Ginsenoside Rb1 inhibits tube-like structure formation of endothelial cells by regulating pigment epithelium-derived factor through the oestrogen beta receptor.

BACKGROUND AND PURPOSE: Angiogenesis is a crucial step in tumour growth and metastasis. Ginsenoside-Rb1 (Rb1), the major active constituent of ginseng, potently inhibits angiogenesis in vivo and in vitro. However, the underlying mechanism remains unknown. We hypothesized that the potent anti-angiogenic protein, pigment epithelium-derived factor (PEDF), is involved in regulating the anti-angiogenic effects of Rb1. EXPERIMENTAL APPROACHES: Rb1-induced PEDF was determined by real-time PCR and western blot analysis. The anti-angiogenic effects of Rb1 were demonstrated using endothelial cell tube formation assay. Competitive ligand-binding and reporter gene assays were employed to indicate the interaction between Rb1 and the oestrogen receptor (ER). KEY RESULTS: Rb1 significantly increased the transcription, protein expression and secretion of PEDF. Targeted inhibition of PEDF completely prevented Rb1-induced inhibition of endothelial tube formation, suggesting that the anti-angiogenic effect of Rb1 was PEDF specific. Interestingly, the activation of PEDF occurred via a genomic pathway of ERbeta. Competitive ligand-binding assays indicated that Rb1 is a specific agonist of ERbeta, but not ERalpha. Rb1 effectively recruited transcriptional activators and activated an oestrogen-responsive reporter gene. Furthermore, Rb1-mediated PEDF activation and the subsequent inhibition of tube formation were blocked by the ER antagonist ICI 182,780 or transfection of ERbeta siRNA, indicating ERbeta dependence. CONCLUSIONS AND IMPLICATIONS: Here we show for the first time that the Rb1 suppressed the formation of endothelial tube-like structures through modulation of PEDF via ERbeta. These findings demonstrate a novel mechanism of the action of this ginsenoside that may have value in anti-cancer and anti-angiogenesis therapy.

Pigment epithelium-derived factor supports normal development of photoreceptor neurons and opsin expression after retinal pigment epithelium removal.

Dysfunction of the retinal pigment epithelium (RPE), its loss, or separation from the underlying neural retina results in severe photoreceptor degeneration. Pigment epithelium-derived factor (PEDF) is a glycoprotein with reported neuroprotective and differentiation properties that is secreted in abundance by RPE cells. The "pooling" of PEDF within the interphotoreceptor matrix places this molecule in a prime physical location to affect the underlying neural retina. The purpose of this study was to analyze the morphogenetic activity of PEDF in a model of photoreceptor dysmorphogenesis induced by removal of the RPE. Eyes were dissected from embryonic Xenopus laevis, and the RPE was removed before culturing in medium containing PEDF, PEDF plus anti-PEDF antibodies, or medium alone. Control retinas were maintained with an adherent RPE. Light and electron microscopic analysis was used to examine retinal ultrastructure. Opsin was localized immunocytochemically and quantified as an index of outer segment membranous material and photoreceptor protein expression. Removal of the RPE resulted in an aberrant assembly of photoreceptor outer segments, loss of fine subcellular ultrastructure in photoreceptors, and a reduction in opsin protein levels when compared with control retinas. The addition of PEDF prevented the dysmorphic photoreceptor changes induced by RPE removal. In particular, photoreceptor ultrastructure, outer segment membrane assembly, and steady-state levels of opsin were equivalent to control conditions. Anti-PEDF antibodies completely blocked the morphogenetic activity of PEDF. These results indicate that PEDF is able to mimic the supportive role of the RPE on photoreceptors during the final stages of retinal morphogenesis.

Pigment epithelium-derived factor (PEDF) protects motor neurons from chronic glutamate-mediated neurodegeneration.

Although pigment epithelium-derived factor (PEDF) is a neurotrophic factor that may aid the development, differentiation, and survival of adjacent neural retinae, the wider distribution of PEDF mRNA in the central nervous system suggested to us that this factor could have pleiotropic neurotrophic and neuroprotective effects on nonretinal neurons. We examined the distribution of PEDF mRNA and its transcript in the spinal cord. By immunohistochemistry and western blot analysis using an antihuman PEDF antiserum of known specificity, we found that PEDF protein is present in spinal cord, cerebrospinal fluid, and skeletal muscle and that its mRNA appears concentrated in motor neurons of the human spinal cord. These observations indicate that PEDF could have potential autocrine and paracrine effects on motor neurons, as well as being target-derived. We analyzed the pharmacologic utility of PEDF in a postnatal organotypic culture model of motor neuron degeneration and proved it is highly neuroprotective. The effect was biologically important, significantly sparing the spinal cord's gross organotypic morphological appearance and preserving motor neuron choline acetyltransferase (ChAT). PEDF alone did not increase ChAT, indicating that the observed effect is neuroprotective, not merely an upregulation of motor neuron ChAT. Further, PEDF preserved motor neuron number, proving a survival effect. We hypothesize that PEDF may play important roles in the survival and maintenance of spinal motor neurons in their neuroprotection against acquired insults in postnatal life. It should be developed further as a therapeutic strategy for motor neuron diseases such as amyotrophic lateral sclerosis (ALS).

Neurotrophic activity of interphotoreceptor matrix on human Y79 retinoblastoma cells.

A neurotrophic activity of adult monkey and bovine interphotoreceptor matrix (IPM) was examined by using cultured human Y79 retinoblastoma cells as a model system. The cells were stimulated for 7 days in suspension culture with soluble IPM components and then attached to poly-D-lysine substratum. IPMs from both species induced greater than 80% neuronal differentiation of Y79 cell aggregates after 11 days of attachment as adjudged morphologically by the extension of lengthy, neurite-like processes. Immunocytochemical studies indicate that differentiated Y79 cells had an increased level of expression of neuron-specific enolase and a concomitant decreased expression of glial fibrillary acidic protein. This neurotrophic activity cannot be ascribed to nerve growth factor, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, or transforming growth factor beta. Although the nature of the factor and its cellular source have yet to be characterized, it may be related to a recently described neurotrophic protein secreted by human fetal retinal pigment epithelial cells in culture. Our findings provide evidence supporting the neuroblastic potential of the Y79 cell line and indicate that the IPM contains a potent neurotrophic activity. Such factors may be important to normal differentiation and maintenance of function of the neural retina.

Neuronal differentiation of retinoblastoma cells induced by medium conditioned by human RPE cells.

Tumor cells can be induced to differentiate in vitro by biochemical manipulation of their culture environment. In the studies described here, the effects of medium conditioned by human retinal pigmented epithelial (RPE) cells on Y79 human retinoblastoma cells have been examined. RPE-conditioned medium in conjunction with laminin and a poly-D-lysine substratum is observed to induce neuronal differentiation of Y79 cells. The cells extend long cellular processes and exhibit immunologically detectable neurotypic properties. In contrast, control Y79 cells not exposed to medium conditioned by RPE cells exhibit only infrequent neuronal phenotypes. This response of Y79 cells to RPE-conditioned medium indicates that factors secreted by RPE cells can act as inducers of neuronal differentiation in retinoblastoma cells and suggest that such factors may be of importance in the development and differentiation of the neural retina.

In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor.

PURPOSE: To determine whether pigment epithelium-derived factor (PEDF) exhibits neurotrophic and neuroprotective activities in vivo for photoreceptor cells. METHODS: Sprague-Dawley albino rats were injected intravitreally with 2 microg PEDF or a mixture of 1 microg basic fibroblast growth factor (bFGF)/1 microg PEDF in a volume of 1 microl phosphate-buffered saline (PBS). Animals were exposed to constant light for different periods at an illuminance level of 1200 to 1500 lux. The electroretinogram (ERG) waveforms of both eyes in the same animal were simultaneously recorded to evaluate functional protection. The morphologic protection was evaluated by quantitative histology. RESULTS: Intravitreal injection of PEDF before exposure to constant light resulted in significant morphologic and functional protection of photoreceptor cells in the retina of light-damaged rats. This protection depended on the duration and severity of light damage. The protection was eliminated by extending the light exposure to 10 days. Injection of PEDF at 0, 1, and 2 days after constant light exposure did not provide significant protection above that seen in PBS-injected eyes. Combination of PEDF with bFGF improved functional protection of photoreceptor cells. CONCLUSIONS: The data demonstrate that PEDF protected photoreceptor cells against light damage. This is significant, because it may open new avenues for the study of molecular mechanisms underlying degenerative processes. This, in turn, may lead to the development of therapeutic strategies for the prevention and treatment of degenerative diseases of the retina.

Effects of medium conditioned by retinal pigmented epithelial cells on neurotransmitter phenotype in retinoblastoma cells.

We previously reported that medium conditioned by retinal pigmented epithelial cells can induce cellular differentiation in human retinoblastoma cells. Extensive neurite outgrowth, increased expression of neuronal marker molecules and decreases expression of glial marker molecules are characteristic of the differentiated phenotype. In the studies described here, we examine whether modulations in the expression of potential neurotransmitter molecules, catecholamines and indolealkyl amines, might be associated with the differentiation of retinoblastoma cells. Concentrations of serotonin, 5-hydroxyindoleacetic acid, 3-methoxytyrosine, homovanillic acid, and 3-methoxy-4-hydroxyphenylacetic acid in extracts of differentiated and undifferentiated retinoblastoma cells were assessed by HPLC. The results show that serotonin and its metabolite, 5-hydroxyindoleacetic acid, are characteristically present in undifferentiated cells. Dopa metabolites, 3-methoxytyrosine, homovanillic acid and 3-methoxy-4-hydroxy-phenylacetic acid, are uniquely present in differentiated cells. It appears that differentiation of retinoblastoma cells induced by factors secreted by retinal pigmented epithelial cells involves a switch from a serotonergic phenotype to one dominated by metabolites of dopa. These findings may provide clues about the factors that control retinoblastoma cells and metastasis.

Four polymorphic variations in the PEDF gene identified during the mutation screening of patients with Leber congenital amaurosis.

PURPOSE: Leber congenital amaurosis (LCA) has been mapped to chromosome 17p13.1. From the candidate genes mapped to this region, thus far, only Retinal Guanylate Cyclase (RetGC), has been found to have pathogenic LCA mutations, in families from North African origin. However, early reports, demonstrated eight LCA families linked to 17p13.1, but only four of them showed mutations in RetGC. Mapped in proximity to this locus is the candidate gene Pigment Epithelium Derived actor (PEDF), a factor implicated in photoreceptor differentiation and neuronal survival. Our purpose in this study was to identify mutations and polymorphisms in the PEDF gene in LCA patients of diverse ethnic origin. METHODS: Automated genotyping with four 17p13.1 markers flanking the PEDF gene was performed to assess homozygosity and PCR-SSCP combined with direct sequencing was used to detect mutations in the PEDF gene in 17 LCA patients. RESULTS: Homozygosity of markers D17S796 and D17S804 was found and four new intragenic basepair alterations were discovered: a Met72Thr polymorphism in exon 3 (T331C), a Thr130Thr polymorphism in exon 4 (T506C), a G to A transition in intron 5 (nine base pairs upstream from splice acceptor site), and a Tyr321Tyr polymorphism in exon 7 (C1079T) were detected. CONCLUSIONS: We report the discovery of four new polymorphic alterations in the PEDF gene in LCA patients and exclude by RFLP analysis the PEDF gene as a common cause of Leber congenital amaurosis. These single nucleotide polymorphisms will aid in future linkage analysis of complex multifactorial diseases involving retinal and RPE dysfunctions.

Organization, evolutionary conservation, expression and unusual Alu density of the human gene for pigment epithelium-derived factor, a unique neurotrophic serpin.

PEDF is a neurotrophic serpin that promotes a neuronal phenotype and augments neuronal cell survival. The isolation, sequence and structural analysis of the human PEDF gene and its promoter along with its evolutionary conservation and expression in human tissues are now described. The gene spans approximately 16 kb and is divided among 8 exons and 7 introns, the junctions of which conform to the AG/GT consensus rule. PEDF appears to fall into the ovalbumin/PAI-2 subgrouping of serpins and is structurally far different from GDN/PN-1, the only other neurotrophic serpin reported to date. The immediate 5'-flanking region is dominated by a dense cluster of Alu repeats in which are embedded several promoter consensus sequences. A CAAT box is present at -43. The putative promoter region is also far different from that reported for GDN/PN-1. Comparable hybridization signals of 23 kb EcoRI fragments containing the PEDF gene are observed by Southern blot analysis in all primate, mammal and avian species examined; conservation is particularly evident among the primates. Northern blot analysis confirms the presence of the PEDF transcript in a broad range of human fetal and adult tissues including almost all brain areas examined, underscoring differences with GDN/PN-1 which, in the adult brain, is only expressed in glia and a subset of neurons.

PEDF (pigment epithelium-derived factor) promotes increase and maturation of pigment granules in pigment epithelial cells in neonatal albino rat retinal cultures.

Pigment Epithelium-Derived Factor (PEDF), purified from human retinal pigment epithelial (RPE) cell culture medium, is a neurotrophic factor which potentiates the differentiation of human Y-79 retinoblastoma cells and increases the survival of cerebellar granule cells. To investigate the effects of PEDF on non-transformed retinal cells, we used primary cultures of neonatal albino rat retinas, where the three principal cell types of the retinal layers (neuronal, glial and epithelial) were all present and focussed our attention on RPE cells, which are of special relevance for retinal pathophysiology. PEDF had a dramatic effect on these cells. They showed a modified phenotype, with larger dimensions, higher cytoplasmic spreading, presence of phagocytic vacuoles, development of wide intercellular contacts, and increase and maturation of pigment granules. These results suggest that PEDF may have a role in regulating RPE cell differentiation.

Upregulation of pigment epithelium-derived factor after laser photocoagulation.

PURPOSE: To determine the changes in the expression of pigment epithelium-derived factor in cultured human retinal pigment epithelial cells and rat retinas after laser photocoagulation. METHODS: Experimental study of laser photocoagulation on human retinal pigment epithelial cells in culture and on adult rats. Reverse transcription-polymerase chain reaction and semiquantitative polymerase chain reaction analysis were used. RESULTS: After photocoagulation, the mRNA expression of pigment epithelium-derived factor was upregulated in human retinal pigment epithelial cells at 6 hours and then gradually decreased. Compared with controls, significantly higher levels of pigment epithelium-derived factor were observed in rat retinas from 6 to 24 hours after laser photocoagulation (P <.005), and they were still higher than before photocoagulation at 2 weeks. CONCLUSION: An upregulation of pigment epithelium-derived factor in retinal pigment epithelial cells and in the retina after photocoagulation suggests that pigment epithelium-derived factor plays a role in inhibiting neovascularization by its antiangiogenic activity.

Pigment epithelium-derived factor in the vitreous is low in diabetic retinopathy and high in rhegmatogenous retinal detachment.

PURPOSE: To report the levels of pigment epithelium-derived factor in the vitreous of patients with diabetic retinopathy, rhegmatogenous retinal detachment, and idiopathic macular hole. METHODS: Using enzyme-linked immunosorbent assay, we measured the levels of pigment epithelium-derived factor in the vitreous of 34 eyes of 33 patients who underwent vitrectomy for the treatment of diabetic retinopathy (17 eyes of 16 patients), rhegmatogenous retinal detachment (10 eyes), and idiopathic macular hole (seven eyes). RESULTS: The vitreal concentration of pigment epithelium-derived factor was 1.15 +/- 0.23 microg/ml (mean +/- standard error) in eyes with diabetic retinopathy, 3.28 +/- 0.69 microg/ml in rhegmatogenous retinal detachment, and 1.71 +/- 0.39 microg/ml in idiopathic macular hole. The pigment epithelium-derived factor level in rhegmatogenous retinal detachment was significantly higher than that in diabetic retinopathy (P =.0008) and idiopathic macular hole (P =.034). For eyes with diabetic retinopathy, the pigment epithelium-derived factor level was 0.88 +/- 0.21 microg/ml in proliferative diabetic retinopathy and 2.43 +/- 0.37 microg/ml in nonproliferative diabetic retinopathy (P =.0083). Additionally, the pigment epithelium-derived factor level in active diabetic retinopathy (0.70 +/- 0.22 microg/ml) was significantly lower than the level in inactive diabetic retinopathy (1.79 +/- 0.35 microg/ml; P =.018). CONCLUSIONS: These results suggest that pigment epithelium-derived factor inhibits angiogenesis and that lower levels of pigment epithelium-derived factor may be related to the angiogenesis in diabetic retinopathy and result in active proliferative diabetic retinopathy. The results also suggest that higher levels of pigment epithelium-derived factor in the eyes with rhegmatogenous retinal detachment may act as a neuroprotective agent for the detached retina.

Pigment epithelium derived factor as a neuroprotective agent against ischemic retinal injury.

PURPOSE: Pigment epithelium-derived factor (PEDF) is a protein shown to have neurotrophic activity. The purpose of this study was to determine whether PEDF is neuroprotective of retinal neurons that are exposed to transient ischemia-reperfusion. METHODS: Transient retinal ischemia was produced by increasing the intraocular pressure for 45 min in albino rats eyes. Immediately after reperfusion, PEDF was injected intravitreally into the experimental eyes. Injury was evaluated morphologically and by measuring the thickness of the inner retinal layers (IRL) and by counting the number of retinal ganglion cells (RGC) in epon embedded sections. RESULTS: Morphologic and morphometric analysis of the thickness of the IRL and the counting of RGC demonstrated that PEDF injected immediately after reperfusion protected the eyes partially but significantly from the ischemic injury. CONCLUSIONS: Intravitreal injection of PEDF even after the ischemia can ameliorate retinal injury. PEDF may be useful in preventing neuronal degeneration in the inner retina resulting from ischemia.

[Biological role of a neurotrophic factor fragment from pigment epithelium: structure-functional homology with a differentiation factor for the HL-60 cell line]. / Biologicheskaia rol' fragmenta neirotrfnogo faktor iz pigmentnogo épiteliia: strukturno-funktsional'naia gomologiia s faktorom differentsirovki kletok linii HL-60.

It was shown that the full-size neurotrophic factor from pigment epithelium (PEDF) induces the cell differentiation of the human promyelocyte leukemia cell line HL-60. A structural analysis of PEDF revealed in its C-terminal region a six-membered peptide fragment PEDF-(352-357) (PEDF-6) whose sequence is highly homologous to the 41-46 fragment of the active site of the human leukocyte differentiation factor HLDF (HLDF-6). The biological effect of PEDF and synthetic peptides PEDF-6 and HLDF-6 on the HL-60 cells and the early gastrula ectoderm of Xenopus laevis embryos was studied. On the basis of the structural and functional homologies of HLDF, PEDF, and their homologous peptides and the computer models of the spatial structures of the full-size PEDF and the PEDF with the C-terminal fragment split off tby the cleavage of the Leu380-Thr381 bond in the serpin loop, a hypothesis on the functional role of the serpin loop in PEDF was put forward.

[Effect of homologous peptides of differentiation factors HLDF and PEDF on preimplantation development of mice in vitro]. / Vliianie gomologichnykh peptidov factorov differentsirovki HLDF i PEDF na doimplantatsionnoe razvitie myshei in vitro.

We studied the effect of synthetic peptides PEDF-6 and HLDF-6 on preimplantation development of mouse embryos in vitro. PEDF-6 peptide corresponds to fragment 351-356 and of pigment epithelium-derived differentiation factor (PEDF), while HLDF-6 peptide corresponds to fragment 84-89 of differentiation factor HLDF isolated from HL-60 cell line. Despite high homology, these peptides had different effects on the early development. PEDF-6 had no effect on the cleavage of 2-4-cell embryos but decelerated blastocyst formation from such embryos and disturbed their structure. In the presence of HLDF-6 the blastomeres divided more actively as compared to the control and a higher number of embryos developed to the blastocyst stage. The effects of both peptides were stage-specific: the affect the embryos at early cleavage stages and, apparently, determine their further development at that moment although do not directly affect formation of the blastocysts.

PEDF in angiogenic eye diseases.

Most diseases that cause blindness do so as a result of neovascularization. Angiogenesis is a complex process regulated in adult tissues by a large interacting network of molecules. In pathological conditions the checks and balances of the angiogenesis system go awry and endothelial cells of the microvasculature, proliferate, migrate, and form new but leaky vessels that invade the tissue. Hemorrhaging vessels cause edema and damage to surrounding tissues, particularly the retina. Microvascular lesions often cause severe retinal detachment and loss of vision. In this review, the value of an important endogenous anti angiogenic molecule, PEDF, is discussed in relationship to its ability to prevent retinal cell death and counter the abnormal vessel growth induced by VEGF in the eye. Its control of a neuroprotective and an antineovascular regulatory axis that determines cell fate, and its possible use in combination therapeutic strategies for ocular neovascular diseases are also reviewed.

Expression of angiogenesis factors in human umbilical vein endothelial cells and their regulation by PEDF.

The VEGFs and FGF-2 stimulate angiogenesis. Pigment epithelium-derived factor (PEDF) and thrombospondin-1 (TSP-1) strongly inhibit angiogenesis. Human umbilical vein endothelial cells (HUVECs) expressed VEGF-A, -B, -C, the VEGF receptors R1, R2, and R3, PEDF, FGF-2, and TSP-1, but VEGF-D transcripts were barely detectable. Hypoxia reduced the transcript levels of VEGF-C and its cognate receptor, VEGF-R3. PEDF blocked the effect of CoCl(2) on these two factors. The expression of VEGF-A and -B as well as VEGF-R1 and VEGF-R2 remained unchanged after exposure to hypoxia, PEDF, or both. There was a marked reduction in TSP-1 transcripts in CoCl(2) treated cultures and PEDF blocked this reduction. PEDF induced a small increase in FGF-2 transcripts in HUVECs, but there was no change in FGF-2 expression in HUVECs exposed to hypoxia or hypoxia plus PEDF. PEDF may control neovascularization, in part, by restoring the negative effects of hypoxia on the expression of a potent angiogenesis inhibitor, TSP-1. PEDF may also modulate vascular leakage by maintaining the transcriptional levels of the vascular homeostasis factors, VEGF-C and VEGF-R3 in hypoxic conditions.

Vitamin A up-regulates the expression of thrombospondin-1 and pigment epithelium-derived factor in retinal pigment epithelial cells.

Vitamin A is essential for the visual system. It is metabolized in the retina and the resulting product, retinoic acid (RA), greatly affects the structure and functions of retinal pigment epithelial (RPE) cells. RPE cells produce a variety of extracellular matrix (ECM) proteins and angiogenic factors, both of which are expressed at varying levels in the normal RPE layer. In this study, we investigated the effect of all-trans-retinoic acid on the production of an ECM protein, thrombospondin-1 (TSP-1), and two angiogenic factors, pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) by RPE cells. RA increased the release of TSP-1 and PEDF, but not that of VEGF, from human RPE cells in vitro. In vitamin A-deficient mice, the expression of TSP-1 and PEDF in the RPE layer considerably decreased compared with that of normal control mice. The vitamin A deficiency hardly affected the accumulation of VEGF in the RPE layer. These findings suggest that vitamin A modulates the structure and anti-angiogenic functions of the RPE layer partly by up-regulating the expression of the angiogenesis-related ECM protein, TSP-1, and the anti-angiogenic factor, PEDF.