Pathological retinal neovascularization and choroidal neovascularization are major causes of vision loss in a variety of clinical conditions, such as retinopathy of prematurity, age-related macular degeneration, and diabetic retinopathy. Pigment epithelial-derived factor (PEDF) has been found to be the most potent natural, endogenous inhibitor of neovascularization, but its application is restricted because of its instability and short half-life. Polyethylene glycol (PEG) has been used as a drug carrier to slow clearance rate for decades. The present study investigated PEGylated-PEDF for the first time and evaluated its long-term effects on preventing angiogenesis in vitro and in vivo. PEG showed lower cytotoxicity to human umbilical vein endothelial cells (HUVECs). In vitro, PEGylated-PEDF inhibited HUVEC proliferation, migration, tube formation, and vascular endothelium growth factor secretion and induced HUVEC apoptosis in a dose-dependent manner, and it showed a statistically significant difference compared with the PEDF treatment group. In vivo, PEGylated-PEDF had a long-lasting effect in both plasma and retinal concentrations. In an oxygen-induced retinopathy model, one intravitreous injection of PEGylated-PEDF after mouse pups were moved into room air resulted in a significant difference in the inhibition of retinal neovascularization, which decreased the nonperfusion area, compared with the PEDF-treated group. Our present study demonstrated for the first time the long-term inhibitory effects of PEGylated-PEDF on the prevention of neovascularization in vitro and in vivo. These data suggest that PEGylated-PEDF could offer an innovative therapeutic strategy for preventing retinal neovascularization.
Retinal neovascularization develops in various retinopathies associated with retinal ischemia, inflammatory diseases, chronic retinal detachment, etc. (Gariano, 2010). These disorders include retinopathy of prematurity (ROP) (Chen and Smith, 2007), diabetic retinopathy (Cheung et al., 2010), and age-related macular degeneration (Mousa and Mousa, 2010). Complications resulting from uncontrolled retinal angiogenesis account for the major causes of severe and irreversible loss of vision throughout the world. In recent decades, numerous studies have noted that retinal neovascularization is driven by the production of proangiogenic growth factors including vascular endothelium growth factor (VEGF), basic fibroblast growth factor, placental-like growth factor, and transforming growth factor-β (Chappelow and Kaiser, 2008). Thus, antiangiogenic drugs, which target proangiogenic growth cytokines, came into being. VEGF, which belongs to the platelet-derived growth factor supergene family, plays a critical role in angiogenesis. Overexpression of VEGF exacerbates neovascularization, whereas withdrawal of VEGF or blocking VEGF or the receptors causes the suppression of vascular growth and regression (Mousa and Mousa, 2010).
Pigment epithelial-derived factor (PEDF) has been reported to be the most potent endogenous, natural antiangiogenic agent (Dawson et al., 1999). Previous research has revealed that PEDF is down-regulated in ROP, and vitreous injection of exogenous PEDF reduces areas of nonvascularization and induces apoptosis of vascular endothelial cells that have been stimulated to generate retinal neovascularization (Tombran-Tink et al., 1991; Gao et al., 2002). However, the application of PEDF is limited, because of the route of delivery and difficulty in sustaining an active state; as such, there is great demand for maintaining PEDF tissue level for an extended period of time.
Polyethylene glycol (PEG), with the structure of HO-CH2-(CH2-O-CH2-)n-CH2-OH (n = number of entities), is a polyether compound with many applications in medicine (Reddy, 2000; Pai et al., 2009). PEG has been the focus of extensive research as a carrier of therapeutic proteins, because of its longer half-life, stable plasma and tissue concentrations, superior physical and thermal stability, greater biocompatibility, and lower biodegradability (Yowell and Blackwell, 2002; Parveen and Sahoo, 2006). PEGylation is the act of covalently coupling a PEG structure to another larger molecule, such as a protein (which is then referred to as PEGylated) (Abuchowski et al., 1977). In the 1990s, PEG-modified adenosine deaminase was manufactured by Dow Chemical (Midland, MI) for pharmaceutical use (Parveen and Sahoo, 2006). PEGylated interferon alfa-2a and -2b have become commonly used injectable treatments for hepatitis C infection (Wedemeyer et al., 2002). In addition, the first FDA-approved, intravitreously injected, antiangiogenic, anti-VEGF medication, pegaptanib (brand name Macugen; Pfizer, New York), is also PEGylated (Gragoudas et al., 2004).
Because PEGylation can potentially address some of the problems associated with the delivery of PEDF, in the present study we investigated PEGylated-PEDF for the first time and evaluated its antiangiogenic effect, both in vitro and in vivo. We explored whether PEDF covalent with PEG can play a long-term inhibiting role in human umbilical vein endothelial cells (HUVECs) in vitro and prevent retinal neovascularization in oxygen-induced retinopathy (OIR) mice models in vivo. The encouraging results of our study provide an innovative strategy for the therapy of retinal neovascularization.
Materials and Methods
Construction of Expression Plasmid pET28a(+)-PEDF.
The human PEDF-encoding gene was subcloned into the expression vector pET28a(+) plasmid (Novagen, Darmstadt, Germany) with a DNA Ligation Kit (Takara, Kyoto, Japan). Competent Escherichia coli JM109 cells (Takara) were transformed with pET28a(+)-PEDF plasmids, and positive clones were selected by the blue/white screening method and confirmed by restriction enzyme analysis with BamHI and XhoI.
Expression of Recombinant PEDF in E. coli BL21 (DE3).
Purified recombinant vector pET28a(+)-PEDF plasmid was transformed into E. coli BL21 cells (DE3; Agilent Technologies, Santa Clara, CA) with the aid of the inducer isopropyl-d-thiogalactopyranoside, and the E. coli cells were harvested by centrifugation. PEDF was expressed as inclusion body protein products, and it underwent purification, renaturation, and repurification procedures.
Recombinant human PEDF was dissolved in 30 mM sodium acetate solution, pH 5.5, and its concentration was adjusted to 3 to 8 mg/ml. PEG (molecular mass 20 kDa; Sigma, St. Louis, MO) was added to the PEDF solution in a 1:2 PEG-to-PEDF molar ratio, while reducing agent CH3BNNa was added at a final concentration of 20 mM. The pH was adjusted to 5.1 to 5.3, the reaction solution was stirred at room temperature for 4 to 6 h, and it was allowed to rest at 4°C for 10 h.
After 20-kDa PEG was covalent to recombinant PEDF in the N terminus, a cation exchange column was used to separate and purify the PEGylated-PEDF. Based on the strength of charge in modified and unmodified protein, PEDF, PEGylated-PEDF was eluted. Collected proteins were detected by SDS-polyacrylamide gel electrophoresis.
Cells and Animals.
HUVECs were obtained from the American Tissue Culture Collection (Manassas, VA) and preserved in our laboratory (Yu et al., 2003). The HUVECs were cultured in DMEM with 10% fetal bovine serum (FBS; Hyclone, Grand Island, NY) in a 37°C humidified incubator with 5% CO2 atmosphere.
Neonatal mice (C57BL/6J) and Wistar rats (∼200–225 g) were obtained from Peking University animal center and raised in the animal room of Peking University People's Hospital. This study adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and was performed in accordance with the guidelines provided by the Animal Care Use Committee of Peking University (Beijing, China). The animals were housed with free access to laboratory food and water ad libitum and kept in a 12-h light/dark cycle.
Plasma Concentration Detection.
Eight healthy Wistar rats were divided into two groups. A single injection of recombinant modified and unmodified PEDF was administered through the tail vein at a dose of 1 mg/kg body weight. PEDF plasma concentrations were measured 5, 10, 20, and 40 min and 1, 2, 4, 8, 16, 24, 48, 72, 96, 120, 168, 216, and 264 h after injection by detection of tail vein blood serum, using a PEDF enzyme-linked immunosorbent assay (ELISA) kit (Adlitteram Diagnostic, San Diego, CA).
Retinal PEDF Concentration Detection.
Thirty-six healthy C57 pups were injected with PEDF or PEGylated-PEDF intravitreously on postnatal day 12 (P12). All of the drugs were injected with 1.5 μl PEDF or PEGylated-PEDF, and the concentration was 5 μg/μl. Three pups from each group were euthanized at 12, 24, 48, 72, 96, and 120 h after injection, and the retinas were separated for PEDF detection. The concentration of PEDF in the clarified supernatant was measured by ELISA (Adlitteram Diagnostic). All ELISA experiments were performed in triplicate and repeated three times.
HUVEC Proliferation Assays.
HUVECs were used to evaluate the effects of PEGylated-PEDF on angiogenesis in vitro by using the Cell Counting Kit-8 (CCK-8; Dojindo, Shanghai, China) assay. In brief, the HUVECs were synchronized in DMEM at a density of 1 × 104 per well in 96-well plates overnight without FBS. PEDF (∼10−1 to 10−7 mg/ml), PEGylated-PEDF (∼10−1 to 10−7 mg/ml), and PEG (∼1.0–1.0 × 10−2 mg/ml), diluted into a series of 1:10 concentrations, were incubated for another 24, 48, 72, 96, and 120 h. After adding 10 μl of CCK-8 to each well, the cells were incubated at 37°C for another ∼30–60 min. Absorbance was measured with an ELISA plate reader at a wavelength of 450 nm. Each experiment was performed in five wells and duplicated at least three times.
To study the angiogenic inhibitory effects of PEGylated-PEDF under the stimulation of VEGF, 20 ng/ml VEGF were added to the culture medium. PEDF, PEGylated-PEDF, and PEG, in the same concentrations as tested above, were incubated for another 24, 48, and 72 h, and CCK-8 assays were performed as stated previously.
HUVEC Cell Migration Assay.
Migration was assayed by Transwell (Corning Life Sciences, Lowell, MA) with a pore size of 8.0 μm as described previously (Huang et al., 2010). In brief, 2 × 104 cells were placed in the top part of a Transwell in 200 μl of serum-free medium. DMEM (containing 10% FBS) with 10−2 mg/ml, 10−4 mg/ml, and 10−6 mg/ml PEDF or PEGylated-PEDF, and 1.0, 0.01, and 0.001 mg/ml PEG were placed in the bottom chamber, for a final volume of 600 μl. All migration assays were conducted at 37°C for 6 h. At the end of the assay, the cells were fixed in 4% paraformaldehyde and stained with 4,6-diamidino-2-phenylindole (Roche Diagnostics, Indianapolis, IN) for 15 min. The cells that had not migrated were removed with a cotton swab, and the membrane was imaged. Cells from five random fields of view were counted.
VEGF Detection by Enzyme-Linked Immunosorbent Assay.
The HUVECs were seeded in 96-well plates (1 × 104 per well) and incubated at 37°C overnight. PEDF (∼10−1 to 10−7 mg/ml) or PEGylated-PEDF (∼10−1 to 10−7 mg/ml) was added to the wells after removing the DMEM. After 48 and 72 h of incubation, the cell culture supernatant was harvested and centrifuged to remove cellular debris. VEGF protein secreted by HUVEC cells in the culture medium was measured with an VEGF ELISA Kit (Boster, Wuhan, China), according to the manufacturer's instructions.
Flow Cytometry Analysis of HUVEC Apoptosis.
Apoptosis was measured with a FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Diego, CA), according to the manufacturer's instructions. In brief, HUVECs (1 × 106) were seeded in six-well plates and incubated for 24, 48, and 72 h with 10−2 mg/ml, 10−4 mg/ml, 10−6 mg/ml PEDF, PEGylated-PEDF, or controls. Then, the cells were detached with EDTA, washed in cold phosphate-buffered saline (PBS), and stained with Annexin V-FITC and propidium iodide (PI), according to the manufacturers' instructions. Flow cytometry analysis was immediately performed (excitation 488 nm; emission 530 nm). The samples were analyzed by using a flow cytometer (FACSCalibur; BD Bioscience) with CellQuest software (BD Biosciences). Then, 104 cells were collected and divided into four groups: dead cells (Annexin V−/PI+; UL), late apoptotic cells (Annexin V+/PI+; UR), viable cells (Annexin V−/PI−; LL), and early apoptotic cells (Annexin V+/PI−; LR). The apoptotic rate was calculated as the percentage of early apoptotic cells (LR) plus late apoptotic cells (UR).
Tube Formation Study.
Tube formation study is a convenient and quantifiable assay for testing the angiogenic/antiangiogenic properties of compounds on vascular endothelial cells. This assay measures the ability of endothelial cells to form capillary-like structures. Upon plating at subconfluent densities with the appropriate extracellular matrix support, endothelial cells attach and generate mechanical forces on the surrounding extracellular support matrix to create tracks or guidance pathways that facilitate cellular migration. The resulting cords of cells eventually form hollow lumens. In our study, tube formation assay was used to assess the antiangiogenic potential of PEGylated-PEDF in vitro. Aliquots (150 μl) of Matrigel (BD Biosciences) solution were poured into 48-well plates, and then they were incubated at 37°C for 30 min in a 5% CO2 incubator. HUVECs (5 × 104 per well) treated with PEDF (10−2 mg/ml, 10−4 mg/ml, and 10−6 mg/ml), PEGylated-PEDF (10−2 mg/ml, 10−4 mg/ml, and 10−6 mg/ml), or controls, were seeded on the Matrigel and cultured in DMEM for 12 h. The networks in Matrigel from five randomly chosen fields were counted and photographed under a microscope. The experiments were performed in triplicate and repeated three times.
Induction of Oxygen-Induced Retinopathy Mouse Model and Assessment of Angiography.
Beginning on P7, the C57BL/6 pups were exposed to hyperoxia (75% oxygen) for 5 days. On P12, the animals were brought back to normoxia (room air) to induce retinal neovascularization until P17 (Smith et al., 1994). At P12, the OIR mice were injected intravitreally with PEDF, PEGylated-PEDF, PEG, and PBS. The volume of the vitreous body injection was 1.5 μl, and the concentration of PEDF, PEGylated-PEDF, and PEG was 5 μg/μl. At P18, the mice were deeply anesthetized intraperitoneally with chloral hydrate (0.2 ml/10 g body weight) and then perfused through the left ventricle with 0.5 ml of PBS containing 50 mg of 2 × 106 molecular weight fluorescein-dextran-FITC (Sigma, St. Louis, MO). The eyes were removed and fixed in 4% paraformaldehyde for 30 min, and the retinas were flat-mounted by four peripheral retinal cuts (Supplemental Fig. 1). The retinas were viewed by fluorescence microscopy (Axiophot; Carl Zeiss Inc., Thornwood, NY) and photographed. The nonperfused areas were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD). The ratio was determined by the nonperfusion area compared with the area of the whole retina (Supplemental Fig. 1). The data were analyzed by two people, one of whom was completely blind to the study groups, and the average was considered to be the final value for statistics.
Data analysis was performed by using statistical software, Prism 5 (GraphPad Software Inc., San Diego, CA). All data are presented as mean ± S.E.M. and have been evaluated for normality of distribution. Differences were evaluated with analysis of variance, followed by the Student-Newman-Keuls test for multiple comparisons and the Student's t test for pairwise comparisons. For ELISA results, the data were analyzed by two-way analysis of variance, followed by Bonferroni post tests for comparisons among the two groups. p < 0.05 was considered to be a statistically significant difference.
The recombinant vector pET28a(+)-PEDF was confirmed by restriction enzyme BamHI and XhoI digestion. Recombinant bacteria BL21 (DE3)/pET28a(+)-PEDF were cultured with the aid of isopropyl-d-thiogalactopyranoside. After purification, renaturation, and repurification, the purity of PEDF reached 95%, and the molecular weight was 44,417.8. The PEDF protein sequence is shown in Table 1. According to the process described under Materials and Methods, 20-kDa PEG was covalent to recombinant PEDF in the N terminus at a ratio of 70% (Fig. 1). After the cation exchange column was used to separate and purify PEGylated-PEDF, the collected proteins were detected by SDS-polyacrylamide gel electrophoresis. The purification process map is shown in Fig. 2.
Long-Lasting Effects of PEGylated-PEDF in Rat Plasma Concentration and Mice Retina.
To determine the in vivo drug metabolism rate in systemic blood circulation, plasma concentration was measured in the Wistar rats; the results are shown in Table 2. The half-life of recombinant PEDF is 4.5 h, whereas the half-life of modified PEGylated-PEDF is 78 h (Fig. 3). At the same time, we detected the concentration of recombinant PEDF and PEGylated-PEDF in the retinas of healthy mouse pups. As shown in Fig. 4, recombinant PEDF had a low concentration after 48 h, whereas PEGylated-PEDF had a long-lasting retinal concentration, even at 120 h, at a concentration of 320 ng/ml.
Effects of PEGylated-PEDF on HUVEC Proliferation Study.
A HUVEC proliferation study was used to evaluate the antiangiogenic effect of PEGylated-PEDF in vitro. HUVECs were incubated for 48, 72, 96, and 120 h at various concentrations (∼10−1 to 10−7 mg/ml). PEG had no effect on the proliferation of HUVECs at concentrations lower than 0.1 mg/ml compared with blank controls (DMEM + 10% FBS) (p > 0.05) (Fig. 5). Thus, the maximum concentration of PEGylated-PEDF we chose was 10−1 mg/ml. As shown in Fig. 5 and Table 3, the PEGylated-PEDF-treated groups were statistically different compared with the PEDF-treated groups until 96 h at ∼10−2 to 10−5 mg/ml and 120 h at ∼10−3 to 10−5 mg/ml. To further evaluate the inhibitory effect of PEGylated-PEDF on VEGF stimulation, we added 20 ng/ml VEGF to the cell culture medium and found that PEGylated-PEDF also exhibited better inhibitory effects on HUVEC proliferation (Fig. 6 and Table 4).
Effects of PEGylated-PEDF on HUVEC Migration Study.
A HUVEC migration study was assessed by using Transwell. As shown in Figs. 7 and 8, the number of cells that crossed the membrane in the PEGylated-PEDF-treated HUVEC groups was significantly lower than the number in the PEDF and DMEM + 10% FBS groups at both 10−4 and10−6 mg/ml (p < 0.05).
Effects of PEGylated-PEDF on HUVEC Apoptosis Study.
FACS was used to evaluate early and late apoptosis effects. As shown in Table 5 and Fig. 9, after incubation with PEDF and PEGylated-PEDF combined with 10% FBS for 24, 48, and 72 h, the early and late apoptotic HUVECs showed significant differences in the 10−2 and 10−4 mg/ml-treated groups, with the percentage of apoptotic cells (UR+LR) significantly higher than the PEDF-treated cells (p < 0.05).
Effects of PEGylated-PEDF on Tube Formation.
Matrigel assay is one of the most widely used methods for evaluating the reorganization of angiogenesis in vitro. In our study, both PEDF and PEGylated-PEDF showed impaired capacity to form a regular network, as expected: HUVECs in both groups could not form hollow lumens (Figs. 10 and 11). Although there was no significant difference between the PEGylated-PEDF- and PEDF-treated groups in lumen formation, the PEGylated-PEDF-treated HUVECs showed statistical differences in length compared with the PEDF-treated groups and presented round morphology and no branches at all, whereas the PEDF-treated group had a tendency to form tubes.
Effects of PEGylated-PEDF on the Prevention of VEGF Secretion.
Correlated with the antiangiogenic effect in vitro (tube formation), PEDF- and PEGylated-PEDF-treated HUVECs showed a decrease in VEGF secretion levels in a time-dependent and dose-dependent manner. As shown in Fig. 12, after treatment for 48 h VEGF was down-regulated in the PEDF groups until 10−5 mg/ml, whereas the PEGylated-PEDF group inhibited VEGF secretion even at 10−7 mg/ml; the difference between the groups is significant (p < 0.01). After treatment for 72 h, the PEGylated-PEDF-treated group showed good inhibitory effects on VEGF secretion, which was significantly different from the PEDF-treated group (p < 0.01).
PEGylated-PEDF Protects Against OIR Retinal Vessel Loss.
Overexpressed PEDF genes have been shown to inhibit angiogenesis and inflammation in the OIR model (Park et al., 2011). To determine whether PEGylated-PEDF had a better effect on the OIR mouse model than PEDF, PEGylated-PEDF, PEDF, and PEG were injected intravitreally into the right eyes of retinopathy rats at P12 (immediately after the animals were returned from hyperoxia to normoxia) and age-matched normal rats. Consistent with other previous observations (Park et al., 2011), the PEDF injection substantially reduced the neovascularized area by 19.0 ± 1.9%, which is significantly different compared with the untreated control results of 28.3 ± 0.6% (p < 0.0001) (Fig. 13). In the PEGylated-PEDF-treated group, the neovascularization area was reduced by 12.6 ± 1.5%, which is significantly different compared to the untreated controls (p < 0.0001) and the PEDF-treated group (p < 0.05). Thus, these experiments demonstrated that PEGylated-PEDF protected postnatal mouse retina from hyperoxia-induced vaso-obliteration more efficiently than did PEDF.
Antiangiogenic treatment is one of the most effective therapeutic strategies in the management of retinal neovascularization disease, including ROP, age-related macular degeneration, and diabetic retinopathy, which are found throughout the world and can cause irreversible blindness (Drack, 2006; Mousa and Mousa, 2010). Several agents have been demonstrated to be helpful in decreasing the amount of neovascularization (Mousa and Mousa, 2010). Among these potential medications, PEDF is considered to be the most potent natural angiogenic inhibitor endogenously expressed in the retina (Tombran-Tink et al., 1991; Dawson et al., 1999).
PEDF, a 50-kDa secreted glycoprotein that belongs to the noninhibitory serpin family, was first discovered in conditioned medium from cultured fetal retinal pigment epithelial cells (Tombran-Tink et al., 1991). Subsequent studies revealed PEDF as a potent and versatile endogenous, antiangiogenic, neuroprotective, antivasopermeabable, glia-static, antitumorigenic agent (Bouck, 2002), which can inhibit the growth of new blood vessels in conditions of hypoxia/ischemia and can promote neuron growth during injury; its exact mechanisms, however, are not well known (Dawson et al., 1999; Barnstable and Tombran-Tink, 2004).
PEDF occurs naturally in the eye, is able to block multiple inducers of angiogenesis, and has been shown to be an essential contributor to the maintenance of the avascularity status in healthy ocular tissues (Dawson et al., 1999; Barnstable and Tombran-Tink, 2004). Several studies have shown that the inhibition effect of PEDF in angiogenesis is caused by inhibiting endothelial cell proliferation and migration and inducing its apoptosis (Dawson et al., 1999; Park et al., 2011). Further study has revealed that PEDF exerts its antiangiogenic function through such activities as decreasing VEGF, down-regulating hypoxia-inducible factor 1α, and suppressing inflammation effects (Tombran-Tink et al., 1991; Park et al., 2011). Although PEDF exhibits effective therapeutic potential, its application is limited by its short half-life, unstable pharmacology, and administration pathway. To explore PEDF application, the gene therapy method, such as adeno-associated virus vector-mediated PEDF, has been used by many laboratories (Streck et al., 2005; Park et al., 2011; He et al., 2012). However, because of its potential oncology-inducing property, immunogenicity, uncertain quantitative expression, and lower production rate, the application is limited. Other studies also have loaded the PEDF gene in poly(d,l-lactide-coglycolide acid) nanoparticles for PEDF delivery, but the critical drawback of a poly(d,l-lactide-coglycolide acid) microsphere delivery system for proteins is activity loss during formulation (Pai et al., 2009).
It is known that the major challenge of using proteins or peptides is their poor in vivo stability, retention, and inactivation by the immune system or by the action of proteolytic enzymes (Abuchowski et al., 1977). Rapid elimination leads to frequent and excessive administration, which is improvident and causes nonspecific toxicity. Thus, controlled release, making it accumulate to effective levels and metabolizing it with minimum toxicity and without intolerable adverse effects, becomes particularly important. A crucial strategy for controlled release is the use of polymers (Qiu and Bae, 2006). Conjugation of proteins with polymers reduces recognition by the immune system and decreases the clearance rate (Abuchowski et al., 1977). Currently, PEG is one of the most widely used polymers for the modification of protein therapeutics, with many applications from industrial manufacturing to medicine (Pai et al., 2009). Because it is inert, inexpensive, and has low toxicity and increased solubility, PEG has been approved by the FDA for drug modification for several years (Harris and Chess, 2003). PEGylation (i.e., the attachment of PEG to proteins, peptides, or other drugs) is an upcoming methodology for drug development that has the potential to improve the pharmacokinetic and pharmacodynamic properties of the administered drug (Parveen and Sahoo, 2006). Several PEGylated products have been available on the market, such as PEG-adenosine deaminase (Hershfield et al., 1987), PEG-l-asparaginase (Graham, 2003), PEG-interferon-α (Heathcote et al., 2000), and PEG-human growth hormone (Drake and Trainer, 2003). In addition, pegaptanib (brand name Macugen), the first FDA-approved intravitreously used anti-VEGF medication, is also PEGylated (Gragoudas et al., 2004).
In the present study, we developed PEGylated-PEDF for the first time (Figs. 1 and 2) and evaluated its antiangiogenic effects both in vitro and in vivo. We confirmed that PEG covalent to PEDF had longer-term effects than unmodified PEDF in both plasma concentration (Fig. 3) and retinal concentration (Fig. 4). In vitro, PEGylated-PEDF showed significant proliferation and migration inhibitory effects on HUVECs compared with controls; the inhibitory effects were dose-dependent (Figs. 5 and 7), which corresponds to previous reports (Dawson et al., 1999). Even in VEGF stimulation medium, PEGylated-PEDF showed significant proliferation inhibitory effects (Fig. 6). We also evaluated the antiangiogenic property of PEGylated-PEDF in vitro with a tube formation study. The results showed that modified PEDF interferes with network formation and could be associated with the apoptosis of vascular endothelial cells (Fig. 8) and the inhibition of VEGF secretion (Fig. 10). In vivo, we used the OIR model to evaluate the antiangiogenic effects of PEGylated-PEDF. The OIR model closely resembles ROP. Because retinal hypoxia is the direct cause of neovascularization and increased VEGF is a major mediator, the pathological mechanism of this model is similar to that of ROP. In our in vivo study, PEGylated-PEDF showed significant inhibitory effects on neovascularization in the OIR model.
In summary, our study found for the first time that PEG covalent to PEDF could effectively inhibit the growth of neovascularization. Our data suggest that the PEGylated-PEDF delivery system could be an extremely promising approach for the long-term treatment of retinopathy of prematurity. The use of PEGylated-PEDF was a safe and effective drug delivery system for therapy of retinal angiogenic diseases, and it may be an innovative approach for future therapeutic strategies against other angiogenesis as well.
Participated in research design: Bai, Huang, and Li.
Conducted experiments: Bai, Du, Xu, and Zhou.
Contributed new reagents or analytic tools: Xu.
Performed data analysis: Bai, Zhou, and Yu.
Wrote or contributed to the writing of the manuscript: Bai, Huang, and Yu.
This work was supported by the National Basic Research Program of China's 973 Program [Grant 2011 CB510200].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- retinopathy of prematurity
- oxygen-induced retinopathy
- pigment epithelial-derived factor
- polyethylene glycol
- vascular endothelium growth factor
- human umbilical vein endothelial cell
- Dulbecco's modified Eagle's medium
- enzyme-linked immunosorbent assay
- propidium iodide
- fetal bovine serum
- Food and Drug Administration
- postnatal day
- Cell Counting Kit-8
- fluorescein isothiocyanate
- phosphate-buffered saline
- late apoptotic cells
- early apoptotic cells
- dead cells
- viable cells
- Received February 1, 2012.
- Accepted April 6, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics