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Pigment epithelium-derived factor (PEDF) is a cytoprotective protein for the retina. We hypothesize that this protein acts on neuronal survival and differentiation of photoreceptor cells in culture. The purpose of the present study was to evaluate the neurotrophic effects of PEDF and its fragments in an in vitro model of cultured primary retinal neurons that die spontaneously in the absence of trophic factors. We used Wistar albino rats. Cell death was assayed by immunofluorescence and flow cytometry through TUNEL assay, propidium iodide, mitotracker, and annexin V. Immunofluorescence of cells for visualizing rhodopsin, CRX, and antisyntaxin under confocal microscopy was performed. Neurite outgrowth was also quantified. Results show that PEDF protected photoreceptor precursors from apoptosis, preserved mitochondrial function and promoted polarization of opsin enhancing their developmental process, as well as induced neurite outgrowth in amacrine neurons. These effects were abolished by an inhibitor of the PEDF receptor or receptor-derived peptides that block ligand/receptor interactions. While all the activities were specifically conferred by short peptide fragments (17 amino acid residues) derived from the PEDF neurotrophic domain, no effects were triggered by peptides from the PEDF antiangiogenic region. The observed effects on retinal neurons imply a specific activation of the PEDF receptor by a small neurotrophic region of PEDF. Our findings support the neurotrophic PEDF peptides as neuronal guardians for the retina, highlighting their potential as promoters of retinal differentiation, and inhibitors of retinal cell death and its blinding consequences. Cover Image for this issue: https://doi.org/10.1111/jnc.15089., (© 2021 International Society for Neurochemistry. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Retinoprotective proteins play important roles for retinal tissue integrity. They can directly affect the function and the survival of photoreceptors, and/or indirectly target the retinal pigment epithelium (RPE) and endothelial cells that support these tissues. Retinoprotective proteins are used in basic, translational and in clinical studies to prevent and treat human retinal degenerative disorders. In this review, we provide an overview of proteins that protect the retina and focus on pigment epithelium-derived factor (PEDF), and its effects on photoreceptors, RPE cells, and endothelial cells. We also discuss delivery systems such as pharmacologic and genetic administration of proteins to achieve photoreceptor survival and retinal tissue integrity.

Here, we evaluated the effects of PEDF (pigment epithelium-derived factor) and PEDF peptides on cone-photoreceptor cell damage in a mouse model of focal LED-induced phototoxicity (LIP) in vivo. Swiss mice were dark-adapted overnight, anesthetized, and their left eyes were exposed to a blue LED placed over the cornea. Immediately after, intravitreal injection of PEDF, PEDF-peptide fragments 17-mer, 17-mer[H105A] or 17-mer[R99A] (all at 10 pmol) were administered into the left eye of each animal. BDNF (92 pmol) and bFGF (27 pmol) injections were positive controls, and vehicle negative control. After 7 days, LIP resulted in a consistent circular lesion located in the supratemporal quadrant and the number of S-cones were counted within an area centered on the lesion. Retinas treated with effectors had significantly greater S-cone numbers (PEDF (60%), 17-mer (56%), 17-mer [H105A] (57%), BDNF (64%) or bFGF (60%)) relative to their corresponding vehicle groups (≈42%). The 17-mer[R99A] with no PEDF receptor binding and no neurotrophic activity, PEDF combined with a molar excess of the PEDF receptor blocker P1 peptide, or with a PEDF-R enzymatic inhibitor had undetectable effects in S-cone survival. The findings demonstrated that the cone survival effects were mediated via interactions between the 17-mer region of the PEDF molecule and its PEDF-R receptor.

The purpose of the study is to evaluate the protective properties of PEDF peptide fragments on rd10 mouse models of retinal degeneration ex vivo. Human recombinant PEDF and synthetic peptides were used. Rd10 retinal explants as well as wild-type retinal explants treated with zaprinast to mimic the rd10 photoreceptor cell death were employed. PEDF protein was intravitreally administered into rd10 mice. Outer nuclear layer thickness measurements in retinal sections, TUNEL labeling in retinal explants, western blots and immunofluorescence with retinal samples were performed. PEDF protein levels in the RPE of rd10 mice decreased with age (P15 - P25). Levels of PEDF receptor PEDF-R declined in the photoreceptor inner segments from rd10 relative to wild-type mice at P25. PEDF administration increased the outer nuclear layer thickness of rd10 retinas in vivo and decreased the number of TUNEL + nuclei of photoreceptors in rd10 retinal explant cultures, both relative to untreated controls. Peptides containing the PEDF neurotrophic region decreased the number of TUNEL + photoreceptors in both rd10 and zaprinast-induced cell death ex vivo models, while peptides without the neurotrophic region and/or lacking affinity for PEDF-R were ineffective in protecting photoreceptors. Thus, retinal explants are a valuable system to evaluate PEDF activity. Short peptides with the photoreceptor-protective property of PEDF may prove useful for the development of therapeutic agents for photoreceptor protection in retinal degenerations., (Published by Elsevier Ltd.)

Pigment epithelium-derived factor (PEDF) is involved in signal transduction cascades necessary for protection of the retina. The interaction between PEDF and retinal cells elicits neuroprotective effects in vitro and in vivo. The direct substrates and signaling mechanisms involved in the survival response derived from such interaction are beginning to be revealed. It is of interest to elucidate cell death pathways that are crucial for the retinoprotective response of PEDF for the identification of targets that interfere with retina degeneration with potential therapeutic value. Here we review the molecular pathways triggered by PEDF that are involved in retinal survival activity.

Calcium ions play a critical role in neuronal cell death. Pigment epithelium-derived factor (PEDF) is a promising neuroprotective protein for photoreceptor cells but the mechanisms mediating its effects against retinal degeneration are still not well characterized. We addressed this question in the rd1 degenerating mouse retina that bears a mutation in the Pde6b gene encoding one subunit of the phosphodiesterase enzyme. Loss of phosphodiesterase activity in rod photoreceptor cells increases cyclic guanosine monophosphate (cGMP) levels leading to a rise in intracellular calcium. Short-term treatments with recombinant human PEDF protein decreased intracellular calcium in photoreceptors in vivo. Taking advantage of calcium pump blockers, we defined that PEDF signaling acts on PMCA calcium pumps to lower intracellular calcium. PEDF restrained cell death pathways activated by high calcium levels and engaging calpains, BAX and AIF. The neurotrophic effects were mediated by the PEDF receptor (PEDF-R), encoded by the PNPLA2 gene. Finally, peptides containing the neurotrophic domain of PEDF targeted these same cell death pathways in vivo. The findings reveal rescue from death of degenerating photoreceptor cells by a PEDF-mediated preservation of intracellular calcium homeostasis.

Oxidative stress has been implicated in neurodegenerative diseases, such as age-related macular degeneration. Hydrogen peroxide and sodium iodate can mediate oxidative injury. Sodium iodate induces a selective retinal degeneration targeting the RPE. We describe a method of chronic sodium iodate-mediated injury on RPE cells that may serve to evaluate protective factors against oxidative stress. Cytotoxicity and cell viability curves of ARPE-19 cells with sodium iodate were generated. The antioxidant pigment epithelium-derived factor decreased sodium iodate-mediated cytotoxicity without affecting ARPE-19 cell viability. A cell culture system to evaluate protection against oxidative stress injury with PEDF is discussed.

Purpose: Protease nexin-1 (PN-1), a serpin encoded by the SERPINE2 gene, has serine protease inhibitory activity and neurotrophic properties in the brain. PN-1 inhibits retinal angiogenesis; however, PN-1's neurotrophic capacities in the retina have not yet been evaluated. Pigment epithelium-derived factor (PEDF) is a serpin that exhibits neurotrophic and antiangiogenic activities but lacks protease inhibitory properties. The aim of this study is to compare PN-1 and PEDF., Methods: Sequence comparisons were performed using computer bioinformatics programs. Mouse and bovine eyes, human retina tissue, and ARPE-19 cells were used to prepare RNA and protein samples. Interphotoreceptor matrix lavage was obtained from bovine eyes. Gene expression and protein levels were evaluated with reverse-transcription PCR (RT-PCR) and western blotting, respectively. Recombinant human PN-1, a version of PN-1 referred to as PN-1[R346A] lacking serine protease inhibitory activity, and PEDF proteins were used, as well as synthetic peptides designed from PEDF and PN-1 sequences. Survival activity in serum-starved, rat-derived retinal precursor (R28) cells was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) cell death assays. Bcl2 levels were measured with RT-PCR., Results: PN-1 is analogous in primary and tertiary structure to PEDF. A region in PN-1 shares homology with the neurotrophic active region of PEDF, a 17-residue region within alpha helix C. The native human retina, ARPE-19 cells, and murine RPE and retina expressed the gene for PN-1 ( SERPINE2 and Serpine2 mRNA). The retina, ARPE-19 cell lysates, and bovine interphotoreceptor matrix contained PN-1 protein. The addition of PN-1, PN-1[R346A], or the 17mer peptide of PN-1 to serum-starved retina cells decreased the number of TUNEL-positive nuclei relative to the untreated cells, such as PEDF. PN-1, PN-1[R346A], and PN-1-17mer treatments increased the Bcl2 transcript levels in serum-starved cells, as seen with PEDF., Conclusions: PN-1 and PEDF share structural and functional features, and expression patterns in the retina. These serpins' mechanisms of action as cell survival factors are independent of serine protease inhibition. We have identified PN-1 as a novel factor for the retina that may play a neuroprotective role in vivo, and small peptides as relevant candidates for preventing retinal degeneration.

Pigment epithelium-derived factor (PEDF) is a natural protein of the retina with demonstrable neurotrophic properties, found in the interphotoreceptor matrix in intimate contact with photoreceptors. This review summarizes the effects of PEDF on photoreceptors in several animal models of retinal degeneration.

The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78-121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98-114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98-114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Bronchopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. Studies show the importance of proangiogenic factors for alveolarization, but the importance of antiangiogenic factors is unknown. We proposed that hyperoxia increases the potent angiostatin, pigment epithelium-derived factor (PEDF), in neonatal lungs, inhibiting alveolarization and microvascularization. Wild-type (WT) and PEDF(-/-) mice were exposed to room air (RA) or 0.9 fraction of inspired oxygen from Postnatal Day 5 to 13. PEDF protein was increased in hyperoxic lungs compared with RA-exposed lungs (P < 0.05). In situ hybridization and immunofluorescence identified PEDF production primarily in alveolar epithelium. Hyperoxia reduced alveolarization in WT mice (P < 0.05) but not in PEDF(-/-) mice. WT hyperoxic mice had fewer platelet endothelial cell adhesion molecule (PECAM)-positive cells per alveolus (1.4 ± 0.4) than RA-exposed mice (4.3 ± 0.3; P < 0.05); this reduction was absent in hyperoxic PEDF(-/-) mice. The interactive regulation of lung microvascularization by vascular endothelial growth factor and PEDF was studied in vitro using MFLM-91U cells, a fetal mouse lung endothelial cell line. Vascular endothelial growth factor stimulation of proliferation, migration, and capillary tube formation was inhibited by PEDF. MFLM-91U cells exposed to conditioned medium (CM) from E17 fetal mouse lung type II (T2) cells cultured in 0.9 fraction of inspired oxygen formed fewer capillary tubes than CM from T2 cells cultured in RA (hyperoxia CM, 51 ± 10% of RA CM, P < 0.05), an effect abolished by PEDF antibody. We conclude that PEDF mediates reduced vasculogenesis and alveolarization in neonatal hyperoxia. Bronchopulmonary dysplasia likely results from an altered balance between pro- and antiangiogenic factors.

Pigment epithelium-derived factor (PEDF) can delay and prevent the death of photoreceptors in vivo. We investigated the survival activity of PEDF on cone photoreceptor-derived 661W cells in culture, the presence of PEDF receptor (PEDF-R) in these cells and the activation of prosurvival Akt. Cell death was induced by light exposure in the presence of 9-cis retinal. Cell viability assays showed that PEDF increased the number of 661W cells exposed to these conditions. Western blots showed that PEDF-treated 661W cells had a higher ratio of phosphorylated Akt to total Akt than untreated cells. The PEDF receptor PEDF-R was immunodetected in the plasma membrane fractions of 661W cells. The results demonstrated that PEDF can protect 661W cells against light-induced cell death and suggest that the binding of PEDF to cell surface PEDF-R triggers a prosurvival signaling pathway.

AMD (age-related macular degeneration) is a neurodegenerative disease causing irreversible central blindness in the elderly. Apoptosis and inflammation play important roles in AMD pathogenesis. PEDF (pigment epithelium-derived factor) is a potent neurotrophic and anti-inflammatory glycoprotein that protects the retinal neurons and photoreceptors against cell death caused by pathological insults. We studied the effects of PEDF on focal retinal lesions in DKO rd8 (Ccl2(-/-)/Cx3cr1(-/-) on C57BL/6N [Crb1(rd8)]) mice, a model for progressive, focal rd (retinal degeneration). First, we found a significant decrease in PEDF transcript expression in DKO rd8 mouse retina and RPE (retinal pigment epithelium) than WT (wild-type, C57BL/6N). Next, cultured DKO rd8 RPE cells secreted lower levels of PEDF protein in the media than WT. Then the right eyes of DKO rd8 mice were injected intravitreously with recombinant human PEDF protein (1 μg), followed by a subconjunctival injection of PEDF (3 μg) 4 weeks later. The untreated left eyes served as controls. The effect of PEDF was assessed by fundoscopy, ocular histopathology and A2E {[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetra-enyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl) 1E,3E,5E,7E-hexatrienyl]-pyridinium} levels, as well as apoptotic and inflammatory molecules. The PEDF-treated eyes showed slower progression or attenuation of the focal retinal lesions, fewer and/or smaller photoreceptor and RPE degeneration, and significantly lower A2E, relative to the untreated eyes. In addition, lower expression of apoptotic and inflammatory molecules were detected in the PEDF-treated than untreated eyes. Our results establish that PEDF potently stabilizes photoreceptor degeneration via suppression of both apoptotic and inflammatory pathways. The multiple beneficial effects of PEDF represent a novel approach for potential AMD treatment.

The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu(159)-Met(325)) with affinity similar to the full-length PEDF-R (Met(1)-Leu(504)). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile(193)-Leu(232)) and P1 (Thr(210)-Leu(249)) peptides. Recombinant C-terminal truncated PEDF-R4 (Met(1)-Leu(232)) and internally truncated PEDF-R and PEDF-R4 (ΔHis(203)-Leu(232)) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His(203)-Leu(232) region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.

The potent actions of pigment epithelium-derived factor (PEDF) on tumour-associated cells, and its extracellular localization and secretion, stimulated research on this multifunctional serpin. Such studies have identified several PEDF receptors and downstream signalling pathways. Known cellular PEDF responses have expanded from the initial discovery that PEDF induces retinoblastoma cell differentiation to its anti-angiogenic, antitumorigenic and antimetastatic properties. Although the diversity of PEDF activities seems to be complex, they are consistent with the varied mechanisms that regulate this multimodal factor. If PEDF is to be used for cancer management, a deeper appreciation of its many functions and mechanisms of action is needed.

Recently, we have shown that the antiangiogenic pigment epithelium-derived factor (PEDF) can bind the catalytic β-subunit of F1-ATP synthase and inhibit endothelial cell surface ATP synthase activity. This factor can additionally restrict tumor growth, invasion and metastasis, and can directly induce death on several tumor cell types. Active cell surface ATP synthase is also present in certain tumor cells and its ATP product is considered a stimulus for tumor growth. The present study aimed to elucidate the biological implications of the interactions between the extracellular PEDF and tumor cell surface ATP synthase. Incubation of T24 human urinary bladder carcinoma cells in media containing human recombinant PEDF protein for 48-96 h dramatically decreased cell viability in a concentration-dependent fashion as monitored by real-time cell impedance with a microelectronic system, microscopic imaging and biomarkers of live cells. Intact tumor cells exhibited cell surface ATP synthesis activity, which was inhibited by piceatannol, a specific inhibitor of F1/F0-ATP synthase. Immunoblotting revealed that the β subunit of F1-ATP synthase was present in plasma membrane fractions of these cells. Interestingly, pre-incubation of tumor cells with PEDF inhibited the activity of cell surface ATP synthase in a concentration-dependent fashion. The PEDF-derived peptide 34-mer decreased tumor cell viability and inhibited extracellular ATP synthesis to the same extent as full-length PEDF. Moreover, ATP additions attenuated both the PEDF-mediated decrease in tumor cell viability and the inhibition of endothelial cell tube formation. The results lead to conclude that PEDF is a novel inhibitor of tumor cell surface ATP synthase activity that exhibits a cytotoxic effect on tumor cells, and that the structural determinants for these properties are within the peptide region 34-mer of the PEDF polypeptide. The data strongly suggest a role for the interaction between the 34-mer region of PEDF and tumor cell-surface ATP synthase in promoting tumor cell death.

Purpose: Pigment epithelium-derived factor (PEDF) is a multifunctional serpin. The purpose of this study is to identify PEDF protein forms and investigate their biological activities on tumor cell lines., Methods: Recombinant human PEDF proteins were purified by cation- and anion-exchange column chromatography. They were subjected to SDS-PAGE, IEF, deglycosylation, heparin affinity chromatography, and limited proteolysis. Cell viability, real-time electrical impedance of cells, and wound healing assays were performed using bladder and breast cancer cell lines, rat retinal R28, and human ARPE-19 cells., Results: Two PEDF protein peaks were identified after anion-exchange column chromatography: PEDF-1 eluting with lower ionic strength than PEDF-2. PEDF-1 had higher pI value and lower apparent molecular weight than PEDF-2. Both PEDF forms were glycosylated, bound to heparin, and had identical patterns by limited proteolysis. However, PEDF-2 emerged as being highly potent in lowering cell viability in all tumor cell lines tested, and in inhibiting tumor and ARPE-19 cell migration. In contrast, PEDF-1 minimally affected tumor cell viability and cell migration but protected R28 cells against death caused by serum starvation., Conclusion: Two distinct biochemical forms of PEDF varying in overall charge have distinct biological effects on tumor cell viability and migration. The existence of PEDF forms may explain the multifunctional modality of PEDF.

Brain metastases are a significant cause of morbidity and mortality for patients with cancer, yet preventative and therapeutic options remain an unmet need. The cytokine pigment epithelium-derived factor (PEDF) is downregulated in resected human brain metastases of breast cancer compared with primary breast tumors, suggesting that restoring its expression might limit metastatic spread. Here, we show that outgrowth of large experimental brain metastases from human 231-BR or murine 4T1-BR breast cancer cells was suppressed by PEDF expression, as supported by in vitro analyses as well as direct intracranial implantation. Notably, the suppressive effects of PEDF were not only rapid but independent of the effects of this factor on angiogenesis. Paralleling its cytotoxic effects on breast cancer cells, PEDF also exerted a prosurvival effect on neurons that shielded the brain from tumor-induced damage, as indicated by a relative 3.5-fold reduction in the number of dying neurons adjacent to tumors expressing PEDF. Our findings establish PEDF as both a metastatic suppressor and a neuroprotectant in the brain, highlighting its role as a double agent in limiting brain metastasis and its local consequences., (©2012 AACR.)

Traditional forms of cancer therapy, which include chemotherapy, have largely been overhauled due to the significant degree of toxicity they pose to normal, otherwise healthy tissue. It is hoped that the use of biological agents, most of which are endogenously present in the body, will lead to safer treatment outcomes, without sacrificing efficacy. The finding that pigment epithelium-derived factor (PEDF), a naturally-occurring protein, is a potent angiogenesis inhibitor has become the basis for studying the role of PEDF in tumours that are highly resistant to chemotherapy. The determination of the direct role of PEDF against cancer paves the way for understanding and developing PEDF as a novel drug. This review focuses on the patent applications behind testing the anticancer therapeutic effect of PEDF via its receptors as an antiangiogenic agent and as a direct anticancer agent. The majority of the PEDF patents describe the antiangiogenic ability and usage of recombinant vectors as the mode of treatment delivery. PEDF's therapeutic potential against different diseases and the discovery of its receptors open possibilities for improving PEDF-based peptide design and drug delivery modes.

Pigment epithelium-derived factor (PEDF) is a secreted serpin that exhibits a variety of interesting biological activities. The multifunctional PEDF has neurotrophic and antiangiogenic properties, and acts in retinal differentiation, survival, and maintenance. It is also antitumorigenic and antimetastatic, and has stem cell self-renewal properties. It is widely distributed in the human body and exists in abundance in the eye as a soluble extracellular glycoprotein. Its levels are altered in diseases characterized by retinopathies and angiogenesis. Its mechanisms of neuroprotection and angiogenesis are associated with receptor interactions at cell-surface interfaces and changes in protein expression. This serpin lacks demonstrable serine protease inhibitory activity, but has binding affinity to extracellular matrix components and cell-surface receptors. Here we describe purification protocols, methods to quantify PEDF, and determine interactions with specific molecules, as well as neurotrophic and angiogenesis assays for this multifunctional protein., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pigment epithelium-derived factor (PEDF) is an endogenously produced glycoprotein with a spectrum of biological roles across diverse pathologies. Recent research has focused on the biochemical properties of PEDF and its associated receptors. This review discusses the recent developments in PEDF biochemistry and how this new knowledge will help progress our understanding of PEDF as a molecular mediator for anti-angiogenesis and -tumorigenesis. Additionally, pathophysiological roles for PEDF in healing and tissue homeostasis are being revealed and our enhanced understanding of the interactions between PEDF and its receptors may yet prove useful in propelling PEDF towards clinical application.

Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a approximately 60 kDa PEDF-binding protein in bovine retina with Bos taurus F(1)-ATP synthase beta-subunit, and that F(1)F(o)-ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F(1). Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F(1). Real-time binding as determined by surface plasmon resonance demonstrated that yeast F(1) interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F(1) and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F(1). Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of approximately 60 kDa. Antibodies to F(1)beta-subunit specifically captured PEDF-binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F(1), these results show that PEDF is a ligand for endothelial cell-surface F(1)F(o)-ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity.

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 (Asp(44)-Asn(77)) and 44-mer (Val(78)-Thr(121)) 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), a multifunctional protein, acts in retinal differentiation, survival and maintenance by interacting with high affinity receptors on the surface of target cells. We have recently identified PEDF-R, a new member of the patatin-like phospholipase domain-containing 2 (PNPLA2) family with characteristics of a PEDF receptor. The PEDF-R sequence reveals a patatin-like phospholipase domain toward its amino-end, and four transmembrane domains interrupted by two extracellular loops and three intracellular regions along its polypeptide sequence. This newly identified protein is present on the surface of retina and RPE cells, and has the expected transmembrane topology. It has specific and high binding affinity for PEDF, and exhibits a potent phospholipase A(2) activity that liberates fatty acids. Most importantly, PEDF binding stimulates the enzymatic phospholipase A(2) activity of PEDF-R. In summary, PEDF-R is a novel component of the retina that is a phospholipase-linked membrane protein with high affinity for PEDF. The results suggest a molecular pathway by which PEDF ligand/receptor interactions on the cell surface could generate a cellular signal. These conclusions enhance our understanding of the role of PEDF as a neurotrophic survival factor.

Pigment epithelium-derived factor (PEDF) is a multifunctional serpin with antitumorigenic, antimetastatic, and differentiating activities. PEDF is found within tissues rich in the glycosaminoglycan hyaluronan (HA), and its amino acid sequence contains putative HA-binding motifs. We show that PEDF coprecipitation with glycosaminoglycans in media conditioned by human retinoblastoma Y-79 cells decreased after pretreatments with hyaluronidase, implying an association between HA and PEDF. Direct binding of human recombinant PEDF to highly purified HA was demonstrated by coprecipitation in the presence of cetylpyridinium chloride. Binding of PEDF to HA was concentration-dependent and saturable. The PEDF-HA interactions were sensitive to increasing NaCl concentrations, indicating an ionic nature of these interactions and having affinity higher than PEDF-heparin. Competition assays showed that PEDF can bind heparin and HA simultaneously. PEDF chemically modified with fluorescein retained the capacity for interacting with HA but lacked heparin affinity, suggesting one or more distinct HA-binding regions on PEDF. The HA-binding region was examined by site-directed mutagenesis. Single-point and cumulative alterations at basic residues within the putative HA-binding motif K189A/K191A/R194A/K197A drastically reduced the HA-binding activity without affecting heparin- or collagen I binding of PEDF. Cumulative alterations at sites critical for heparin binding (K146A/K147A/R149A) decreased HA affinity but not collagen I binding. Thus these clusters of basic residues (BXBXXBXXB and BX3AB2XB motifs) in PEDF are functional regions for binding HA. In the spatial PEDF structure they are located in distinct areas away from the collagen-binding site. The HA-binding activity of PEDF may contribute to deposition in the extracellular matrix and to its reported antitumor/antimetastatic effects.

Purpose: To investigate the effects of dexamethasone (DEX) on pigment epithelium-derived factor (PEDF) cDNA and secreted protein in human trabecular meshwork (TM)., Methods: Anterior segment organ cultures were perfused with 0.1 microM DEX (OD) and vehicle (OS). Primary human TM cells (HTM) were treated with DEX under similar conditions. PEDF mRNA and secreted PEDF protein were quantitated by RT-PCR and Western blot., Results: PEDF mRNA and secreted PEDF protein levels were significantly higher in DEX over vehicle-treated cultures. In contrast, DEX decreased the activity of a 92-kDa gelatinolytic zymogen in organ culture effluents., Conclusion: DEX action in the human TM might include a PEDF-mediating pathway.

Pigment epithelium-derived factor (PEDF) is an extracellular multifunctional protein belonging to the serpin superfamily with demonstrable neurotrophic, gliastatic, neuronotrophic, antiangiogenic, and antitumorigenic properties. We have previously provided biochemical evidence for high affinity PEDF-binding sites and proteins in plasma membranes of retina, retinoblastoma, and CNS cells. This study was designed to reveal a receptor involved in the biological activities of PEDF. Using a yeast two-hybrid screening, we identified a novel gene from pigment epithelium of the human retina that codes for a PEDF-binding partner, which we term PEDF-R. The derived polypeptide has putative transmembrane, intracellular and extracellular regions, and a phospholipase domain. Recently, PEDF-R (TTS-2.2/independent phospholipase A(2) (PLA(2))zeta and mouse desnutrin/ATGL) has been described in adipose cells as a member of the new calcium-independent PLA(2)/nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) family that possesses triglyceride lipase and acylglycerol transacylase activities. Here we describe the PEDF-R gene expression in the retina and its heterologous expression by bacterial and eukaryotic systems, and we demonstrate that its protein product has specific and high binding affinity for PEDF, has a potent phospholipase A(2) activity that liberates fatty acids, and is associated with eukaryotic cell membranes. Most importantly, PEDF binding stimulates the enzymatic phospholipase A(2) activity of PEDF-R. In conclusion, we have identified a novel PEDF-R gene in the retina for a phospholipase-linked membrane protein with high affinity for PEDF, suggesting a molecular pathway by which ligand/receptor interaction on the cell surface could generate a cellular signal.

Purpose: To investigate the in vitro, ex vivo, and in vivo transscleral-retinal pigment epithelium (RPE) permeability of PEDF and structurally related proteins for the exploration of novel routes of protein delivery to the retina., Methods: Monkey RPE cells were cultured on permeable supports to separate apical and basal compartments. Porcine scleral tissue was placed in Ussing chambers to separate uveal and orbital compartments. Transepithelial resistance and voltage were measured by an electrical resistance system, and paracellular tracer flux was evaluated with trypan blue. Subconjunctival administration in rat eyes was by injections of soluble protein or by implantation of polyvinyl alcohol devices containing protein. Fluorescein-conjugated (Fl-) PEDF and ovalbumin were determined by spectrofluorometry, laser scanning, immunoblotting, epifluorescence, and confocal microscopy. Permeability was assessed by fluoresceinated-protein flux., Results: Transepithelial resistance, impermeability to trypan blue, and confocal microscopy confirmed functional and structural tight junction formation of RPE cells cultured on permeable supports. Full-length Fl-PEDF and Fl-ovalbumin proteins diffused through RPE cell monolayers from either the apical or basal side. Fl-ovalbumin diffused through scleral tissues at constant rates. Subconjunctival Fl-PEDF or Fl-ovalbumin administration in vivo revealed movement of full-length protein into the choroid and retina as early as 1 hour., Conclusions: The sclera and RPE were permeable in vitro, ex vivo, and in vivo to PEDF and ovalbumin proteins. These large proteins can traverse through the sclera-RPE to reach the retina. In addition, these data prompt the proposal that subconjunctival protein delivery may represent a feasible and minimally invasive route for PEDF administration in the clinic.

Purpose: Pigment epithelium-derived factor (PEDF), a protein secreted by the retinal pigment epithelium (RPE), acts on retinal survival and angiogenesis. Because hypoxia and VEGF regulate matrix metalloproteinases (MMPs), their effects on PEDF proteolysis were explored., Methods: Mouse models for retinopathy of prematurity (ROP) were used. Cultured monkey RPE cells were exposed to low oxygen and chemical hypoxia mimetics. PEDF and VEGF mRNA levels in RPE were determined by RT-PCR. MMPs were assessed by zymography, DQ-gelatin degradation solution assays, and MMP immunostaining. PEDF proteolysis was assayed in solution and followed by SDS-PAGE and immunostaining. MMP induction by VEGF was performed in baby hamster kidney (BHK) cells. Retinal R28 cell survival, ex vivo chick embryonic aortic vessel sprouting, and directed in vivo angiogenesis assays were performed., Results: Levels of PEDF in RPE/choroid significantly decreased in the ROP model. Hypoxia decreased PEDF levels in the media conditioned by RPE cells, with no significant change in PEDF mRNA. Conversely, PEDF proteolysis, gelatinolytic activities of approximately 57-kDa and approximately 86-kDa zymogens, and MMP-2 immunoreactivities increased with hypoxia. Addition of VEGF to BHK cells caused a time and dose-related upregulation of approximately 57-kDa zymogens and of DQ-gelatinolytic and PEDF-degrading activity. The PEDF-degrading activity and approximately 57-kDa zymogens in the BHK media shared MMP protease inhibition patterns and MMP-2 immunoreactivities with those in the vitreous. Limited proteolysis with MMP-2 and -9 degraded PEDF in a Ca(+2)-dependent fashion. MMP-mediated proteolysis of PEDF abolished the retinal survival and antiangiogenic activities of the PEDF protein., Conclusions: Hypoxia and VEGF can downregulate PEDF through proteolytic degradation. PEDF is a novel substrate for MMP-2 and -9. These results reveal a novel posttranslational mechanism for downregulating PEDF, and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios, in angiogenesis and/or in neuronal death.

Pigment epithelium-derived factor (PEDF) is an extracellular protein derived from the retinal pigment epithelium (RPE), a tissue formed by polarized cells that release growth and trophic factors in a directional fashion. We have investigated the distribution and directional release of PEDF protein by the monkey RPE. We established primary cultures of monkey RPE cells that expressed the PEDF gene, and that synthesized and secreted the PEDF protein. Northern analysis of RPE cultures and monkey ocular tissues showed that PEDF transcripts were highly expressed in RPE as compared with several other monkey ocular tissues, being even more abundant in cultured cells than they were in the native RPE. The differentiated RPE cells in culture secreted protein that shared the immunological, biochemical and biological characteristics of PEDF. The overall PEDF levels in the RPE conditioned media reached 6.5 mg ml- after 8 days in culture (i.e. 1.1 pg of PEDF per RPE cell). RPE cells were cultivated on permeable supports as monolayers forming a barrier between apical and basal compartments. Apical and basal culture media were sampled at three or four-day intervals for 18 cycles, and the PEDF content was quantified. Most of the PEDF protein was significantly higher in the apical than in the basal medium (>4 times) at the initial recovery intervals, to be detected only in the apical medium at the latter intervals. In the native monkey eye, the concentration of soluble PEDF in the interphotoreceptor matrix (144 nM) was 7-fold and 25-fold greater than in vitreous and aqueous, respectively. PEDF was abundant in the interphotoreceptor matrix surrounding rod and cone outer segments, and was detectable at lower levels in the RPE as visualized by confocal microscopy. We concluded that PEDF synthesized by the RPE is secreted preferentially from the apical surface and is distributed apically to the RPE bordering the outer segments of photoreceptors. PEDF can be a useful marker for RPE polarization and differentiation. The polarization of RPE may be an important mechanism to control PEDF secretion and our results offer interesting possibilities on regulation of PEDF.

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (serpin) family, is a survival factor for various types of neurons. We studied the mechanisms by which human PEDF protects motor neurons from degeneration, with the goal of eventually conducting human clinical trials. We first searched for a molecular region of human PEDF essential to motor neuron protection. Using a spinal cord culture model of chronic glutamate toxicity, we show herein that a synthetic 44 mer peptide from an N-terminal region of the human PEDF molecule that lacks the homologous serpin-reactive region contains its full neuroprotective activity. We also investigated the presence and distribution of PEDF receptors in the spinal cord. Using a fluoresceinated PEDF probe, we show that spinal motor neurons contain specific binding sites for PEDF. Kinetics analyses using a radiolabeled PEDF probe demonstrate that purified rat motor neurons contain a single class of saturable and specific binding sites. This study indicates that a small peptide fragment of the human PEDF molecule could be engineered to contain all of its motor neuron protective activity, and that the neuroprotective action is likely to be mediated directly on motor neurons via a single class of PEDF receptors. The data support the pharmacotherapeutic potential of PEDF as a neuroprotectant in human motor neuron degeneration.

The present study compared susceptibilities of Sprague Dawley (SD) and Brown Norway (BN) rats with ischemia-induced retinal neovascularization. An exposure to constant hyperoxia followed by normoxia induced significant retinal neovascularization in BN rats but not in SD rats, as demonstrated by fluorescein retinal angiography, measurement of avascular area, and count of preretinal vascular cells. These results indicate a rat strain difference in susceptibility to retinal neovascularization. To understand the molecular basis responsible for the strain difference, we have measured the levels of pigment epithelium-derived factor (PEDF), an angiogenic inhibitor, and vascular endothelial growth factor (VEGF), a major angiogenic stimulator in the retina. The hyperoxia-treated BN rats showed a significant reduction in retinal PEDF, but they showed a substantial increase of VEGF at both the protein and RNA levels, resulting in an increased VEGF-to-PEDF ratio. Hyperoxia-treated SD rats showed changes in PEDF and VEGF levels that were less in magnitude and of shorter duration than in BN rats. In age-matched normal BN and SD rats, however, there was no detectable difference in the basal VEGF-to-PEDF ratio between the strains. These observations support the idea that different regulation of angiogenic inhibitors and stimulators under ischemia are responsible for the differences in susceptibility to ischemia-induced retinal neovascularization in SD and BN rats.

Pigment epithelium-derived factor (PEDF), a recently defined retinal trophic factor and anti-angiogenic factor for the eye, is also present in the CNS and is a motor neuron protectant. We asked whether PEDF levels in CSF are altered in patients with amyotrophic lateral sclerosis (ALS). Pigment epithelium-derived factor protein was detected by quantitative western blot analysis with a PEDF-specific antiserum. Levels of PEDF in CSF, expressed as a ratio to total CSF protein, were significantly elevated 3.4-fold in 15 patients with ALS compared with neurologic disease controls (p < 0.0003). This increase does not seem likely to reflect up-regulation of PEDF synthesis in muscle in response to denervation, as CSF PEDF was not elevated in severe denervating diseases other than ALS. Nor does the increase represent some non-specific release in neurodegeneration, as CSF PEDF was not elevated in other neurodegenerative diseases. While the mechanism of this presumably reactive increase is not known, the distinctive, surprisingly elevated level of PEDF in the CSF may be an autoprotective reaction in ALS.

Purpose: The neurotrophic activity of pigment epithelium-derived factor (PEDF), an extracellular factor present in the retina, is mediated by binding to cell-surface receptors in responsive cell cultures. In the present study, the expression of PEDF receptors in native neural retinas from adult steers was examined., Methods: Binding reactions were performed with (125)I-PEDF and fluoresceinated PEDF using plasma membranes, detergent-soluble membrane proteins, or cryosections of retina from adult bovine eyes. Radioligand-binding and competition analyses were performed with a computer-assisted program. Ligand blot analysis of detergent-soluble membrane proteins was performed with (125)I-PEDF followed by autoradiography. Ligand-affinity column chromatography of detergent-soluble membrane proteins was performed with PEDF-coupled resin followed by SDS-PAGE. Binding of fluoresceinated PEDF to retina cryosections was detected by confocal microscopy., Results: Radioligand-binding assays showed that (125)I-PEDF bound in a specific and saturable fashion to one class of sites on retina membranes (K(d) = 2.5-6.5 nM; maximum binding [B(max)] = 1-48 x 10(10) sites/retina). A peptide of 44 amino acids (44-mer), identified as the receptor-binding region of PEDF, competed efficiently for (125)I-PEDF binding to retina membranes with kinetics similar to the full-length PEDF. Ligand blot analysis and ligand-affinity chromatography revealed a specific and high-affinity PEDF-binding protein of approximately 85 kDa in retina plasma membranes. Confocal microscopy showed that fluorescein-conjugated PEDF stained exclusively the inner segments of photoreceptors and cells of the ganglion cell layer in retinal cryosections., Conclusions: Altogether, these data conclusively demonstrate the existence of PEDF receptors discretely distributed on the surface of cells from the adult neural retina of bovine eyes. Furthermore, they provide evidence for the direct action of PEDF on photoreceptor and ganglion cell neurons and an anatomic basis for studies to assess PEDF neurotrophic effects on the adult retina.

We have previously shown that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cells (CGCs), by blocking apoptotic death, and can also protect these cells against glutamate-induced neurotoxicity. In preparation for gene therapy studies, pseudotyped HIV-1-based lentiviral vectors containing the PEDF gene, as well as either green fluorescent protein or beta-galactosidase, were prepared. These bicistronic vectors are unique in that they express two genes efficiently under one promoter. Primary cell cultures of CGCs from postnatal day 8 rats were infected with the vectors encoding PEDF. RT-PCR demonstrated expression of mRNA and Western blot analysis confirmed that infected CGCs secrete PEDF protein to the medium. Assays for cell survival demonstrated that PEDF-infected cells were significantly more protected compared with mock-infected controls for 6-8 days in culture, as well as against induced apoptosis. The PEDF vectors expressing tat (trans-acting transcription factor) provided more protection than the tat(-) vectors. These results demonstrate that while the lentiviral vectors expressing PEDF are as neuroprotective as the protein itself for CGCs, the vectors have the advantage of providing long-lasting expression of PEDF protein, which will be more effective in in vivo studies. The present results suggest that this system may be useful for gene therapy for neurodegenerative disorders., (Copyright 2001 S. Karger AG, Basel)

Purpose: The clusterin gene encodes a multi-functional protein that has been identified in different tissues, including a number of different eye tissues, primarily in the mouse and to a much lesser extent in humans. Clusterin has been implicated in a number of cellular processes such as lipid transport, membrane integrity, apoptosis, and neurodegeneration, all of which could be important to the biology of the eye. In the current communication, we provide data that confirms the expression of clusterin in a number of different human eye tissues and establishes the expression profile of this gene in monkey derived eye tissues. The issue that we sought to examine is whether a broad profile of clusterin expression in the eye is consistent in primates (monkey and human)., Methods: The majority of our study was done using monkey eye tissues. Where possible, we have used human tissues in order to confirm published findings. Northern and western analysis was performed using tissues derived from monkey eyes. In situ hybridization and immunochemistry were carried out on human eye sections., Results: Clusterin mRNA is expressed in primate lens, cornea, limbus, sclera, orbital muscle, ciliary body, retina, RPE/choroid, and RPE cells in culture. Western analysis revealed that two major groups of clusterin exist in the eye, a high molecular weight group (>100 kDa) and a second group consisting of at least five clusterin species that are all approximately 80 kDa. Analysis of conditioned media from RPE cells cultured on permeable supports suggests that different forms of clusterin display alternative patterns of secretion., Conclusions: Clusterin is expressed in a broad range of eye tissues in both human and monkey, suggesting that this is a characteristic feature in primates. We demonstrate for the first time that a diverse number of clusterin isoforms were observed in monkey eye tissues by western analysis. Meanwhile, the molecular size of clusterin mRNA detected in the array of tissues are identical in size, suggesting that the nature of the diversity in clusterin forms is due to post-translational modifications. In addition, new insights were made in defining clusterin expression in ciliary body, cornea, and the retinal pigment epithelium.

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor superfamily produced by retinal pigment epithelial cells in the developing and adult retina. In vitro, it induces neuronal differentiation of retinoblastoma cells and promotes survival of cerebellar granule neurons. The pedf gene is closely linked to an autosomal-dominant locus for retinitis pigmentosa, suggesting that PEDF could be a survival factor for photoreceptors. We have investigated this possibility by injecting PEDF into the eyes of homozygous retinal degeneration (rd) and retinal degeneration slow (rds) mice, two mutants displaying apoptotic photoreceptor loss. This procedure resulted in a transient delay of photoreceptor loss in the rd mouse and a reduction in apoptotic photoreceptor profiles in the rds mouse. We conclude that PEDF can act as a survival-promoting factor for photoreceptors in vivo and could potentially be useful for the treatment of photoreceptor diseases., (Copyright 1999 Academic Press.)

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor (serpin) superfamily that has been shown previously to promote the survival and/or differentiation of rat cerebellar granule neurons and human retinoblastoma cells in vitro. However, in contrast to most serpins, PEDF has no inhibitory activity against any known proteases, and its described biological activities do not appear to require the serpin-reactive loop located toward the carboxy end of the polypeptide. Because another serpin, protease nexin-1, has been shown to promote the in vivo survival and growth of motor neurons, the authors investigated the potential neurotrophic effects of PEDF on spinal cord motor neurons in highly enriched cultures and in vivo after injury. Here, it is shown that native bovine and recombinant human PEDF promoted the survival and differentiation (neurite outgrowth) of embryonic chick spinal cord motor neurons in vitro in a dose-dependent manner. A truncated form of PEDF that lacks approximately 62% of the carboxy end of the polypeptide comprising the homologous serpin-reactive loop also exhibited neurotrophic activities similar to those of the full-length protein. Furthermore, the data here showed that PEDF was transported retrogradely and prevented the death and atrophy of spinal motor neurons in the developing neonatal mouse after axotomy. These results indicate that PEDF exerts trophic effects on motor neurons, and, together with previous reports, these findings suggest that this protein may be useful as a pharmacologic agent to promote the development and maintenance of motor neurons. J. Comp. Neurol. 412:506-514, 1999. Published 1999 Wiley-Liss, Inc.

Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a secreted serpin identified in extracellular matrixes. We show that PEDF extractions from the interphotoreceptor matrix are more efficient with increasing NaCl concentrations, indicating that ionic interactions mediate its association with this polyanionic matrix. We have used affinity chromatography and ultrafiltration to probe for direct binding of PEDF to glycosaminoglycans/polyanions. Correctly folded PEDF bound to immobilized heparin, chondroitin sulfate-A, -B, -C, and dextran sulfate columns and eluted from each with an increase in NaCl concentration. However, in the presence of urea, the protein lost its affinity for heparin. Binding of PEDF to heparan sulfate proteoglycan in solution was in a concentration-dependent fashion (half-maximal specific binding EC50 = 40 micrograms/mL) and was sensitive to increasing NaCl concentrations. The glycosaminoglycan-binding region was analyzed using chemical modification and limited proteolysis. PEDF chemically modified on lysine residues by biotinylation lost its capacity for interacting with heparin, implicating the involvement of PEDF lysine residues in heparin binding. Cleavage of the serpin-exposed loop with chymotrypsin did not affect the heparin-binding property. A limited proteolysis product containing residues 21-approximately 260 bound to heparin with similar affinity as the intact PEDF. Homology modeling of PEDF based on the X-ray crystal structures of antithrombin III and ovalbumin shows a region at the center of beta-sheet A-strands 2 and 3- and helix F that has a basic electrostatic surface potential and is densely populated with lysines exposed to the surface (K134, K137, K189, K191, H212, and K214) that are available to interact with various glycosaminoglycans/polyanions. This region represents a novel site for glycosaminoglycan binding in a serpin, which in PEDF, is distinct and nonoverlapping from the PEDF neurotrophic active region.

We have shown previously that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cell neurons in culture, as well as protecting them against glutamate toxicity. In this study we have examined effects of PEDF on apoptotic cell death. We find that the granule cells die of apoptosis throughout the culture period, what we have termed "natural" apoptosis. PEDF prevents this natural apoptosis if added to immature cells, within the first 2 days in vitro (DIV), and the effect is maintained for up to DIV12. However, PEDF has no effect if added to mature cells at DIV5. Similar results are obtained when apoptosis is induced by shifting the cells from a serum- and 25 mM KCl-containing medium to serum-free medium with 5 mM KCl. PEDF most effectively blocks induced apoptosis in immature cells (DIV2) when added 24 hr prior to the change of medium, but still provides some protection when added simultaneously. However, 24 hr pretreatment with PEDF has a minimal effect when apoptosis is induced in mature DIV6 cells; addition at the same time is completely ineffective. Two polypeptide fragments of PEDF, only one of which contains the serine-protease inhibitory site, are equally active, supporting previous results which suggest that the neurotrophic effects of PEDF are not mediated by protease inhibition. We conclude that PEDF protects immature but not mature granule cells against both natural and induced apoptosis.

PEDF, a member of the serpin superfamily of proteins related through their highly conserved folded conformation, has neurotrophic properties, including promotion of neurite-outgrowth and neuronal survival. Previously, we have purified and characterized PEDF protein from extracellular matrixes of bovine eyes. Here, we show the cDNA sequence and expression analysis of bovine PEDF. Northern analysis of RNA from bovine retinal pigment epithelium (RPE) and neural retina using a human PEDF cDNA fragment reveals expression of the PEDF gene only for RPE. Sequence analysis of a cDNA clone isolated from bovine RPE predicts a polypeptide of 416 amino acid residues that shares 88.6% and 85% amino acid identity with human and mouse PEDF, respectively. It has an N-terminal signal peptide, a consensus glycosylation site and homology with serpins including the conserved residues required for maintaining the serpin tertiary structure. Cell-free expression of the bovine PEDF cDNA by in vitro transcription and translation yields a precursor polypeptide of 45,000-Mr that immunoprecipitates with an antibody to human PEDF. Expression analysis in stably transfected baby hamster kidney cells shows that the recombinant bovine protein is secreted to the culture media as a mature 50,000-Mr protein, which induces neurite-outgrowth on retinoblastoma cells, like the naturally-occurring PEDF protein. Thus, the bovine PEDF cDNA isolated here codes for a functional soluble secreted PEDF glycoprotein.

Pigment epithelium-derived factor (PEDF), a neurotrophic agent first identified in conditioned medium from cultured human retinal pigment epithelial cells, induces neuronal differentiation with neurite outgrowth in Y-79 retinoblastoma cells and has a neurotrophic survival effect on cerebellar granule cells in culture. In the present study, we investigated the effects of human recombinant PEDF (rPEDF) on proliferation and activation of microglia and astrocytes isolated from newborn rat brain. rPEDF treatment caused microglia to round up morphologically, increased their metabolic activity (measured by both MTS conversion and acid phosphatase activity), but blocked proliferation (mitosis). This blocking effect could be demonstrated in cultures stimulated to proliferate by addition of granulocyte-macrophage colony stimulating factor. The effect of rPEDF on microglial metabolic activity showed a dose-response relationship both in serum-containing medium and in chemically defined medium and was blocked with anti-PEDF antibody. rPEDF had no direct effect on the metabolic activity or proliferation of cultured astrocytes but blocked their proliferation in astrocyte-microglia co-cultures. Proliferation of isolated astrocytes was also blocked by conditioned medium from microglia treated with PEDF (PMCM). The effect of PMCM on astrocytes was not blocked by an antibody to transforming growth factor-beta. These results demonstrate that PEDF activates microglial metabolism while blocking proliferation and suggest that a soluble factor(s) released by rPEDF-stimulated microglia blocks the proliferation of astrocytes. Thus, PEDF could play an important role in regulation of glial function and proliferation in the central nervous system.

Pigment epithelium-derived factor (PEDF) is a survival factor for cerebellar granule cells in culture. In the present study, we have investigated the ability of a recombinant form of PEDF (rPEDF) to protect against glutamate neurotoxicity. When rPEDF was added to cerebellar granule cell cultures 30 min before addition of 100 microM glutamate, glutamate-induced neuronal death was significantly reduced. The protective effect of rPEDF was dose-dependent in the range from 0.023 to 7.0 nM (1-500 ng/ml), with a half-maximal dose of 0.47 nM. An antibody to rPEDF blocked this protective effect. Measurement of intraneuronal free calcium levels demonstrated that rPEDF raised the basal calcium content. However, after the elevation of intracellular calcium in response to administration of glutamate, rPEDF reduced the plateau level seen in the presence of glutamate. These data show that PEDF can protect neurons against glutamate-induced neurotoxicity, possibly via a calcium-related pathway. The finding that only 30 min of preincubation is required for the neuroprotective effect, significantly faster than other known neurotrophic factors, suggests that PEDF may be useful clinically as a neuroprotective agent in the CNS.

Pigment epithelium-derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full-length glycosylated human recombinant PEDF at high levels (> 20 micrograms/ mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full-length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation.

Purpose: Experiments were designed to identify proteolytic activities that cleave pigment epithelium-derived factor (PEDF), a member of the serpin (serine protease inhibitor) family., Methods: Proteins in vitreous humor from bovine eyes were analyzed by Western blot with antiserum to human recombinant PEDF protein. Protein fractionation was by ammonium sulfate saturation and by S-Sepharose column chromatography. Proteolytic activities were determined by gelatin zymography and by solution assays against PEDF or chromogenic peptide substrates., Results: PEDF protein was identified and purified to near homogeneity from vitreous humor of bovine eyes. Limited proteolysis showed that the vitreal protein has a protease-sensitive region at its serpin-exposed peptide loop. Proteolytic activities that cleave the PEDF 49.5 kDa-polypeptide were identified only when proteins from these extracts were separated by 45% to 70% ammonium sulfate fractionation (P70). The degradation product had an apparent molecular weight of 46 kDa. This result is consistent with cleavage at the serpin-exposed loop. The PEDF-cleavage activity in P70 was inhibited specifically by the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), but not by aprotinin, EDTA, or pepstatin. The vitreal P70 extracts contained 49- and 53-kDa gelatinolytic activities that also were inhibited by AEBSF and not by EDTA, aprotinin, or pepstatin. The PEDF-cleavage activity did not hydrolyze substrates for thrombin, factor Xa, alpha-chymotrypsin, trypsin, or plasmin, nor did it immunoreact with antibody to urokinase plasminogen activator., Conclusions: These data indicated that vitreous has a serine-proteolytic activity associated with a novel 49/53-kDa enzyme that cleaves the PEDF protein in a serpinase fashion. In addition to cleavage in vitro, these proteases might play a role in modulating PEDF in vivo.

Pigment epithelium-derived factor (PEDF), a neurite-promoting factor, has an amino acid primary structure that is related to members of the serine protease inhibitor (serpin) family. Controlled proteolysis of native PEDF (50 kDa) with either trypsin, chymotrypsin, elastase, or subtilisin yields in each case one major limited product of 46 kDa as analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analysis of the isolated 46-kDa products indicates a favored cleavage region located toward the C-terminal end of PEDF. A proteolyzed PEDF protein reaction mixture reveals two overlapping sequences: that of the N terminus of intact PEDF and that of an internal region, consistent with cleavage of PEDF about position 382. These data indicate that PEDF protein has a globular conformation with one protease-sensitive exposed loop that contains the homologous serpin-reactive site. Cleavage within the reactive-site loop of PEDF does not cause a conformational change in the molecules (the stressed (S)-->relaxed (R) transition) and results in heat denaturation identical to its native counterpart. This lack of conformational change is also seen upon cleavage within the reactive-site loop of the noninhibitory serpin ovalbumin. Furthermore, the PEDF neurite-promoting function is not lost with cleavage of the exposed loop. Recombinant PEDF polypeptide fragments with larger truncations from the C-terminal end show neurotrophic activity. Our results clearly indicate that integrity of the PEDF homologous serpin reactive center is dispensable for neurotrophic activity. Thus, the PEDF induction of neurites must be mediated by a mechanism other than serine protease inhibition. Altogether our data indicate that PEDF belongs to the subgroup of noninhibitory serpins and that its N-terminal region confers a neurite-promoting activity to the protein. The neurotrophic active site of PEDF is separated from the serpin reactive-site loop, not only in the primary structure, but also in the folded protein structure.

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein and a member of the serine protease inhibitor superfamily. Here we describe the identification of PEDF in bovine eyes and optimization of its purification from this natural source. We have developed a polyclonal antibody to recombinant human PEDF, Ab-rPEDF, that immunoreacts in a specific, sensitive, and linear fashion with PEDF protein, and furthermore, blocks its neurotrophic activity. We show that Ab-rPEDF specifically recognizes a 49,500-M(r) polypeptide on Western transfers of a wash of the extracellular matrix between the retinal pigment epithelium and the neural retina-termed interphotoreceptor matrix (IPM). PEDF is present as approximately 1% of total soluble IPM protein. Starting with an IPM wash, PEDF protein is purified 164-fold to near homogeneity by ammonium sulfate fractionation and cation-exchange chromatography, with a recovery of 47%. The highly purified protein has an apparent M(r) of 49,500 +/- 1,500 as assessed by SDS-polyacrylamide gel electrophoresis, and a native pI of 7.0-7.7. It elutes as a single peak on gel-filtration chromatography with a retention time immediately behind that of ovalbumin (43,000 M(r)). N-glycosidase treatment indicates that each PEDF molecule has a 5% carbohydrate content attached to internal asparagine residue(s). Amino terminal sequence of the purified PEDF reveals removal of an amino-terminal peptide region for the mature protein. Purified PEDF has neurotrophic activity on human retinoblastoma cells, as previously observed for IPM. The neurotrophic activities of both PEDF and IPM are blocked by antiserum Ab-rPEDF. Altogether, PEDF is present in the bovine IPM as a soluble, extracellular, monomeric glycoprotein that by itself confers neurotrophic activity to the IPM. Thus, native PEDF isolated and purified as described here should prove useful for biochemical studies as well as other approaches.

Pigment epithelium-derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y-79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose-response relationship, with a larger effect in chemically defined medium than in serum-containing medium [ED50 = 30 ng/ml (0.70 nM) in chemically defined medium and 100 ng/ml (2.3 nM) in serum-containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme-linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.