5% CO2 for 24 to 36 hours. Microvessels sprouting from each aortic ring were photographed, and the images were saved as TIFF files. Vessel sprouting and ring surfaces in the photographs were measured using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). Vessel/ring ratios were calculated and plotted in a spreadsheet (Excel; Microsoft, Redmond, WA). 
Immunohistochemistry
Eyes were fixed for 1 hour in 4% paraformaldehyde, washed with phosphate-buffered saline (PBS) 1×, and cryoprotected. Eyes were embedded in two parts optimum cutting temperature compound (Tissue-Tek, Sakura-Finetek, Torrance, CA) and one part sucrose 20%, frozen, and cut into 10-μm sections. Immunohistochemical labeling was performed by incubating the sections overnight at 4°C in 5% goat serum with rabbit polyclonal PEDF antibody (catalog no. PED613; BioProducts MD, Middletown, MD) at 1 μg/mL. Alexa 488-conjugated goat anti-rabbit secondary antibodies (Invitrogen-Molecular Probes, Eugene, OR) at 6.6 μg/mL and at 1 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen-Molecular Probes) were applied for 30 minutes at 23°C. Grid confocal microscopy was used to visualize sections (Apotome; Carl Zeiss, Oberkochen, Germany). Files were imported into a graphics editing program (Photoshop; Adobe, Mountain View, CA) and were converted to PSD format for layout purposes. 
Laser-Induced CNV
All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Laser-induced CNV, flat-mount preparations, and lesion evaluation were performed according to established methods and as described. 39 Experimental CNV was induced by laser breakage of Bruch's membrane in Brown Norway rats. Neovessels were visualized with confocal microscopy using choroid/RPE flat-mounts labeled with Alexa Fluor 568-isolectin IB4 (Vector Laboratories, Burlingame, CA) to identify endothelial cells, and neovessel volumes were quantified using high-performance 3D imaging software (Volocity; PerkinElmer, Wellesley, MA), as described. 39  
Subconjunctival Injections
Protein or peptide stock solutions were diluted in PBS and filtered sterilized. Dilutions were prepared so that 2 μL would contain the indicated dosage per animal. Two microliters were injected into the subconjunctiva of each eye while the animal under general anesthesia for restraint. Samples were administered daily. In one set of experiments, injections began immediately after laser injury and were repeated until day 4. Doses of 10, 1, 0.1, and 0.01 pmol rhuPEDF, 34-mer, and 44-mer and doses of 0.1 and 0.01 pmol angiostatin were used per injection. Lasered eyes with no injection, PBS injection, and angiostatin were used as controls. The animals were euthanatized by CO2 exposure at day 7. In another set of experiments, daily rhuPEDF injections started at the seventh day after laser and continued until day 11 after laser. The animals were euthanatized by CO2 exposure at day 14 after laser. 
In preliminary experiments, one eye from each animal was treated with subconjunctival PEDF protein, and its contralateral eye was used as a nontreated control. We observed that the CNV volumes from nontreated controls showed no statistical significant difference when compared with those from animals in which both eyes were nontreated controls (noninjected). Similarly, the CNV volumes from PEDF-treated eyes had no statistical significant difference when compared with those from animals in which both eyes had been treated with similar doses of PEDF, which were lower than their corresponding nontreated controls (Supplementary Fig. S1). In subsequent experiments, both eyes of each rat were nontreated, vehicle treated, or treated with given dosages of protein to minimize the number of animals used in the study and to meet requirements for statistically significant data. 
Statistical Analysis
Statistical analysis was assessed using one-way ANOVA and Dunnett's multiple comparison test. 
Results
Inhibition of Ex Vivo Vessel Sprouting by PEDF Cleaved at Its Serpin-Exposed Loop
The potential of PEDF polypeptide fragments to inhibit vessel sprouting was evaluated ex vivo using aortic ring vessel sprouting assays (Fig. 1). Purified full-length PEDF (rhuPEDF) inhibited vessel sprouting induced by ECGS within the concentration range of 0.1 to 300 nM. The values for relative vessel sprouting (vessel/ring ratio) decreased with 0.1- to 30-nM rhuPEDF additions in a dose-response fashion (Fig. 1B). More than 80% inhibition of vessel sprouting was observed with 10 to 30 nM rhuPEDF (Fig. 1B). However, inhibition was less effective with 100 nM and 300 nM rhuPEDF; only 50% inhibition was observed with the latter concentration. The half-maximal inhibition (IC50) estimated for the concentration range between 0.1 and 30 nM rhuPEDF appeared to be lower than 0.1 nM rhuPEDF. A second set of assays with a different batch of aortic rings and a rhuPEDF concentration range between 0.1 and 100 nM showed that rhuPEDF inhibited vessel sprouting in a dose-response fashion with an estimated IC50 value between 0.4 and 1 nM (20–50 ng/mL rhuPEDF). Angiostatin, an antiangiogenic protein, 40,41 was used as a positive control, and ovalbumin, a serpin with unknown antiangiogenic activity, was used as negative control. As expected, angiostatin at 0.05 nM and 0.5 nM was an effective inhibitor against the angiogenic inducer (ECGS) in this assay (Fig. 1B), and ovalbumin at 10 nM did not inhibit vessel sprouting (JA, data not shown, 2004). 
Figure 1.
 
Inhibition vessel sprouting by PEDF. (A) Representative photographs of vessel sprouting in the presence and absence of 30 nM PEDF. ECGS (400 μg/mL) was used as an angiogenesis promoter. (B) Concentration response of the inhibitory activity of PEDF on vessel sprouting. Relative vessel sprouting was calculated from surface values of vessels and ring. Each point corresponds to the average of four replicate assays. The bars represent standard deviations. Closed circles: PEDF; closed squares: angiostatin (angiogenic inhibitor control). A plot of the relative vessel sprouting, represented by the vessel/ring surface ratio, as function of protein concentration is shown. Inset: concentration response of PEDF and angiostatin at lower concentrations (0–0.5 nM). Horizontal dotted line: negative control was without ECGS. Positive control was with ECGS but without PEDF or angiostatin.
Figure 1.
 
Inhibition vessel sprouting by PEDF. (A) Representative photographs of vessel sprouting in the presence and absence of 30 nM PEDF. ECGS (400 μg/mL) was used as an angiogenesis promoter. (B) Concentration response of the inhibitory activity of PEDF on vessel sprouting. Relative vessel sprouting was calculated from surface values of vessels and ring. Each point corresponds to the average of four replicate assays. The bars represent standard deviations. Closed circles: PEDF; closed squares: angiostatin (angiogenic inhibitor control). A plot of the relative vessel sprouting, represented by the vessel/ring surface ratio, as function of protein concentration is shown. Inset: concentration response of PEDF and angiostatin at lower concentrations (0–0.5 nM). Horizontal dotted line: negative control was without ECGS. Positive control was with ECGS but without PEDF or angiostatin.
PEDF can be cleaved at its serpin-exposed loop by limited proteolysis. 27,29 Chymotrypsin was used in limited proteolysis reactions of rhuPEDF, and the serine protease inhibitor AEBSF was added to stop the reaction. Figure 2A shows that limited chymotrypsin-mediated proteolysis of rhuPEDF yielded a polypeptide product of 46 kDa, which corresponds to the PEDF core as shown previously. 27,29 The small PEDF-derived peptide of approximately 4 kDa was not detectable. Figure 2B shows that the cleaved rhuPEDF protein at 1 nM, 10 nM, and 100 nM, like the unmodified rhuPEDF protein, inhibited vessel sprouting ex vivo (P ≤ 0.05). The additions of AEBSF (0.8, 8, or 80 μM) did not affect the antiangiogenic activity of rhuPEDF. A second set of assays showed similar results and indicated that 100 μM AEBSF alone did not inhibit vessel sprouting induced by ECGS (JA, unpublished findings, 2004). These results indicate that the serpin-exposed loop located toward the carboxy-end of the PEDF polypeptide was dispensable for antiangiogenic activity and that the PEDF core protein was antiangiogenic. 
Figure 2.
 
Antiangiogenic activity of PEDF cleaved at its serpin-exposed loop. (A) Limited proteolysis of PEDF with chymotrypsin (CT +) at a protease/substrate ratio of 1:100 (wt/wt) incubated for 30 minutes at 25°C. Reactions were stopped with 0.5 mM AEBSF, a serine protease inhibitor, and products were resolved by SDS-PAGE. Coomassie blue–-stained 10% to 20% polyacrylamide gel is shown. M, molecular weight standards; PpB, phosphorylase B; BSA, bovine serum albumin; ova, ovalbumin; CA, carbonic anhydrase; Lys, lysozyme. (B) Effects of chymotrypsin-treated PEDF on vessel sprouting. In the x-axis, controls (negative was without ECGS; positive was with ECGS and without effectors) and effectors were additions of reaction mixtures after incubation at 25°C containing the concentrations of each indicated component (PEDF, chymotrypsin, and/or AEBSF) to rings with ECGS. The y-axis shows the relative area of vessel sprouting per ring (vessel/ring surface ratio). Each bar corresponds to the average of four replicates ± SD.
Figure 2.
 
Antiangiogenic activity of PEDF cleaved at its serpin-exposed loop. (A) Limited proteolysis of PEDF with chymotrypsin (CT +) at a protease/substrate ratio of 1:100 (wt/wt) incubated for 30 minutes at 25°C. Reactions were stopped with 0.5 mM AEBSF, a serine protease inhibitor, and products were resolved by SDS-PAGE. Coomassie blue–-stained 10% to 20% polyacrylamide gel is shown. M, molecular weight standards; PpB, phosphorylase B; BSA, bovine serum albumin; ova, ovalbumin; CA, carbonic anhydrase; Lys, lysozyme. (B) Effects of chymotrypsin-treated PEDF on vessel sprouting. In the x-axis, controls (negative was without ECGS; positive was with ECGS and without effectors) and effectors were additions of reaction mixtures after incubation at 25°C containing the concentrations of each indicated component (PEDF, chymotrypsin, and/or AEBSF) to rings with ECGS. The y-axis shows the relative area of vessel sprouting per ring (vessel/ring surface ratio). Each bar corresponds to the average of four replicates ± SD.
Ex Vivo Vessel Sprouting Inhibition by PEDF Peptide 34-mer
Our results suggest that a region outside the serpin-exposed loop contained determinants for the PEDF antiangiogenic function. The potential antiangiogenic effects of 34-mer and 44-mer peptides, derived from regions toward the amino terminus of human PEDF, were assessed in the aortic ring vessel sprouting assay. Figure 3 shows that the 34-mer peptide at concentrations of 1, 10, and 100 nM inhibited ex vivo vessel sprouting, whereas the 44-mer was antiangiogenic only at concentrations of 100 nM. Nonlinear regression analysis of these data estimated a lower IC50 for the 34-mer (∼1.2 nM) than for the 44-mer peptide (∼13 nM). These results indicate that the region composed of amino acid positions 44 to 77 of human PEDF (34-mer) contained a structural determinant for vessel sprouting inhibition of the full-length PEDF polypeptide. 
Figure 3.
 
Effects of PEDF-derived peptides on vessel sprouting. (A) Representative photographs of vessel sprouting in the presence and absence of 10 nM peptide 34-mer. (B) Vessel sprouting assays with 1-, 10-, and 100-nM concentrations of PEDF-derived peptides (x-axis). Negative control was without ECGS; positive control was with ECGS and without peptides. Additions to rings with ECGS are indicated in the x-axis. The relative area of vessel sprouting (y-axis) as a function of peptide concentration was plotted. Each bar corresponds to the average of four replicate assays ± SD.
Figure 3.
 
Effects of PEDF-derived peptides on vessel sprouting. (A) Representative photographs of vessel sprouting in the presence and absence of 10 nM peptide 34-mer. (B) Vessel sprouting assays with 1-, 10-, and 100-nM concentrations of PEDF-derived peptides (x-axis). Negative control was without ECGS; positive control was with ECGS and without peptides. Additions to rings with ECGS are indicated in the x-axis. The relative area of vessel sprouting (y-axis) as a function of peptide concentration was plotted. Each bar corresponds to the average of four replicate assays ± SD.
PEDF Immunostaining in Choroid on Laser-Induced Injury
PEDF protein distribution in CNV lesions was examined in choroid/RPE/retina complexes from rats after 1, 3, 5, and 7 days of laser injury and in those without injury. PEDF immunostaining was intense in the interphotoreceptor matrix of nonlasered eyes (Fig. 4). It was also detected in the RPE and retinal ganglion cell layer at a lower intensity (JA, data not shown, 2009). The observed distribution is in agreement with a previously reported PEDF distribution in RPE/choroid/retina from human, monkey, and rat eyes. 22,4244 However, the PEDF label decreased significantly in the interphotoreceptor matrix and RPE after laser injury (see Fig. 4 for eyes at 1, 3, 5, and 7 days after laser). These observations imply that PEDF protein levels declined in laser-induced CNV as early as 1 day after injury. They are in agreement with other studies on PEDF expression at ≥3 days after injury of experimental CNV. 4446  
Figure 4.
 
Immunolocalization of PEDF in laser-induced CNV. Retina/RPE/choroid cryosections from rats without laser injury and 1, 3, 5, and 7 days after injury stained with anti-PEDF primary antibodies followed by highly absorbed-Alexa 488 (PEDF, green) secondary antibodies. (blue, DAPI). ONL, outer nuclear layer.
Figure 4.
 
Immunolocalization of PEDF in laser-induced CNV. Retina/RPE/choroid cryosections from rats without laser injury and 1, 3, 5, and 7 days after injury stained with anti-PEDF primary antibodies followed by highly absorbed-Alexa 488 (PEDF, green) secondary antibodies. (blue, DAPI). ONL, outer nuclear layer.
Suppression and Regression of Laser-Induced CNV by PEDF Protein
Subconjunctival injections of rhuPEDF protein were evaluated for the suppression of laser-induced CNV in rats. Daily injections of protein solutions started immediately after laser injury. Negative controls were noninjected or vehicle PBS injections, and positive controls were angiostatin injections. Representative projections illustrate that PEDF and angiostatin decreased CNV lesion volume compared with controls (Fig. 5B). Quantitative analysis showed that doses of 0.01, 0.1, 1, and 10 pmol rhuPEDF per day for 5 days suppressed CNV lesion volume by 22%, 52% (P < 0.001), 17%, and 6% CNV, respectively, compared with control animals (both noninjected and vehicle PBS injected) (Fig. 5C). CNV lesion volumes of vehicle PBS-injected animals had median values similar to those of noninjected counterparts. Daily subconjunctival injections of 0.01 and 0.1 pmol angiostatin decreased CNV complex volumes by 13% (P ≤ 0.002) and 40%, respectively, compared with controls. 
Figure 5.
 
Full-length PEDF inhibited the growth of CNV complex lesions. (A) Scheme to illustrate the protocol for PEDF injections for the suppression of neovessel growth. (B) Representative flat-mount projections from confocal microscope Z-series 7 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (C) Box-and-whisker plot representations (four experiments) of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel lesion volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 163). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 0.1 pmol. *Significant or **highly significant difference compared with no injections and PBS.
Figure 5.
 
Full-length PEDF inhibited the growth of CNV complex lesions. (A) Scheme to illustrate the protocol for PEDF injections for the suppression of neovessel growth. (B) Representative flat-mount projections from confocal microscope Z-series 7 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (C) Box-and-whisker plot representations (four experiments) of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel lesion volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 163). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 0.1 pmol. *Significant or **highly significant difference compared with no injections and PBS.
To evaluate the effects of PEDF on CNV regression, daily subconjunctival rhuPEDF injections were begun 7 days after laser injury and continued for 5 days. We have previously described that by postlaser day 7, the CNV lesions are developed in the selected CNV rat model. 39 Figure 6 summarizes the quantification of CNV lesion volumes of the treated rats. It shows that rhuPEDF-treated animals had a significant decrease in volume of developed CNV complexes, with doses of 0.01 pmol/d (31%; P < 0.007), 0.1 pmol/d (42%; P < 0.006), 1 pmol/d (50%; P < 0.001), and 10 pmol/d (36.5%; P < 0.01) compared with vehicle (PBS) controls. Together these results implied that PEDF protein can be effective in the suppression of CNV development and in the regression of developing CNV complexes. 
Figure 6.
 
Full-length PEDF regressed preformed CNV complex lesions. (A) Scheme to illustrate protocol of PEDF injections for regression of fully formed neovesssels. (B) Representative flat-mount projections from epifluorescence microscope 14 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (C) Box-and-whisker plot representations of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel complex volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 143). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 1 pmol. *Significant or **highly significant difference compared with no injections and PBS.
Figure 6.
 
Full-length PEDF regressed preformed CNV complex lesions. (A) Scheme to illustrate protocol of PEDF injections for regression of fully formed neovesssels. (B) Representative flat-mount projections from epifluorescence microscope 14 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (C) Box-and-whisker plot representations of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel complex volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 143). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 1 pmol. *Significant or **highly significant difference compared with no injections and PBS.
Suppression of Laser-Induced CNV by 34-mer Peptide
Synthetic peptides 34-mer and 44-mer were also evaluated for potential inhibition of CNV. Daily subconjunctival injections of 0.1, 1, and 10 pmol 34-mer peptide suppressed CNV lesion volume by 47% (P ≤ 0.001), 35%, and 20% when compared with vehicle (PBS) injections (Fig. 7). The 44-mer had minimum or no effect on neovessel formation. These results demonstrate that 34-mer can be effective in suppressing experimental CNV and imply the location of a structural determinant for CNV inhibition located within residue positions 44 to 77 of the human PEDF polypeptide. 
Figure 7.
 
Peptide 34-mer suppressed the growth of CNV complex lesions. (A) Representative flat-mount projections from confocal microscope 7 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (B) Box-and-whisker plot representations of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel complex volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 170). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 0.1 pmol. *Highly significant difference compared with PBS injections.
Figure 7.
 
Peptide 34-mer suppressed the growth of CNV complex lesions. (A) Representative flat-mount projections from confocal microscope 7 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (B) Box-and-whisker plot representations of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel complex volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 170). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 0.1 pmol. *Highly significant difference compared with PBS injections.
Discussion
Angiogenesis, defined as the sprouting of new blood vessels from preexisting vasculature, is critical in CNV development. In this study, we showed that rhuPEDF or PEDF-derived peptide 34-mer can inhibit vessel sprouting and attenuate choroidal neovascularization. We have previously shown that rhuPEDF can traverse the sclera from the subconjunctiva, reaching the choroid/RPE complex. 35 The present results demonstrate that subconjunctival administration of rhuPEDF or 34-mer can suppress the development and can even provoke the regression of rat CNV lesions. However, optimum doses are to be determined for efficacy in each assay because higher doses become inefficient. These conclusions indicate that, after subconjunctival injections, PEDF and 34-mer, as well as angiostatin, traverse the sclera and reach the choroid/RPE as functional inhibitors of angiogenesis. 
The results have implications on the structure-function relationships of PEDF. The amino acid residue leucine at the homologous serpin reactive site P1 (Leu382) reveals a potential for the serpin PEDF to act as an inhibitor of leucyl-endopeptidases (such as chymotrypsin and cathepsin G). 21,47 Circumstantial evidence discards the involvement of serine protease inhibition in PEDF antiangiogenic effects: (1) PEDF behaves like a substrate rather than an inhibitor of chymotrypsin and chymotrypsin-like proteases 47 ; (2) antiangiogenic activity of serine protease inhibitors antichymotrypsin 48 and AEBSF (unpublished results; JA 2004) is lacking; and (3) chymotrypsin does not have demonstrable proangiogenic or antiangiogenic activities. More conclusively, in this study we show that the serpin-exposed loop of PEDF is dispensable for its antiangiogenic activity. Together with antiangiogenic activity of cleaved serpins maspin, 23 angiotensinogen, 24 antithrombin III, 25 and protease C inhibitor, 49 it is clearly demonstrated that the molecular pathway by which these serpins block angiogenesis is independent of serine protease inhibition. 
The results also show that even when approximately 85% from the carboxy-end is removed (i.e., 34-mer), PEDF retains the antivessel sprouting and anti-CNV activities of the full-length polypeptide. They are in agreement with the inhibitory effects of 34-mer (but not 44-mer) on HUVEC migration and cell growth and on corneal angiogenesis. 34 Although the 34-mer appears less effective than rhuPEDF (full-length or cleaved) for antivessel sprouting, the dose-response curves and optimum dosages (0.1 pmol/d) for anti-CNV are similar for both rhuPEDF and 34-mer. Thus, a functional region for suppressing CNV and vessel sprouting resides within Asp44-Thr77 of PEDF, whereas the neurotrophic 44-mer (Val78-Thr121) is not an efficient antiangiogenic peptide. In the 3D configuration of PEDF, 26 the antiangiogenic structural determinant forms an α-helix surrounded by several amino acid residues with negatively charged side chains on the face with acidic surface potential. This region is next to the collagen-binding site. 30 The opposite face has basic surface potential and contains a hyaluronan-binding region. 1,50 It is presumed that the acidic, negatively charged nature of the 34-mer environment is available to attract positively charged molecules. It has been recently reported that the laminin receptor is involved in the antiangiogenic activity of PEDF and binds 34-mer. 51 Another possible receptor is PEDF-R, a lipase-linked cell membrane receptor for PEDF. 52 Sequence comparison between 34-mer and TIMP-3, a naturally occurring antiangiogenic factor in the choroid/RPE, 53 reveals alignment of a region with potential structural determinants for the inhibition of CNV (Supplementary Fig. S2). This region is also present in P18, a recently identified shorter peptide with antiangiogenic activity derived from 34-mer. 54 Although the identity of the receptor for 34-mer that mediates CNV angiostatic effects remains to be determined, the present study identifies 34-mer as an inhibitory region of PEDF for CNV. 
Given the decrease in PEDF levels at the site of laser-induced CNV lesion (Fig. 4) and in the human choroid of patients with age-related macular degeneration, 8 it is proposed that local administration of PEDF (full-length or 34-mer) can serve to balance and restore the deficit of the antiangiogenic agent to the CNV region. It is clear that the increase in local levels of PEDF protein by intraocular PEDF gene transfer is what prevents CNV. 1518 There are obvious advantages in administering highly purified protein over heterogeneous agents. From our previous study on transscleral-RPE permeability of PEDF and ovalbumin proteins, 35 we estimate that <1% of the subconjunctivally injected material reaches the rat choroid/RPE by the first hour and decreases to <0.1% by 24 hours. Subsequent daily protein injections would result in cycles of rhuPEDF levels in the choroid/RPE during the 5 days of administration (1–0.1 fmol in the 1st and 24th hours after injection, respectively, with a 0.1 pmol rhuPEDF dosage), followed by no increase in the last 2 days before evaluation, during which the rats were not injected. 
We compared dosages among agents used in our study (Supplementary Fig. S3) and those reported elsewhere. Curves for rhuPEDF and 34-mer showed an inverted bell-shape effect for CNV suppression, with dose-dependent decreases of CNV volumes until the optimum dose was reached (0.1 pmol/d) and then inverted dependence as the dosage increased. The antivessel sprouting effects of rhuPEDF also insinuated reversal with ≥100 nM rhuPEDF. Although PEDF appeared less potent than angiostatin in inhibiting vessel sprouting (Fig. 1), its efficacy for inhibiting endothelial (HUVEC and HMVEC) cell migration 22 and suppressing CNV was somewhat higher than that of angiostatin (0.01 and 0.1 pmol/d). Comparison between rhuPEDF curves for CNV suppression and regression reveals a 10-fold difference in dosage, suggesting higher PEDF efficiencies in preventing CNV progress than in regressing preformed CNV lesions. Although the highest 44-mer concentration (100 nM) examined in the ex vivo assay may be below the range with reversal effects, the observation that peptide 44-mer exhibits CNV stimulatory effects at the highest dose (10 pmol/d) suggests a potential contribution of this region to the proangiogenic activity at higher PEDF concentration per dosage. Apte et al. 20 have reported that low doses of PEDF protein administered subcutaneously suppress CNV, in contrast to higher doses, which stimulate angiogenesis. We have calculated their maximum dosages to be 17.3-fold the highest ones used in the present study (173 pmol PEDF administered in skin at constant flow per day for 7 days compared with 10 pmol subconjunctival PEDF injection each day for 5 days), whereas anti-CNV effects were demonstrated with one-fourth of that (estimated as 43.2 pmol/d). We conclude that optimum effects are obtained with less protein administered subconjunctivally, representing an economic advantage over subcutaneous administration. Yang et al. 55 also observed a reversal dosage effect for angiostatin, with low dosages decreasing hepatic micrometastasis but higher dosages being inefficient. Both groups of investigators show evidence of increased VEGF production as the concentration of the antiangiogenic factors is raised (e.g., >100 nM PEDF). VEGF induction may explain the inverted bell-shape dosage effects of PEDF observed in the present study. In summary, although caution is to be exerted when dosing PEDF-based compounds, use of purified recombinant human PEDF protein or synthetic PEDF-derived peptide 34-mer through a local route may prove beneficial to inhibit CNV. 
Supplementary Materials
 
 
 
The authors thank Ben Burkham, Marlen Diaz, Travis Aleman, and Marcus Tremblay for technical assistance with ex vivo neovascularization and tube formation assays, Julian Lajoy for assistance with quantification of CNV complex volumes, and Monika Deshpande and Preeti Subramanian for interesting discussions and proofreading the manuscript. 
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Figure 1.
 
Inhibition vessel sprouting by PEDF. (A) Representative photographs of vessel sprouting in the presence and absence of 30 nM PEDF. ECGS (400 μg/mL) was used as an angiogenesis promoter. (B) Concentration response of the inhibitory activity of PEDF on vessel sprouting. Relative vessel sprouting was calculated from surface values of vessels and ring. Each point corresponds to the average of four replicate assays. The bars represent standard deviations. Closed circles: PEDF; closed squares: angiostatin (angiogenic inhibitor control). A plot of the relative vessel sprouting, represented by the vessel/ring surface ratio, as function of protein concentration is shown. Inset: concentration response of PEDF and angiostatin at lower concentrations (0–0.5 nM). Horizontal dotted line: negative control was without ECGS. Positive control was with ECGS but without PEDF or angiostatin.
Figure 1.
 
Inhibition vessel sprouting by PEDF. (A) Representative photographs of vessel sprouting in the presence and absence of 30 nM PEDF. ECGS (400 μg/mL) was used as an angiogenesis promoter. (B) Concentration response of the inhibitory activity of PEDF on vessel sprouting. Relative vessel sprouting was calculated from surface values of vessels and ring. Each point corresponds to the average of four replicate assays. The bars represent standard deviations. Closed circles: PEDF; closed squares: angiostatin (angiogenic inhibitor control). A plot of the relative vessel sprouting, represented by the vessel/ring surface ratio, as function of protein concentration is shown. Inset: concentration response of PEDF and angiostatin at lower concentrations (0–0.5 nM). Horizontal dotted line: negative control was without ECGS. Positive control was with ECGS but without PEDF or angiostatin.
Figure 2.
 
Antiangiogenic activity of PEDF cleaved at its serpin-exposed loop. (A) Limited proteolysis of PEDF with chymotrypsin (CT +) at a protease/substrate ratio of 1:100 (wt/wt) incubated for 30 minutes at 25°C. Reactions were stopped with 0.5 mM AEBSF, a serine protease inhibitor, and products were resolved by SDS-PAGE. Coomassie blue–-stained 10% to 20% polyacrylamide gel is shown. M, molecular weight standards; PpB, phosphorylase B; BSA, bovine serum albumin; ova, ovalbumin; CA, carbonic anhydrase; Lys, lysozyme. (B) Effects of chymotrypsin-treated PEDF on vessel sprouting. In the x-axis, controls (negative was without ECGS; positive was with ECGS and without effectors) and effectors were additions of reaction mixtures after incubation at 25°C containing the concentrations of each indicated component (PEDF, chymotrypsin, and/or AEBSF) to rings with ECGS. The y-axis shows the relative area of vessel sprouting per ring (vessel/ring surface ratio). Each bar corresponds to the average of four replicates ± SD.
Figure 2.
 
Antiangiogenic activity of PEDF cleaved at its serpin-exposed loop. (A) Limited proteolysis of PEDF with chymotrypsin (CT +) at a protease/substrate ratio of 1:100 (wt/wt) incubated for 30 minutes at 25°C. Reactions were stopped with 0.5 mM AEBSF, a serine protease inhibitor, and products were resolved by SDS-PAGE. Coomassie blue–-stained 10% to 20% polyacrylamide gel is shown. M, molecular weight standards; PpB, phosphorylase B; BSA, bovine serum albumin; ova, ovalbumin; CA, carbonic anhydrase; Lys, lysozyme. (B) Effects of chymotrypsin-treated PEDF on vessel sprouting. In the x-axis, controls (negative was without ECGS; positive was with ECGS and without effectors) and effectors were additions of reaction mixtures after incubation at 25°C containing the concentrations of each indicated component (PEDF, chymotrypsin, and/or AEBSF) to rings with ECGS. The y-axis shows the relative area of vessel sprouting per ring (vessel/ring surface ratio). Each bar corresponds to the average of four replicates ± SD.
Figure 3.
 
Effects of PEDF-derived peptides on vessel sprouting. (A) Representative photographs of vessel sprouting in the presence and absence of 10 nM peptide 34-mer. (B) Vessel sprouting assays with 1-, 10-, and 100-nM concentrations of PEDF-derived peptides (x-axis). Negative control was without ECGS; positive control was with ECGS and without peptides. Additions to rings with ECGS are indicated in the x-axis. The relative area of vessel sprouting (y-axis) as a function of peptide concentration was plotted. Each bar corresponds to the average of four replicate assays ± SD.
Figure 3.
 
Effects of PEDF-derived peptides on vessel sprouting. (A) Representative photographs of vessel sprouting in the presence and absence of 10 nM peptide 34-mer. (B) Vessel sprouting assays with 1-, 10-, and 100-nM concentrations of PEDF-derived peptides (x-axis). Negative control was without ECGS; positive control was with ECGS and without peptides. Additions to rings with ECGS are indicated in the x-axis. The relative area of vessel sprouting (y-axis) as a function of peptide concentration was plotted. Each bar corresponds to the average of four replicate assays ± SD.
Figure 4.
 
Immunolocalization of PEDF in laser-induced CNV. Retina/RPE/choroid cryosections from rats without laser injury and 1, 3, 5, and 7 days after injury stained with anti-PEDF primary antibodies followed by highly absorbed-Alexa 488 (PEDF, green) secondary antibodies. (blue, DAPI). ONL, outer nuclear layer.
Figure 4.
 
Immunolocalization of PEDF in laser-induced CNV. Retina/RPE/choroid cryosections from rats without laser injury and 1, 3, 5, and 7 days after injury stained with anti-PEDF primary antibodies followed by highly absorbed-Alexa 488 (PEDF, green) secondary antibodies. (blue, DAPI). ONL, outer nuclear layer.
Figure 5.
 
Full-length PEDF inhibited the growth of CNV complex lesions. (A) Scheme to illustrate the protocol for PEDF injections for the suppression of neovessel growth. (B) Representative flat-mount projections from confocal microscope Z-series 7 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (C) Box-and-whisker plot representations (four experiments) of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel lesion volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 163). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 0.1 pmol. *Significant or **highly significant difference compared with no injections and PBS.
Figure 5.
 
Full-length PEDF inhibited the growth of CNV complex lesions. (A) Scheme to illustrate the protocol for PEDF injections for the suppression of neovessel growth. (B) Representative flat-mount projections from confocal microscope Z-series 7 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (C) Box-and-whisker plot representations (four experiments) of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel lesion volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 163). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 0.1 pmol. *Significant or **highly significant difference compared with no injections and PBS.
Figure 6.
 
Full-length PEDF regressed preformed CNV complex lesions. (A) Scheme to illustrate protocol of PEDF injections for regression of fully formed neovesssels. (B) Representative flat-mount projections from epifluorescence microscope 14 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (C) Box-and-whisker plot representations of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel complex volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 143). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 1 pmol. *Significant or **highly significant difference compared with no injections and PBS.
Figure 6.
 
Full-length PEDF regressed preformed CNV complex lesions. (A) Scheme to illustrate protocol of PEDF injections for regression of fully formed neovesssels. (B) Representative flat-mount projections from epifluorescence microscope 14 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (C) Box-and-whisker plot representations of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel complex volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 143). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 1 pmol. *Significant or **highly significant difference compared with no injections and PBS.
Figure 7.
 
Peptide 34-mer suppressed the growth of CNV complex lesions. (A) Representative flat-mount projections from confocal microscope 7 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (B) Box-and-whisker plot representations of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel complex volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 170). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 0.1 pmol. *Highly significant difference compared with PBS injections.
Figure 7.
 
Peptide 34-mer suppressed the growth of CNV complex lesions. (A) Representative flat-mount projections from confocal microscope 7 days after laser. The red channel identifies vessels (isolectin IB-4). Conditions are indicated below each projection. (B) Box-and-whisker plot representations of volume of CNV lesions from rats treated with daily administrations of effectors, as indicated on the x-axis. The y-axis represents neovessel complex volume expressed in cubic micrometers. Each point corresponds to one CNV lesion (n = 170). Values inside the boxes correspond to the central 50% of measurements, their internal horizontal bars correspond to median values, and the vertical lines outside the boxes correspond to variances of measurements. The horizontal dotted red lines correspond to the median values (indicated to the right) of PBS and PEDF 0.1 pmol. *Highly significant difference compared with PBS injections.
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
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