Pigment Epithelium-derived Factor Protein Research Articles

Changes in pigment epithelium-derived factor expression following kainic acid induced cerebellar lesion in rat

Pigment epithelium-derived factor (PEDF) is a potent and broad-acting neurotrophic factor that protects various types of cultured neurons against glutamate excitotoxicity and induced apoptosis. The expression pattern and functions of PEDF in the central nervous system (CNS) remain largely undetermined. In this study, we analyzed the spatial and temporal expression of PEDF in normal and kainic acid (KA)-induced lesioned rat cerebellum using immunoblotting, immunohistochemistry and fluorescent in situ hybridization techniques. In normal rat cerebellum, PEDF protein and mRNA were mostly confined and co-localized with calbindin-positive cells in the Purkinje cell layer of the cerebellum, but not with glial fibrillary acidic protein (GFAP)-, 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-, and isolectin B4-positive cells. Injection of KA into the right cellebellum caused severe loss of calbindin-positive Purkinje neurons, and an increased number of GFAP-positive astrocytes and isolectin B4-positive microglia was observed on the ipsilateral side of the lesioned cerebellum. Although the PEDF level on the ipsilateral side of the cerebellum was dramatically decreased 2 days after KA treatment, significantly elevation of PEDF levels was observed at 7 days. In agreement with these results, PEDF protein and PEDF mRNA expression were co-localized with GFAP-positive reactive astrocytes in the ipsilateral side 7 days after KA treatment. Although the mechanism by which PEDF is induced in reactive astrocytes remains unclear, the increase in PEDF expression in injured brain may form part of a compensation mechanism against neuronal degeneration.

Administration of pigment epithelium-derived factor (PEDF) inhibits cold injury-induced brain edema in mice

Brain edema is the most life-threatening complication that occurs as a result of a number of insults to the brain. However, its therapeutic options are insufficiently effective. We have recently found that administration of pigment epithelium-derived factor (PEDF) inhibits retinal hyperpermeability in rats by counteracting biological effects of vascular endothelial growth factor (VEGF). In this study, we investigated whether PEDF could inhibit cold injury-induced brain edema in mice. Cold injury was induced by applying a pre-cooled metal probe on the parietal skull. VEGF and its receptor Flk-1 gene and/or protein expressions were up-regulated in the cold-injured brain. Cold injury induced brain edema, which was reduced by intraperitoneal injection of VEGF antibodies (Abs) or apocynin, an inhibitor of NADPH oxidase. PEDF mRNA and protein levels were up-regulated in response to cold injury. PEDF dose-dependently inhibited the brain edema, whose effect was neutralized by simultaneous treatments with anti-PEDF Abs. Although VEGF and Flk-1 gene and/or protein expressions were not suppressed by PEDF, PEDF or anti-VEGF Abs inhibited the cold injury-induced NADPH oxidase activity in the brain. Further, PEDF treatment inhibited activation of Rac-1, an essential component of NADPH oxidase in the cold-injured brain, while it did not affect mRNA levels of gp91phox, p22phox, or Rac-1. These results demonstrate that PEDF could inhibit the cold injury-induced brain edema by blocking the VEGF signaling to hyperpermeability through the suppression of NADPH oxidase via inhibition of Rac-1 activation. Our present study suggests that PEDF may be a novel therapeutic agent for the treatment of brain edema.

Correlation between levels of pigment epithelium-derived factor and vascular endothelial growth factor in the striatum of patients with Parkinson's disease

Parkinson's disease (PD) is caused by progressive degeneration of nigrostriatal dopaminergic neurons and can potentially be treated by intrastriatal delivery of neurotrophic factors. Pigment epithelium-derived factor (PEDF), which exhibits protective effects on various neuronal populations, is up-/down-regulated in the cerebrospinal fluid in some neurodegenerative conditions. Here we investigated the level of PEDF protein in the striatum and immunoreactivity for PEDF in the substantia nigra (SN) of patients with PD to assess its role in the pathophysiology of PD. We also studied changes in PEDF expression in the striatum of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. We found a transient and rapid up-regulation of PEDF transcripts and a marked increase in immunoreactivity for PEDF protein in response to MPTP administration in mice. However, there were no significant changes in striatal levels of PEDF and immunoreactivity for PEDF in the SN of PD patients compared with age-matched non-PD patients. Intriguingly, the striatal levels of PEDF and vascular endothelial growth factor (VEGF), which has opposite functions to PEDF in terms of angiogenesis and vascular permeability, correlated positively in PD patients. Our results suggest up-regulation of PEDF in response to acute insult to the dopaminergic pathway, but such response might be disturbed in patients with advanced PD. The correlation between PEDF and VEGF striatal levels in PD patients suggests that concerted neurotrophic functions of these factors or structural changes in blood vessel walls play an important role in the pathophysiology of PD.

Expression of pigment-epithelium-derived factor during kidney development and aging

Inhibitors and stimulators of endothelial cell growth are essential for the coordination of blood vessel formation during organ growth and development. In the adult kidney, one of the major inhibitors of angiogenesis is pigment-epithelium-derived factor (PEDF). We have analyzed the expression and distribution of PEDF during various stages of renal development and aging with particular emphasis on the formation of functional glomeruli. We show that PEDF gene expression and protein levels in the kidney significantly increase with age. We have detected PEDF in the mesenchyme and endothelial cells at all developmental stages studied, in all regions of the nephrogenic zone in which the formation of new blood vessels is associated with the development of nephrons and collecting ducts, and in mature podocytes in the adult kidney. Our results are the first to suggest that PEDF is important in early renal postnatal development, that it could be relevant to the maturation of glomerular function and the filtration barrier formed by these cells, and that it may serve as an anti-angiogenic modulator during kidney development.

Hydrogen Peroxide Stimulates Pigment Epithelium-derived Factor Gene and Protein Expression in the Human Hepatocyte Cell Line OUMS-29

Pigment epithelium-derived factor (PEDF) may have a protective role in atherosclerosis and is associated with the presence of components of the metabolic syndrome. Since oxidative stress has been postulated to play an important role in the pathogenesis of vascular injury in the metabolic syndrome, this study investigated the effects of hydrogen peroxide (H2O2) on PEDF in the immortalized human hepatocyte cell line OUMS-29. PEDF gene expression was measured using quantitative real-time reverse transcription-polymerase chain reaction and PEDF protein expression was analysed by Western blot. H2O2 upregulated PEDF mRNA levels and increased PEDF protein production in OUMS-29 cells in time- and dose-dependent manners. The anti-oxidant N-acetylcysteine significantly blocked H2O2-induced PEDF overexpression in OUMS-29 cells. These results suggest that hepatic PEDF levels may be elevated to counteract the effects of oxidative stress. H2O2-induced PEDF overproduction in the liver may act as a negative feedback system against vascular damage in the metabolic syndrome.

Gelatinase‐mediated proteolysis of pigment epithelium‐derived factor (PEDF) is pH dependent

The antiangiogenic and neurotrophic factor PEDF is a member of the SERPIN superfamily of proteins. We have shown before that the majority of proteinases cleave PEDF (50-kDa) at its homologous serpin reactive loop leaving an active limited product of 46-kDa, and that gelatinases (MMP-2 and MMP-9) can degrade PEDF abolishing its biological activities. In this study we characterized the MMP-2 mediated proteolysis of PEDF. While MMP-2 in phosphate buffer degraded PEDF in a calcium-depended manner, activated MMP-2 in Tris buffer pH 7.5 plus calcium only cleaved PEDF to a 46-kDa product. Controlled chymotryptic proteolysis of PEDF in Tris and phosphate buffer plus CaCl2 resulted in a 46-kDa limited product, implying no change in folded conformation of the PEDF protein under these buffer conditions. Because the pH of phosphate buffer decreased with CaCl2, the effect of pH on MMP-2 activity was examined. MMP-2 in Tris/MES buffers had optimum activity at pH 5.5–6, with maximum autodegradation of the MMP-2 polypeptide at this pH range. These results demonstrate that MMP-2 can autoactivate in a pH-dependent fashion and suggest a pH-sensitive MMP-2-mediated degradation of PEDF. This work was supported by the Intramural Research Program of NEI and NCI, NIH.

A PEDF N-terminal peptide protects the retina from ischemic injury when delivered in PLGA nanospheres

The neuroprotective effects of small pigment epithelium-derived factor (PEDF) peptides injected intravitreally as free peptides or delivered in poly(lactide-co-glycolide) (PLGA) nanospheres, were tested in retinal ischemic injury. We induced transient ischemia in C57BL/6 mice by elevating the intraocular pressure to the equivalent of 120 mmHg for 60 min, then injected these eyes with one of the following: PBS, full-length native PEDF, N-terminal peptides—PEDF 136–155 and PEDF 82–121, blank PLGA nanospheres or PLGA loaded with PEDF 82–121 (PLGA-PEDF 82–121). Morphometric analysis and TUNEL assays were used to determine the extent of retinal damage. Transient ischemia caused a rapid reduction in the number of viable cells in the retinal ganglion cell (RGC) layer over 48 h as compared to non-ischemic retinas. About 76% surviving cells in the RGC layer were observed in the full-length PEDF protein treated group, whereas only 32% of cells survived in the PBS group. Thus, PEDF prevented approximately 44% of the cell death in the RGC layer resulting from transient ischemia. PEDF 82–121 peptide was as effective as full-length PEDF when injected as either a free peptide or delivered in PLGA nanospheres. PLGA-PEDF 82–121 showed longer-term protection of the RGC layer with no noticeable side effects at 7 days. PEDF and PEDF 82–121 lessened damage to the IPL as measured by layer thickness. PEDF and PEDF 82–121 also delayed retinal responses to ischemic injury as measured by GFAP immunolabeling in Müller cells. PEDF 82–121 is an effective neuroprotective peptide in retinal ischemia. PLGA-PEDF 82–121 offers greater protection to the retina suggesting that this peptide and the method of delivering therapeutically active drugs have potential clinical advantages for longer-term treatments of retinal diseases.

Pigment Epithelium-Derived Factor Is Estrogen Sensitive and Inhibits the Growth of Human Ovarian Cancer and Ovarian Surface Epithelial Cells

Epithelial ovarian carcinoma is the most lethal gynecological cancer. However, little is known about the molecular mechanisms underlying the disease development and progression. In this study, we found that the expression of pigment epithelium-derived factor (PEDF) was greatly reduced in ovarian tumors and in ovarian cancer cell lines when compared with their normal precursor, ovarian surface epithelium (OSE). In addition, we showed that exogenous PEDF inhibited the growth of cultured human OSE as well as ovarian cancer cell lines, whereas targeted inhibition of endogenous PEDF using small interfering RNA or neutralizing PEDF antibody promoted the growth of these cells, confirming that the growth-inhibitory effect was PEDF specific. We also report for the first time that estrogen is an important upstream regulator of PEDF in human OSE. Treatment of the cultured cells with 17 beta-estradiol (E2) inhibited the expression of PEDF protein and mRNA in a dose- and time-dependent manner, which could be reversed by the specific estrogen receptor antagonist, ICI 182,780, indicating that the regulation was estrogen receptor-mediated. We further showed that this down-regulation of PEDF gene transcription was a direct, primary effect of E2. E2 promoted OSE and ovarian cancer cell growth, whereas simultaneous treatment with E2 and PEDF abrogated the estrogenic growth stimulation of these cells. This study is the first to demonstrate a role of PEDF in OSE biology and ovarian cancer and suggests that the loss of PEDF may e of relevance in carcinogenesis.

Pigment epithelium–derived factor is a niche signal for neural stem cell renewal

Adult stem cells are characterized by self-renewal and multilineage differentiation, and these properties seem to be regulated by signals from adjacent differentiated cell types and by extracellular matrix molecules, which collectively define the stem cell "niche." Self-renewal is essential for the lifelong persistence of stem cells, but its regulation is poorly understood. In the mammalian brain, neurogenesis persists in two germinal areas, the subventricular zone (SVZ) and the hippocampus, where continuous postnatal neuronal production seems to be supported by neural stem cells (NSCs). Here we show that pigment epithelium-derived factor (PEDF) is secreted by components of the murine SVZ and promotes self-renewal of adult NSCs in vitro. In addition, intraventricular PEDF infusion activated slowly dividing stem cells, whereas a blockade of endogenous PEDF decreased their cycling. These data demonstrate that PEDF is a niche-derived regulator of adult NSCs and provide evidence for a role for PEDF protein in NSC maintenance.

Angiostatic Effect of Penetrating Ocular Injury: Role of Pigment Epithelium-Derived Factor

To characterize the angiostatic effect of penetrating ocular injury and to begin to explore its mechanism, with an emphasis on the role of pigment epithelium-derived factor (PEDF). Using the rat model of oxygen-induced retinopathy (OIR), single or multiple dry needle injuries were made, penetrating the globe of one eye; the opposite eye served as a control. Eyes were harvested from rats killed 1, 3, and 6 days after injury, and retinas were dissected and processed for assessment of neovascularization and microglial activation or were processed for genetic and proteomic analysis. Temporal and spatial expression patterns of PEDF were analyzed by in situ hybridization. Penetrating ocular injury resulted in a 30% decrease in neovascular area in the retinas of OIR rats. At day 1 after injury, needle insertion caused a 4.1-fold increase in retinal PEDF mRNA and a 1.5-fold increase in retinal PEDF protein. Vitreous PEDF protein increased 3.4-fold in injured eyes compared with noninjured eyes. In situ hybridization showed an increase in PEDF mRNA in areas surrounding the puncture site. Concentrated vitreous protein from injured eyes caused a 60% decrease in retinal neovascularization when injected into the vitreous cavity of OIR rats. Preincubation of vitreous samples with anti-PEDF partially abolished this efficacy. The pattern of angiostasis resulting from penetrating ocular injury is consistent with the release of an endogenous antiangiogenic factor from the wound site. Preliminary studies show a possible role for PEDF in this effect. Further characterization of this role and the identification of other factors may lead to new therapeutic strategies for angiogenic eye conditions.

809. Suppression of Human Pancreatic Cancer Growth by Lentiviral Vector-Mediated Gene Transfer of Pigment Epithelium Derived Factor (PEDF)

Top of pageAbstract Pancreatic cancer is the fourth leading cause of cancer death. Most patients are diagnosed after metastasis to adjacent organs and blood vessels. Angiogenesis, which is required to for solid tumor growth beyond 1-2 mm3, plays a critical role in pancreatic cancer. Pigment epithelium-derived factor (PEDF) has been identified as the main inhibitor of retinal angiogenesis. Subsequently, PEDF has been shown to critically regulate the vasculature and mass of the pancreas. Here we examined the feasibility of using lentiviral vector-expressed human PEDF gene to treat human pancreatic tumor cell line-derived tumors in a murine model. Mono-cistronic FIV vectors encoding human PEDF or a control protein (eGFP) with each gene linked to neoR via an IRES, were used. PEDF protein produced from transduced cells and direct application of PEDF vector significantly reduced angiogenesis (63% inhibition compared to control) in a capillary formation assay using human umbilical vascular endothelial cells (HUVECs) and in a chick chorioallantoic membrane assay (14% angiogenesis index compared to control index of 64%). Enhanced HUVEC apoptosis was also observed. We examined the therapeutic efficacy of injected PEDF and control vectors on established human pancreatic tumor xenografts in nude mice. Significant anti-tumor effects and prolonged overall survival benefit was observed in PEDF vector-treated mice (> 80% tumor reduction after 1- 2 x 10^7 TU at day 30 post injection). PEDF vector-treated animals survived 52|[plusmn]|2 days compared to 30|[plusmn]|3 days with control vector, n = 16 in each group. Tumors treated with PEDF vector had enhanced apoptosis and vascular endothelial growth factor receptor-1 expression. We also studied tumor formation by stable pancreatic cell lines derived by transduction with PEDF and control vectors and G418 selection. PEDF secreted from these stable tumor cell lines was shown to be biologically active in the in vitro assays and a significant antitumor effect and prolonged overall survival benefit was seen. However, injection of vectors into established tumors had a better therapeutic effect than pre-transducing and selecting cells prior to establishing tumors, and this correlated with up-regulation of VEGFR-1 in the stable tumor cell lines. PEDF is a promising transgene for pancreatic cancer gene therapy.

Pigment Epithelium–Derived Factor Gene Therapy Inhibits Human Pancreatic Cancer in Mice

Pigment epithelium-derived factor (PEDF), which has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, is also expressed in the pancreas. Previously, we have screened the expression of PEDF by immunohistochemical analysis and showed that low expression of PEDF is associated with increased risk of hepatic metastasis and short survival. The purpose of this study was to investigate whether PEDF gene is a potent tumor suppressor and a potential candidate for cancer gene therapy. We investigated both in vitro and in vivo growth characteristics of human pancreatic adenocarcinoma cell lines that were stably transfected to overexpress human PEDF and therapeutic effects of lentivirus-based vectors expressing PEDF on tumor growth in murine s.c. tumor model. We discovered that cells secreted PEDF protein in the media and this exhibited strong inhibitory effects on proliferation and migration of human umbilical vein endothelial cells. The size of PEDF-overexpressing pancreatic adenocarcinoma tumors was significantly smaller than that of control tumors in s.c. tumor models. Moreover, the growth of PEDF-overexpressing pancreatic adenocarcinoma cells was significantly suppressed in comparison with control cells in peritoneal metastasis models. In gene transfer models, intratumoral injection of a lentivirus vector encoding PEDF (LV-PEDF) caused significant inhibition of tumor growth. The antitumor effect observed after treatment with LV-PEDF was associated with decreased microvessel density in tumors. Our data suggest that PEDF may exert a biological effect on tumor angiogenesis and PEDF gene therapy may provide a new approach for treatment of pancreatic adenocarcinoma.

Differential expression of pro- and antiangiogenic factors in mouse strain-dependent hypoxia-induced retinal neovascularization

Clinical observations suggest that genetic factors may influence heterogeneity of angiogenic responses in cardiovascular disease, proliferative diabetic retinopathy, and neoplasia. Experiments among mouse strains using a corneal micropocket assay indicate that extent of angiogenesis may be genetically determined. Here, we established the strain-dependence of hypoxia-induced retinal angiogenesis in multiple mouse strains which paralleled the rank order found for bFGF-induced corneal angiogenesis. Using quantitative real-time RT-PCR, strain-related gene expression differences in retina/choroid between C57BL/6J and 129S3/SvIM, inbred strains with relatively low and high levels of angiogenesis, respectively, after 0, 6, 12, 24, 48, and 96 h hypoxia were determined for vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), angiogenic ligands potently induced by hypoxia, and for pigment epithelium-derived factor (PEDF) and thrombospondin-1 (TSP-1), endogenous broad-spectrum antiangiogenic factors. Indirect ELISA was used to correlate VEGF and PEDF protein levels with mRNA expression. At the onset of hypoxia, both PEDF and TSP-1 levels were increased over 15-fold and VEGF was increased over 10-fold compared to Ang-2 in both strains. At the onset of neovascularization (48 h), both VEGF and Ang-2 mRNA levels were increased in the more angiogenic 129S3/SvIM strain (P<0.02), which was not observed among developmental control animals. PEDF expression was higher in the less angiogenic C57BL/6J strain at 6, 12, 24, and 96 h hypoxia (P<0.03), while TSP-1 expression was higher in C57BL/6J throughout the entire time course of hypoxia (4 days) compared to 129S3/SvIM (P<0.02). Among developmental control animals, PEDF and TSP-1 expression was also increased at P14 and P16 in C57BL/6J strain compared to 129S3/SvIM (P<0.02). Strain-dependent expression of both pro- and antiangiogenic growth factors may determine heterogeneity in the angiogenic response and potentially, susceptibility to angiogenesis-dependent diseases.

Production of active pigment epithelium-derived factor inE. coli

Human pigment epithelium-derived factor (PEDF), a neurotrophic factor, is the most potent natural inhibitor of angiogenesis. To produce the active PEDF, the gene coding for the human PEDF protein was expressed in E. coli. The rPEDF protein was expressed at 457 mg l-1 as a soluble protein. The yield of purified GST fusion protein was 14 mg l-1. Purified rPEDF inhibited tube formation in endothelial cells.

Pigment-Epithelium-Derived Factor Is Upregulated in Photocoagulated Human Retinal Pigment Epithelial Cells

There is much evidence that pigment-epithelium-derived factor (PEDF) is a potent antiangiogenic cytokine which inhibits retinal and choroidal neovascularization by inducing apoptosis in activated vascular endothelial cells. Furthermore, the regulation of PEDF appears to be linked to the regulation of vascular endothelial growth factor (VEGF), one of the most potent inducers of intraocular neovascularization. Previous studies have established that thermal photocoagulation, the mainstay in the therapy of various neovascular diseases of the posterior segment, results in a decrease in intraocular concentrations of VEGF and other angiogenic growth factors, thereby inhibiting active retinal neovascularization. In the current study, we sought to determine whether thermal photocoagulation has the potential to regulate the expression of PEDF in human retinal pigment epithelial (RPE) cells. Cultures of RPE cells were photocoagulated with a 532-nm diode laser. Subsequently, RNA was isolated for RT-PCR, and whole-cell extracts and precipitated cell culture supernatant were subjected to Western blot analysis. According to our results, PEDF mRNA and protein are significantly upregulated after photocoagulation. Moreover, PEDF protein was found to be secreted in the cell culture medium.

Pigment Epithelium-derived Factor as a New Diagnostic Marker for Melanocytic Tumors

Pigment epithelium-derived factor (PEDF), a potent antiangiogenetic factor, has been lately known to correlate well with angiogenic and metastatic potentials of tumor cells. We investigated the expression of PEDF protein in various types of human tumor cells by an immunohistochemical technique using a monoclonal antibody. Consequently, we found the significantly frequent and intense expression of PEDF in human melanocytic tumor cells including malignant melanoma as compared to non-melanocytic ones. We evaluated the diagnostic usefulness of anti-PEDF antibody in melanocytic tumors by comparing its immunoreactive sensitivity with that of other conventional melanocytic markers such as S-100 protein, HMB-45 and Melan-A, and found that PEDF possess the equal ability to others on its sensitivity. We finally concluded that PEDF is a useful diagnostic marker for melanocytic tumors, especially malignant melanomas, by its use in combination with other markers.

Suppression of corneal neovascularization by PEDF release from human amniotic membranes.

Human amniotic membrane (HAM) transplantation is commonly used in corneal surface reconstruction and is known to inhibit neovascularization of this tissue. The purpose of the present study is to reveal the molecular basis underlying antiangiogenic activity of HAM. The effects of HAM protein on proliferation of vascular endothelial cells and corneal epithelial cells were determined by quantifying viable cells using the MTT assay. The presence of pigment epithelium-derived factor (PEDF) in HAM was demonstrated at the protein level by Western blot analysis and immunohistochemistry using a monoclonal antibody specific to human PEDF. The PEDF concentration was measured by a specific ELISA. The expression of PEDF in HAM was confirmed at the RNA level by RT-PCR and DNA sequencing. Soluble proteins from HAM inhibited proliferation of human umbilical vein endothelial cells and bovine retinal capillary endothelial cells (BRCECs) while promoting proliferation of bovine cornea epithelial cells. Moreover, the HAM-induced inhibition of BRCECs was neutralized by a specific anti-PEDF antibody. PEDF protein was identified with an abundance of 103.84 +/- 33.21 ng/mg of soluble proteins, which is comparable to that in the retina, a PEDF-rich tissue. PEDF expression was predominantly localized in the basement membrane of HAM. RT-PCR using specific PEDF primers amplified a single product from HAM RNA. The PCR product has a sequence identical with that of human PEDF. HAM specifically inhibits endothelial cell growth and thus suppresses neovascularization in the cornea. PEDF in HAM has a major role in eliciting this antiangiogenic activity.

In vitro studies of pigment epithelium-derived factor in human Schwann cells after treatment with axolemma-enriched fraction

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with known anti-angiogenic and trophic properties, capable of promoting the survival and growth of Schwann cells (SC). Normal rat SCs and ganglioneuroma-derived human SCs secrete PEDF. The ability of normal SC to secrete a number of trophic factors is controlled by axonal contact. Normal human Schwann cells (HSC) and malignant peripheral nerve sheath tumors (MPNST) cell lines synthesize and secrete PEDF as determined by reverse transcription PCR analysis for PEDF mRNA, immunocytochemistry, and Western blot analysis for PEDF protein. Two MPNST cell lines secreted higher levels of PEDF than did HSC. A 90.3% decrease in PEDF mRNA and a 29.3% decrease in secreted PEDF were observed after treatment of HSC with axolemma-enriched fraction (AEF, 100 microg/ml), a neuronal membrane fraction of the axonal plasma membrane used with cultured SC to mimic axonal contact in vitro. PEDF levels remained unchanged, however, in MPNST-derived SC conditioned media under the same treatment paradigm. These results suggest that MPNST SC lose the ability to downregulate PEDF upon axonal contact, which is characteristic of HSC. The elevated PEDF levels expressed by MPNST cell lines may serve to promote their proliferation and survival.

Antiangiogenic property of pigment epithelium-derived factor in hepatocellular carcinoma.

Pigment epithelium-derived factor (PEDF) is one of the most powerful endogenous antiangiogenic reagents discovered to date. Its antiangiogenic potential in neoplastic disease remains unclear. In this study, we investigated antiangiogenic property of PEDF in hepatocellular carcinoma (HCC), a typical hypervascular tumor. In HCC cell lines, constitutive messenger RNA and protein expression of PEDF varied. Genomic DNA encoding the PEDF gene was the same in the cell lines examined by Southern blotting. In chemically induced hypoxic conditions, secreted PEDF protein was suppressed in contrast to elevation of vascular endothelial growth factor protein. When PEDF was overexpressed by gene transfer, proliferation and migration of endothelial cells were inhibited in conditioned media derived from all HCC cell lines. However, the serum concentration of PEDF, as measured by enzyme-linked immunosorbent assay, was decreased in patients with cirrhosis or HCC complicated by cirrhosis compared to healthy volunteers and patients with chronic hepatitis. According to the endothelial cell proliferation assay, the serum PEDF of patients with HCC had antiangiogenic activity. Moreover, intratumoral injection of a PEDF-expressing plasmid in athymic mouse models caused significant inhibition of preestablished tumor growth. In conclusion, PEDF plays a role in the angiogenic properties of HCC. Reduction of serum PEDF concentration associated with the development of chronic liver diseases may contribute to the progression of HCC. In addition, gene therapy using PEDF may provide an efficient treatment for HCC.

Pigment epithelium-derived factor in the monkey retinal pigment epithelium and interphotoreceptor matrix: apical secretion and distribution

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 n m) 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.