Bacterial endotoxin activates retinal pigment epithelial cells and induces their degeneration through IL-6 and IL-8 autocrine signaling

Inflammation is a major contributing factor to many blinding disorders including uveitis, diabetic retinopathy, and age-related macular degeneration. Here we examined the response of the retinal pigment epithelium (RPE) to physiological levels of lipopolysaccharide (LPS) to understand the role of this epithelium in inflammatory retinal conditions. Expression of a group of inflammatory mediators was identified by gene array analysis and confirmed by PCR and immunocytochemistry in primary human RPE cultures and ARPE19. The effects of LPS on the expression of these cytokines and RPE survival were examined by PCR, Luminex bead, and MTT assays. RPE cells express many cytokine receptors including IL-1R, -4R, -6R, -8RA, IFNAR1, IFNGR1/2 and secrete a range of pro- and anti-inflammatory cytokines including IL-4, -6, -8, -10, -17, IFN-γ, MCP-1, and VEGF. LPS increases IL-13RA1 and IFNAR1, and decreases IL-7R receptor expression. It also increases RPE secretion of IL-4, -6, -8, -10, IFN-γ and MCP-1, and is toxic to RPE cells at LC50 = 17.7 μg/ml. LPS toxicity is mediated by IL-6 and IL-8 through an autocrine feedback loop. Silencing IL-6R and IL-8RA gene expression by siRNA blocks death by their respective ligands or LPS. These findings imply that RPE cells are acutely sensitive to inflammatory stress and that over secretion of IL-6 and IL-8 by this epithelium during inflammatory stimulus may be an underlying factor in the progression of some retinal pathologies. © 2008 Elsevier Ltd. All rights reserved.link_to_subscribed_fulltex

ZIP2 and ZIP4 mediate age-related zinc fluxes across the retinal pigment epithelium

Decreases in systemic and cellular levels of zinc (Zn 2+) during normal aging correlate with several age-related pathologies including age-related macular degeneration. Zn 2+ homeostasis in tissues is not only dependent on dietary intake but also on optimal expression and function of its influx (ZIP) and efflux (ZnT) transporters. We recently showed that many of the Zn 2+ transporters are expressed by the retinal pigment epithelial (RPE) cells. In this study, we present evidence that RPE cells contain less endogenous Zn 2+ with increased aging and transport this ion vectorially with greater transport from the basal to apical direction. Expression of two Zn 2+ influx transporters, ZIP2 and ZIP4, is reduced as a function of RPE age. Gene silencing of ZIP2 and ZIP4 in RPE cells from young donors or their overexpression in cells from older donors confirms that these two transporters are essential in controlling Zn 2+ influx and sequestration in RPE cells. Both transporters are distributed on the basal surface of the RPE where they are likely to control Zn 2+ homeostasis in the outer retina. © Springer Science+Business Media, LLC 2011.link_to_subscribed_fulltex

Expression of ZnT and ZIP zinc transporters in the human RPE and their regulation by neurotrophic factors

PURPOSE. Zinc is an essential cofactor for normal cell function. Altered expression and function of zinc transporters may contribute to the pathogenesis of neurodegenerative disorders including macular degeneration. The expression and regulation of zinc transporters in the RPE and the toxicity of zinc to these cells were examined. METHODS. Zinc transporters were identified in a human RPE cell line, ARPE19, using a 28K human array, and their expression was confirmed by PCR, immunocytochemistry, and Western blot analysis in primary human RPE cultures and ARPE19. Zinc toxicity to ARPE19 was determined using monotetrazolium, propidium iodide, and TUNEL assays, and Zn 2+ uptake was visualized with Zinquin ethyl ester. The effect of various growth factors on zinc transporter expression also was examined. RESULTS. Transcripts for 20 of 23 zinc transporters are expressed in fetal human RPE, 16 of 23 in adult human RPE, and 21 of 23 in ARPE19. Zn transporter proteins were also detected in ARPE19. ZnT5 expression was not observed, whereas ZnT6, ZIP1, and ZIP13 were the most abundantly expressed in all RPE samples. The addition of low concentrations of Zn 2+ to cultures resulted in a dose-dependent increase in intracellular Zn 2+ content in ARPE19, and >30 nM Zn 2+ induced necrosis with an LC 50 of 117.4 nM. Brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor (GDNF), and pigment epithelial-derived neurotrophic factor (PEDF) increased ZIP2 expression, GDNF and PEDF increased ZnT2 expression, and PEDF increased ZnT3 and ZnT8 expression. These neurotrophic factors also promoted Zn 2+ uptake in the RPE. CONCLUSIONS. The array of zinc transporters expressed by the RPE may play a key role in zinc homeostasis in the retina and in ocular health and diseases. Copyright © Association for Research in Vision and Ophthalmology.link_to_subscribed_fulltex

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 17β-estradiol (E 2) 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 E 2. E 2 promoted OSE and ovarian cancer cell growth, whereas simultaneous treatment with E 2 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. Copyright © 2006 by The Endocrine Society.link_to_subscribed_fulltex

Mitochondrial complex i defect induces ROS release and degeneration in trabecular meshwork cells of POAG patients: Protection by antioxidants

Purpose. There is growing evidence that oxidative stress contributes to the progression of primary open-angle glaucoma (POAG), a leading cause of irreversible blindness worldwide. The authors provide evidence that mitochondrial dysfunction is a possible mechanism for the loss of trabecular meshwork (TM) cells in persons with POAG. Methods. TM from patients with POAG (GTM) and age-matched subjects without disease (NTM) were obtained by standard surgical trabeculectomy. Primary TM cultures were treated with one of the following mitochondrial respiratory chain inhibitors: rotenone (ROT, complex I inhibitor), thenoyl-trifluoroacetone (TTFA, complex II inhibitor), myxothiazol or antimycin A (MYX, AM-complex III inhibitors); mitochondrial permeability transition (MPT) inhibitor cyclosporine A (CsA); and antioxidants vitamin E (Vit E) or N-acetylcysteine (NAC). Mitochondrial function was determined by changes in mito-chondrial membrane potential (δψm) and adenosine triphosphate (ATP) production with the fluorescent probes 5,5′6,6′ tetrachloro-1,1′ 3,3′-tetraethylbenzimid azolocarbocyanine iodide (JC-1) and a luciferin/luciferase-based ATP assay, respectively. Reactive oxygen species (ROS) level, determined by H 2-DCF-DA, and cell death' measured by lactate dehydrogenase activity and Annexin V-FITC labeling, were also examined. Results. GTM cells have higher endogenous ROS levels, lower ATP levels, and decreased Aψm and they are more sensitive to mitochondrial complex I inhibition than their normal counterparts. ROT induces a further increase in ROS production, the release of cytochrome c and decreases in ATP level and δψm in GTM cells, eventually leading to apoptosis. Complex II and III inhibition had little effect on the cells. Antioxidants protect against ROT-induced death by inhibiting ROS generation and cytochrome c release. Conclusions. The authors propose that a mitochondrial complex I defect is associated with the degeneration of TM cells in patients with POAG, and antioxidants and MPT inhibitors can reduce the progression of this condition. Copyright © Association for Research in Vision and Ophthalmology.link_to_subscribed_fulltex

Ginsenoside Rb1 inhibits tube-like structure formation of endothelial cells by regulating pigment epithelium-derived factor through the oestrogen β 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 ERβ. Competitive ligand-binding assays indicated that Rb1 is a specific agonist of ERβ, but not ERα. 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 ERβ siRNA, indicating ERβ 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 ERβ. These findings demonstrate a novel mechanism of the action of this ginsenoside that may have value in anti-cancer and anti-angiogenesis therapy. © 2007 Nature Publishing Group All rights reserved.link_to_subscribed_fulltex