ABSTRACT
PURPOSE: Our previous study showed that apelin was increased in the vitreous and fibrotic membranes of patients with proliferative diabetic retinopathy (PDR) in vivo, which suggested that apelin may be involved in the development of PDR. In this study, we investigated whether the expression of apelin was upregulated in human retinal pigment epithelial (RPE) cells in vitro under high glucose conditions. Furthermore, to explore the role of apelin in RPE cells, we investigated the effect of exogenous recombinant apelin on proliferation, migration, and collagen I (a major component of extracellular matrix molecules, associated with PDR) expression and investigated the signaling pathways involved in these processes. METHODS: Real-time PCR and western blot were performed to determine the apelin expression in ARPE-19 cells under high glucose conditions. Exogenous recombinant apelin was used to study the effect of apelin on ARPE-19 cells in vitro. Cell proliferation, migration, and collagen I expression were assessed using an MTT assay, a transwell assay, and real-time PCR analysis. LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and PD98059 (an inhibitor of mitogen-activated protein kinase) were used to help to determine the apelin signaling mechanism. RESULTS: High glucose upregulated apelin expression in RPE cells. Exogenous recombinant apelin activated protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) phosphorylation and promoted proliferation, migration, and collagen I expression in RPE cells. Pretreatment with LY294002 and PD98059 abolished apelin-induced activation of Akt and Erk, proliferation, and collagen I expression. Apelin-induced migration was partially blocked by pretreatment with LY294002 and PD98059. CONCLUSIONS: The expression of apelin was upregulated under high glucose conditions in RPE cells in vitro. Exogenous recombinant apelin increased the biologic activity of RPE cells, as well as the expression of collagen I. Apelin promoted proliferation, migration, and collagen I expression through the PI3K/Akt and MEK/Erk signaling pathways in RPE cells. From these results, we revealed the role of apelin in regulating proliferation, migration, and collagen I expression in RPE cells and the signaling mechanism under these processes, which suggested that apelin may play a profibrotic role in the development of PDR.
Subject(s)
Cell Movement/drug effects , Collagen Type I/genetics , Intercellular Signaling Peptides and Proteins/pharmacology , MAP Kinase Signaling System/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Pigment Epithelium of Eye/cytology , Proto-Oncogene Proteins c-akt/metabolism , Apelin , Cell Proliferation/drug effects , Collagen Type I/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/drug effects , Glucose/pharmacology , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , MAP Kinase Signaling System/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphorylation/drug effects , Pigment Epithelium of Eye/drug effects , Pigment Epithelium of Eye/enzymology , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Fibrosis is characterized by excessive accumulation of scar tissue as a result of exaggerated deposition of extracellular matrix (ECM), leading to tissue contraction and impaired function of the organ. Fibronectin (Fn) is an essential component of the ECM, and plays an important role in fibrosis. One such fibrotic pathology is that of proliferative vitreoretinopathy (PVR), a sight-threatening complication which develops as a consequence of failure of surgical repair of retinal detachment. Such patients often require repeated surgeries for retinal re-attachment; therefore, a preventive measure for PVR is of utmost importance. The contractile membranes formed in PVR, are composed of various cell types including the retinal pigment epithelial cells (RPE); fibronectin is an important constituent of the ECM surrounding these cells. Together with the vitreous, fibronectin creates microenvironments in which RPE cells proliferate. We have successfully developed a dual-action, fully human, fibronectin-specific single chain variable fragment antibody (scFv) termed Fn52RGDS, which acts in two ways: i) binds to cryptic sites in fibronectin, and thereby prevents its self polymerization/fibrillogenesis, and ii) interacts with the cell surface receptors, ie., integrins (through an attached "RGD" sequence tag), and thereby blocks the downstream cell signaling events. We demonstrate the ability of this antibody to effectively reduce some of the hallmark features of fibrosis--migration, adhesion, fibronectin polymerization, matrix metalloprotease (MMP) expression, as well as reduction of collagen gel contraction (a model of fibrotic tissue remodeling). The data suggests that the antibody can be used as a rational, novel anti-fibrotic candidate.
Subject(s)
Antibodies/immunology , Epitopes/immunology , Extracellular Matrix/pathology , Fibronectins/chemistry , Fibronectins/immunology , Protein Engineering , Amino Acid Sequence , Antibodies/chemistry , Cell Adhesion , Cell Movement , Cell Proliferation , Cell Survival , Collagen , Extracellular Matrix/metabolism , Fibrosis/pathology , Humans , Integrins/metabolism , Matrix Metalloproteinases/metabolism , Molecular Sequence Data , Molecular Weight , Pigment Epithelium of Eye/cytology , Pigment Epithelium of Eye/enzymology , Polymerization , Protein Binding , Single-Chain Antibodies/immunologyABSTRACT
Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-α-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-α-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.
Subject(s)
Cell Movement/drug effects , Matrix Metalloproteinase 9/biosynthesis , Pigment Epithelium of Eye/drug effects , Proteins/metabolism , Proto-Oncogene Proteins c-akt/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cell Movement/physiology , Cells, Cultured , Humans , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes , Pigment Epithelium of Eye/enzymology , Pigment Epithelium of Eye/physiology , RNA Interference , Signal Transduction , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Vitreoretinopathy, Proliferative/enzymology , Vitreoretinopathy, Proliferative/pathologyABSTRACT
Mitochondrial DNA mutations and associated defects in cytochrome c oxidase (COX) are proposed to play an important role in human ageing; however there have been limited studies on the frequency of these defects in normal mouse ageing. Here we compare COX-deficiency in two epithelial tissues; the colon and the ciliary epithelium, from human and mouse. The pattern of accumulation of COX-deficiency is similar in both tissues in the two species; however the frequency of colonic crypts with COX-deficiency in aged humans is significantly higher than in aged mice, whereas the levels of COX-deficiency in the ciliary epithelium are higher in the mouse than in humans. This suggests the impact of mitochondrial defects on normal ageing may differ significantly between species.
Subject(s)
Aging/genetics , Aging/metabolism , Cytochrome-c Oxidase Deficiency/genetics , DNA, Mitochondrial/genetics , Animals , Colon/enzymology , Humans , Male , Mice , Mice, Inbred C57BL , Mutation , Pigment Epithelium of Eye/enzymology , Species SpecificityABSTRACT
Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retinal pigment epithelium (RPE) cells. We analysed melanin synthesis in adenoviral-transduced tyrosinase-gene-expressing amelanotic RPE (ARPE) 19 cells to determine whether premelanosome formation is needed for post-natal melanogenesis. The synthesis of melanogenic proteins and melanin granules was investigated by immunocytochemistry and light and electron microscopy. The occurrence of tyrosinase was analysed ultrastructurally by dihydroxyphenylalanine histochemistry. The viability of transduced cell cultures was examined via MTT assay. We found active tyrosinase in small granule-like vesicles throughout the cytoplasm and in the endoplasmic reticulum and nuclear membrane. Tyrosinase was also associated with multi-vesicular and multi-lamellar organelles. Typical premelanosomes, structural protein PMEL17, tyrosinase-related protein 1 and classic melanosomal stages I-IV were not detected. Instead, melanogenesis took place inside multi-vesicular and multi-lamellar bodies of unknown origin. Viability was not affected up to 10 days after transduction. We thus demonstrate a pathway of melanin formation lacking typical hallmarks of melanogenesis.
Subject(s)
Melanins/biosynthesis , Melanosomes/metabolism , Metabolic Networks and Pathways , Organogenesis , Pigment Epithelium of Eye/metabolism , Adult , Cell Line , Cell Survival , Dihydroxyphenylalanine/metabolism , Fluorescence , Humans , Immunohistochemistry , Melanosomes/ultrastructure , Monophenol Monooxygenase/metabolism , Pigment Epithelium of Eye/cytology , Pigment Epithelium of Eye/enzymology , Pigment Epithelium of Eye/ultrastructure , Transduction, GeneticABSTRACT
BACKGROUND: The visual cycle is an enzymatic pathway employed in the vertebrate retina to regenerate the chromophore after its release from light-activated rhodopsin. However, a visual cycle is thought to be absent in invertebrates such as the fruit fly Drosophila melanogaster. RESULTS: We demonstrate that an enzymatic visual cycle exists in flies for chromophore regeneration and requires a retinol dehydrogenase, PDH, in retinal pigment cells. Absence of PDH resulted in progressive light-dependent loss of rhodopsin and retinal degeneration. These defects are suppressed by introduction of a mammalian dehydrogenase, RDH12, which is required in humans to prevent retinal degeneration. We demonstrate that a visual cycle is required in flies to sustain a visual response under nutrient deprivation conditions that preclude de novo production of the chromophore. CONCLUSIONS: Our results demonstrate that an enzymatic visual cycle exists and is required in flies for maintaining rhodopsin levels. These findings establish Drosophila as an animal model for studying the visual cycle and retinal diseases associated with chromophore regeneration.
Subject(s)
Alcohol Oxidoreductases/metabolism , Drosophila melanogaster/physiology , Vision, Ocular , Animals , Animals, Genetically Modified , Drosophila melanogaster/genetics , Pigment Epithelium of Eye/enzymology , Pigment Epithelium of Eye/physiologyABSTRACT
PURPOSE: Cell cycle progression is governed by the coordinated activities of kinases, phosphatases, and the ubiquitin system. The entire complement of ubiquitin pathway components that mediate this process in retinal pigment epithelial (RPE) cells remains to be identified. This study was undertaken to determine whether the human ubiquitin-conjugating enzyme, UBE2E3, is essential for RPE cell proliferation. METHODS: UBE2E3 expression and localization in telomerase-immortalized, human RPE cells was determined with a UBE2E3-specific antibody. The necessity for UBE2E3 in RPE proliferation was determined using small interfering (si)RNA to target the expression of the enzyme. Cell counts and immunolabeling for the proliferation marker Ki-67 and the cyclin-dependent kinase inhibitor p27(Kip1) were performed to assess the consequences of UBE2E3 depletion. A mouse strain harboring a disrupted allele of UbcM2 (the mouse counterpart of UBE2E3) with the coding sequence for beta-galactosidase was used to track the developmental expression of the enzyme in murine RPE cells. RESULTS: UBE2E3 localized in the nucleus of the immortalized RPE cells. Depletion of the enzyme by siRNA resulted in a cell-cycle exit accompanied by a loss of Ki-67, an increase in p27(Kip1), and a doubling in cell area. Rescue experiments confirmed the specificity of the RNA interference. In vivo, UbcM2 was transcriptionally downregulated during RPE development in the mouse. CONCLUSIONS: UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation.
Subject(s)
Cell Proliferation , Pigment Epithelium of Eye/cytology , Pigment Epithelium of Eye/enzymology , Ubiquitin-Protein Ligases/physiology , Animals , Blotting, Western , Cell Count , Cell Cycle , Cell Size , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p27 , Down-Regulation , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation, Enzymologic/physiology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Ki-67 Antigen/metabolism , Mice , Mice, Inbred C57BL , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Ubiquitin-Conjugating Enzymes/geneticsABSTRACT
PURPOSE: Progressive pigment chorioretinopathy is a major long-term complication of mitochondrial trifunctional protein (MTP) defects, disorders of mitochondrial fatty acid beta-oxidation. To better understand the pathogenesis of the retinopathy component, the authors studied expression of the main regulatory protein of the beta-oxidation pathway, carnitine palmitoyltransferase (CPT) 1, and acyl-CoA dehydrogenase (ACAD) 9 in retinal sections and cultured cells. METHODS: Immunoblotting was performed with polyclonal antibodies to ACAD9 and the three isoforms of CPT1. In quantitative real-time PCR (QRT-PCR), predesigned gene-specific probes and primer sets for human CPT1 isoforms were used. In situ hybridization (ISH) and immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of the rat and human eye. RESULTS: The predominant CPT1 mRNA types detected by QRT-PCR in cultured human retinal pigment epithelial cells were of the liver (CPT1A) and brain (CPT1C) isotypes. CPT1A and ACAD9 protein expression was found in cultured human and rat RPE and rat neural retinal precursor cells. ISH of rat retinal sections showed CPT1A and CPT1C expression in the retinal pigment epithelium (RPE), the inner nuclear layer, and the ganglion cell layer. CPT1A expression was also detected in the Müller cell microvilli, and CPT1C expression was detected in the photoreceptor inner segments. ACAD9 immunolabeling was detected in rat and human RPE, human photoreceptor inner segments, and ganglion cell layer. CONCLUSIONS: These findings imply that the mitochondrial fatty acid beta-oxidation pathway probably is active in metabolism of the RPE and certain neuroretinal cell types. Accumulation of 3-hydroxylated intermediates of long-chain fatty acids may contribute to the pathogenesis of retinopathy in MTP deficiencies.
Subject(s)
Acyl-CoA Dehydrogenase, Long-Chain/genetics , Carnitine O-Palmitoyltransferase/genetics , Gene Expression Regulation, Enzymologic/physiology , Multienzyme Complexes/metabolism , Retina/enzymology , Retinal Diseases/enzymology , Acyl-CoA Dehydrogenase, Long-Chain/metabolism , Animals , Brain/enzymology , Carnitine O-Palmitoyltransferase/metabolism , Cell Line , Humans , Immunoblotting , Immunohistochemistry , In Situ Hybridization , Liver/enzymology , Mitochondrial Trifunctional Protein , Muscle, Skeletal/enzymology , Pigment Epithelium of Eye/enzymology , RNA, Messenger/metabolism , Rats , Rats, Wistar , Retinal Ganglion Cells/enzymology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Mutation or loss of MerTK as well as deficiency of alphavbeta5-integrins, gives rise to retinal-degeneration due to inefficient phagocytosis of photoreceptor outer-segment fragments by the retinal pigment epithelium (RPE). This study shows that Gas6 expressed endogenously by human RPE promotes phagocytosis. The RPE expresses Gas6 more highly in vivo and in serum-reduced conditions in vitro than in high-serum conditions, suggesting a negative-feedback control. An antibody-blockage approach revealed that Gas6-expressing RPE phagocytizes photoreceptor outer-segment fragments due to stimulation of MerTK by endogenous Gas6 in vitro. MerTK- and Gas6-antibodies reduced phagocytosis. Blocking L-type Ca(2+)-channels with nifedipine inhibited MerTK dependent phagocytosis in vitro. Application of integrin inhibitory, soluble, RGD-containing peptides or soluble vitronectin reduced L-type Ca(2+)-channel currents in RPE. Herbimycin A, which reduces phosphorylation of integrin receptor-associated proteins and decreases L-type Ca(2+)-channel currents in RPE, eliminates the inhibiting vitronectin effect and abolishes phagocytosis. Thus, Gas6-promoted phagocytosis was inhibited by L-type Ca(2+)-channel blockage, which in turn may be activated by integrin receptor stimulation. These results suggest that L-type Ca(2+)-channels could be regulated downstream of both MerTK and alphavbeta5-integrin, indicating that the binding and uptake mechanisms of phagocytosis are part of a converging pathway.
Subject(s)
Calcium Channels, L-Type/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Phagocytosis , Pigment Epithelium of Eye/metabolism , Antibodies/pharmacology , Benzoquinones/pharmacology , Calcium Channel Blockers/pharmacology , Cells, Cultured , Humans , Integrin alphaV/metabolism , Intercellular Signaling Peptides and Proteins/analysis , Intercellular Signaling Peptides and Proteins/immunology , Lactams, Macrocyclic/pharmacology , Ligands , Nifedipine/pharmacology , Phagocytosis/drug effects , Pigment Epithelium of Eye/drug effects , Pigment Epithelium of Eye/enzymology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/immunology , Receptor Protein-Tyrosine Kinases/metabolism , Rifabutin/analogs & derivatives , c-Mer Tyrosine KinaseABSTRACT
PURPOSE: In the central nervous system (CNS), increased mitochondrial DNA (mtDNA) damage is associated with aging and may underlie, contribute to, or increase the susceptibility to neurodegenerative diseases. Because of the focus on the retinal pigment epithelium (RPE) and choroid as tissue relevant to age-related macular degeneration (AMD), we examined young and aged RPE and choroid, harvested from rodent eyes, for DNA damage and for changes in selected DNA repair enzymes. METHODS: Immunohistochemical labeling and quantitative ELISA for the oxidative DNA damage marker, 8-hydroxy-2'-deoxy-guanosine (8-OHdG), were measured in young and aged rodent RPE and choroid. mtDNA and nuclear DNA (nDNA) damage was determined by quantitative polymerase chain reaction (PCR) by comparing the relative amplification of small and large DNA fragments. Expression of several DNA repair enzymes was measured using real-time quantitative reverse transcription -PCR (qRT-PCR) and immunoblot. RESULTS: Immunohistochemical labeling for 8-OHdG increased in aged rodent RPE and choroid. Quantitative ELISA confirmed increased levels of 8-OHdG. Measurements of nDNA and mtDNA lesions indicated that DNA damage is primarily in mtDNA in aged RPE and choroid. Using qRT-PCR, we found that gene expression of DNA repair enzymes, 8-oxoguanine-DNA glycosylase 1 (OGG1), mutY homolog (MYH), and thymine DNA glycosylase were decreased in an age-dependent pattern in RPE and choroid. However, endonuclease III homolog 1 was not significantly changed in aged RPE and choroid. Using immunoblots, we found that protein levels of OGG1 and MYH were decreased in aged RPE and choroid. CONCLUSIONS: Our results show that there is increased mtDNA damage in aged RPE and choroid, which is likely due to decreased DNA repair capability. mtDNA damage in the RPE and choroid may be a susceptibility factor that underlies the development of AMD.
Subject(s)
Aging/metabolism , Choroid/enzymology , DNA Damage , DNA Repair Enzymes/genetics , DNA, Mitochondrial/metabolism , Down-Regulation/genetics , Pigment Epithelium of Eye/enzymology , 8-Hydroxy-2'-Deoxyguanosine , Aging/physiology , Animals , Cell Nucleus/metabolism , Choroid/cytology , DNA Repair Enzymes/metabolism , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Enzyme Repression , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Pigment Epithelium of Eye/cytology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred BN , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Eyes with age-related macular degeneration (AMD) demonstrate accumulation of specific deposits and extracellular matrix (ECM) molecules under the retinal pigment epithelium (RPE). AMD is about two times more prevalent in aging postmenopausal women. Therefore we studied whether 17beta-estradiol (E(2)) modulates the expression and activity of the trimolecular complex (MMP-2, TIMP-2 and MMP-14), molecules which are of major importance for ECM turnover in RPE. We used cell lines isolated from estrogen receptor knockout mice (ERKO) to determine which ER (estrogen receptor) subtype was important for ECM regulation in RPE cells. We found that mouse RPE sheets had higher baseline MMP-2 activity in the presence of ERbeta. This correlated with higher MMP-2 activity in RPE cell lines isolated from ERKOalpha mice. Exposure to E(2) increased MMP-2 activity in mouse RPE cell lines. In addition E(2) increased transcriptional activation of the MMP-2 promoter through a functional Sp1 site which required the presence of ERbeta, but not ERalpha. E(2) also maintained levels of pro MMP-2, and MMP-14 and TIMP-2 activity after oxidant injury. Since the direct effects of E(2) on MMP-2 transcriptional activation and the regulation of the trimolecular complex after oxidant-induced injury requires ERbeta, this receptor subtype may have a role as a potential therapeutic target to prevent changes in activation of MMP-2.
Subject(s)
Estrogen Receptor alpha/physiology , Estrogen Receptor beta/physiology , Matrix Metalloproteinase 2/metabolism , Pigment Epithelium of Eye/enzymology , Retina/enzymology , Animals , Cell Line , Enzyme Activation/drug effects , Estradiol/pharmacology , Female , Matrix Metalloproteinase 14/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Oxidative Stress/physiology , Pigment Epithelium of Eye/drug effects , Retina/drug effects , Tissue Inhibitor of Metalloproteinase-2/metabolism , TransfectionABSTRACT
Drug delivery to retinal cells has represented a major challenge for ophthalmologists for many decades. However, drug targeting to the retina is essential in therapies against retinal diseases such as age-related macular degeneration, the most common reason of blindness in the developed countries. Retinal cells are chronically exposed to oxidative stress that contributes to cellular senescence and may cause neovascularization in the most severe age-related macular degeneration cases. Various pre- and clinical studies have revealed that heat shock protein 90 (HSP90) inhibitors, such as geldanamycin and radicicol, are promising drugs in the treatment of different malignant processes. In this study, our goal was to compare the effects of 0.1 microM, 1 microM or 5 microM geldanamycin or radicicol on the oxidative stress response, cytotoxicity, and efflux protein activity (a protein pump which removes drugs from cells) in ARPE-19 (human retinal pigment epithelial, RPE) cells. Our findings indicate that geldanamycin and radicicol increased HSP70 and HSP27 expression analyzed by western blotting. Cellular levels of protein carbonyls were increased in response to 0.1 microM (P=0.048 for 24 h, P=0.018 for 48 h) or 5 microM (P=0.030 for 24 h, P=0.046 for 48 h) radicicol but not to geldanamycin analyzed by ELISA assay. In addition, HNE-protein adducts were accumulated in the RPE cells exposed to 0.1 microM or 5 microM radicicol but not to geldanamycin analyzed by western blotting. However, MTT assay revealed that 5 microM geldanamycin reduced cellular viability 20-30% (P<0.05 for 24 h, P<0.01 for 48 h), but this was not observed at any radicicol concentration in RPE cells. Interestingly, the increased oxidative stress response was associated with efflux protein inhibition (20-30%) when the cells were exposed to 1 microM or 5 microM (P<0.05) radicicol, but not in geldanamycin-treated RPE cells. These novel findings help in understanding the influence of HSP90 inhibition and regulatory mechanisms of drug delivery to retinal cells.
Subject(s)
Benzoquinones/pharmacology , Epithelial Cells/drug effects , Lactams, Macrocyclic/pharmacology , Macrolides/pharmacology , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Oxidative Stress/drug effects , Pigment Epithelium of Eye/drug effects , Protein Kinase Inhibitors/pharmacology , Blotting, Western , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/enzymology , Epithelial Cells/metabolism , HSP27 Heat-Shock Proteins , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Humans , Molecular Chaperones , Multidrug Resistance-Associated Proteins/metabolism , Neoplasm Proteins/metabolism , Pigment Epithelium of Eye/enzymology , Pigment Epithelium of Eye/metabolism , Protein Carbonylation/drug effects , Time FactorsABSTRACT
PURPOSE: The retinal pigmented epithelium (RPE) expresses many genes that play important roles in the support and maintenance of photoreceptors. The present study was conducted to develop a system amenable to the dissection of the temporal function of these genes, specifically within RPE cells. Transgenic mice were generated and characterized in which the expression of Cre recombinase could be specifically induced within the RPE. METHODS: Transgenic mice carrying the human vitelliform macular dystrophy-2 (VMD2) promoter (P(VMD2))-directed reverse tetracycline-dependent transactivator (rtTA) and the tetracycline-responsive element (TRE)-directed cre were generated. Inducible Cre expression was achieved by feeding doxycycline to these mice and was characterized by using a Cre-activatable lacZ reporter mouse strain (R26R). RESULTS: A beta-galactosidase assay of rtTA/Cre-R26R mice demonstrated that the basal level of Cre expression without doxycycline induction was negligible. Addition of doxycycline led to induction of RPE-specific Cre expression/function at least from embryonic day 9 to postnatal day 60. The highest induction occurred at approximately postnatal day 4. As measured by ERG and histology, retinal function and morphology were normal in 10-month-old rtTA/Cre mice that were treated with doxycycline at weaning age. CONCLUSIONS: Transgenic mice were generated that express Cre recombinase in the RPE in an inducible fashion. These mice will be useful for studies of the RPE-specific role of genes that are expressed in the RPE as well as other cells, particularly for avoiding embryonic lethality and dissecting the function of genes that play dual roles in development and adulthood.
Subject(s)
Gene Expression Regulation, Enzymologic/physiology , Integrases/genetics , Pigment Epithelium of Eye/enzymology , Animals , Bestrophins , Chloride Channels/genetics , Doxycycline/administration & dosage , Electroretinography , Eye Proteins/genetics , Mice , Mice, Transgenic , Pigment Epithelium of Eye/drug effects , Plasmids , Promoter Regions, Genetic/genetics , beta-Galactosidase/metabolismABSTRACT
PURPOSE: AMP-activated protein kinase (AMPK) has been suggested to be a novel signaling pathway in regulating inflammation. The role of AMPK in retinal pigment epithelial cell inflammatory response is addressed using AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR). METHODS: Protein expression and activation of signaling molecules were detected by immunoblotting. Cytokines were determined by ELISA kits. AMPKalpha expression was knockdown by siRNAs. RESULTS: AICAR inhibited tumor necrosis factor (TNF)-alpha- or interleukin (IL)-1beta-induced production of IL-6, IL-8, and monocyte chemotactic protein (MCP)-1 and of intercellular adhesion molecule (ICAM)-1 expression in human RPE cells. The inhibitory effect on cytokine production and ICAM-1 expression persisted in the RPE cells in which AMPK was knocked down by AMPK siRNA. Moreover, an adenosine kinase inhibitor 5'-iodotubercidin, which effectively abolished AMPK activation caused by AICAR, did not reverse the anti-inflammatory effect of AICAR. In comparison, anti-inflammatory effects of AICAR were mimicked by adenosine but not inosine, the metabolites of AICAR. Finally, with the exception of TNF-alpha-induced IL-6 production, adenosine dialdehyde, an inhibitor of S-adenosylhomocysteine hydrolase, was found to block cytokine production and ICAM-1 expression. CONCLUSIONS: Regardless of the ability of AICAR to activate AMPK, the inhibitory effects of AICAR on cytokine production and ICAM-1 expression were not associated with AMPK. The mechanism of AICAR inhibition may be attributed to the interference of adenosylmethionine-dependent methylation.
Subject(s)
Adenosylhomocysteinase/physiology , Aminoimidazole Carboxamide/analogs & derivatives , Chemokines/metabolism , Interleukin-1beta/antagonists & inhibitors , Pigment Epithelium of Eye/drug effects , Ribonucleotides/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , AMP-Activated Protein Kinases , Adenosine/analogs & derivatives , Adenosine/pharmacology , Aminoimidazole Carboxamide/pharmacology , Cells, Cultured , Chemokine CCL2/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Immunoblotting , Intercellular Adhesion Molecule-1/metabolism , Interleukin-1beta/pharmacology , Interleukin-6/metabolism , Interleukin-8/metabolism , Multienzyme Complexes/metabolism , Pigment Epithelium of Eye/enzymology , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering/genetics , Transfection , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Glucose is the main fuel for energy metabolism in retina. The regulatory mechanisms that maintain glucose homeostasis in retina could include hormonal action. Retinopathy is one of the chemical manifestations of long-standing diabetes mellitus. In order to better understand the effect of hyperglycemia in retina, we studied glycogen content as well as glycogen synthase and phosphorylase activities in both normal and streptozotocin-induced diabetic rat retina and compared them with other tissues. Glycogen levels in normal rat retina are low (46 +/- 4.0 nmol glucosyl residues/mg protein). However, high specific activity of glycogen synthase was found in retina, indicating a substantial capacity for glycogen synthesis. In diabetic rats, glycogen synthase activity increased between 50% and 100% in retina, brain cortex and liver of diabetic rats, but only retina exhibited an increase in glycogen content. Although, total and phosphorylated glycogen synthase levels were similar in normal and diabetic retina, activation of glycogen synthase by glucose-6-P was remarkable increased. Glycogen phosphorylase activity decreased 50% in the liver of diabetic animals; it was not modified in the other tissues examined. We conclude that the increase in glycogen levels in diabetic retina was due to alterations in glycogen synthase regulation.
Subject(s)
Diabetes Mellitus, Experimental/metabolism , Glycogen/metabolism , Retina/metabolism , Animals , Blood Glucose/metabolism , Cerebral Cortex/enzymology , Electrophoresis, Polyacrylamide Gel , Glycogen Phosphorylase/metabolism , Glycogen Synthase/metabolism , Liver/enzymology , Pigment Epithelium of Eye/enzymology , Rats , Rats, Long-Evans , Retina/enzymologyABSTRACT
The retinal pigment epithelium (RPE) constitutes the blood-retinal barrier, whose function is impaired in various pathological conditions, including cerebral malaria, a lethal complication of Plasmodium falciparum infection. Prostaglandin (PG) D(2) is abundantly produced in the brain to regulate sleep responses. Moreover, PGD(2) is a potential factor derived from intra-erythrocyte falciparum parasites. Heme oxygenase-1 (HO-1) is important for iron homeostasis via catalysis of heme degradation to release iron, carbon monoxide and biliverdin/bilirubin, and may influence iron supply to the intra-erythrocyte falciparum parasites. Here, we showed that treatment of human RPE cell lines, ARPE-19 and D407, with PGD(2) significantly increased the expression levels of HO-1 mRNA, in a dose- and time-dependent manner. Transient expression assays showed that PGD(2) treatment increased the HO-1-gene promoter activity through the enhancer sequence, containing a Maf-recognition element. Thus, PGD(2) may contribute to the maintenance of heme homeostasis in the brain by inducing HO-1 expression.
Subject(s)
Epithelial Cells/enzymology , Heme Oxygenase-1/metabolism , Pigment Epithelium of Eye/enzymology , Prostaglandin D2/administration & dosage , Cell Line , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Humans , Pigment Epithelium of Eye/drug effectsABSTRACT
The goals of this study were to evaluate human retinal pigment epithelial cell line (ARPE-19) for cell encapsulation and to optimize the alginate-based microencapsulation. We used immortalized ARPE-19 cells and the transfected sub-line that expresses secreted alkaline phosphatase (SEAP) reporter enzyme. Alginate was cross-linked with different divalent cations (Ca(2+), Ba(2+), Sr(2+) and combination of Ca(2+) and Ba(2+)), coated first with poly-l-lysine (PLL), and then with alginate. Microcapsules with different pore sizes and stability were generated. The pore size of the microcapsules was assessed by the release of encapsulated fluorescein isothiocyanate (FITC)-dextrans. The viability of the cells in the microcapsules was studied in vitro by assessing the secretion rates of SEAP and oxygen consumption by the cells. The best microcapsule morphology, durability and cellular viability were obtained with alginate microcapsules that were cross-linked with Ca(2+) and Ba(2+) ions and then coated with PLL and alginate. Based on FITC-dextran release these microcapsules have porous wall that enables the rapid contents release. The ARPE-19 cells maintained viability in the Ca(2+) and Ba(2+) cross-linked microcapsules for at least 110 days. The alginate microcapsules cross-linked with Ca(2+) and Ba(2+) have sufficiently large pore size for prolonged cell viability and for the release of secreted SEAP model protein (Mw 50 kDa; radius of gyration of 3 nm). ARPE-19 cells show long-term viability and protein secretion within alginate microcapsules cross-linked with Ca(2+) and Ba(2+). This combination may be useful in cell therapy.
Subject(s)
Alginates/chemistry , Cell- and Tissue-Based Therapy/methods , Epithelial Cells , Pigment Epithelium of Eye , Alkaline Phosphatase/metabolism , Cell Line , Cell Membrane Permeability , Cell Survival , Cross-Linking Reagents/chemistry , Dextrans/chemistry , Drug Compounding , Epithelial Cells/cytology , Epithelial Cells/enzymology , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Humans , Molecular Weight , Oxygen/metabolism , Particle Size , Pigment Epithelium of Eye/cytology , Pigment Epithelium of Eye/enzymology , Time FactorsABSTRACT
Retinal pigment epithelial (RPE) cells are the main cell type involved in the pathogenesis of proliferative vitreoretinopathy (PVR). As a result from retinal detachment or surgical procedures, RPE comes in contact with glutamate from serum, glial release and the injured retina. The purpose of this study was to explore a possible role for glutamate in the development of PVR, mediated by the receptor-stimulated activation of the ERK1/2 MAPK pathway, the alteration of cell proliferation and the transdifferentiation of RPE cells, using rat RPE cells in culture as a model system. We demonstrated the expression in these cells of Group I metabotropic-and ionotropic AMPA/KA and NMDA glutamate receptors (GluRs), predominantly of the NMDA subtype, which are targeted to the membrane, and exhibit pharmacological and biochemical characteristics equivalent to those previously established in brain tissue. Proliferation was measured by MTS-reduction colorimetric assay, and actin cytoskeleton dynamics was visualized by immunoflurescence using alpha-sma specific antibodies. Activation of metabotropic, AMPA and NMDA receptors by glutamate induced the time-and dose-dependent phosphorylation of ERK1/2, assessed by Western blot analysis, in parallel to a significant increase in cell proliferation and a decrease in alpha-sma expression and its recruitment into stress fibers. These effects were all prevented by the inhibition of MEK. Hence, results suggest that glutamate could be involved in the generation of PVR, through a GluR-mediated increase in proliferation and phenotypic transformation, cause-effect related to the activation of ERK1/2.
Subject(s)
Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Pigment Epithelium of Eye/cytology , Receptors, Glutamate/physiology , Vitreoretinopathy, Proliferative/pathology , Actins/metabolism , Animals , Cell Proliferation/drug effects , Cells, Cultured , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Enzyme Activation/physiology , Glutamic Acid/pharmacology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Microscopy, Confocal , Pigment Epithelium of Eye/drug effects , Pigment Epithelium of Eye/enzymology , Pigment Epithelium of Eye/pathology , Rats , Rats, Long-Evans , Vitreoretinopathy, Proliferative/enzymology , Vitreoretinopathy, Proliferative/metabolismABSTRACT
PURPOSE: To generate a mouse model in which the Lrat gene is selectively disrupted in the retinal pigment epithelium (RPE). To evaluate the effects on the synthesis of retinyl esters and on the expression of other proteins involved in the continuation of the visual cycle. METHODS: A mouse line in which part of the first exon of the Lrat gene has been flanked by loxP sites, was generated and used in the study (Lrat(L3/L3) mice). Heterozygous mice (Lrat(+/L3)) were crossed with mice expressing Cre-recombinase under control of the tyrosinase-related protein-1 (Tyrp1) promoter, which is active selectively in melanin-synthesizing cells such as RPE cells. Accordingly, mice obtained from these crosses should display an RPE-specific disruption of the Lrat gene (Lrat(rpe-/-)). In addition, by crossing CMV-Cre transgenic mice with Lrat(L3/L3) animals, a germline null Lrat knockout (Lrat(L-/L-) mice) was generated. RNA and protein expression, endogenous retinoid levels, and electroretinogram (ERG) analyses were performed on Lrat(rpe-/-) and Lrat(L-)/(L-) mice, to determine the effects of Lrat disruption. Retinoid levels in nonocular tissues were also analyzed for comparison. RESULTS: Analysis of RPE tissues from Lrat(rpe-/-) mice showed absence of Lrat message, lack of Lrat protein expression and consequently a reduced light response in ERG recordings. In addition, RPE cells from Lrat(rpe-/-) showed a strong reduction in their ability to synthesize all-trans retinyl esters, whereas Lrat activity in other tissues known to process retinol was comparable to control Lrat(L3/L3) animals. The Lrat(L-/L-) mice showed no detectable Lrat message, lack of protein expression, and barely detectable ester formation in RPE cells or several other relevant tissues analyzed. CONCLUSIONS: Three Lrat mouse lines with genetic modifications were generated. The Lrat(L-)/(L-) mice displayed features similar to equivalent models previously reported by others. The second mouse line (Lrat(rpe-/-)) displayed loss of Lrat function only in the RPE. The third line possesses functional Lrat in all tissues, but part of the Lrat coding gene was flanked by loxP sites (Lrat(L3/L3)). This feature allows the disruption of this gene in any tissue of choice, by intercrossing with mice in which Cre-recombinase expression is driven by an appropriate tissue-specific promoter.
Subject(s)
Acyltransferases/genetics , Gene Expression Regulation, Enzymologic/physiology , Pigment Epithelium of Eye/enzymology , Retina/metabolism , Retinoids/metabolism , Acyltransferases/biosynthesis , Animals , Blotting, Western , Electroretinography , Fluorescent Antibody Technique, Indirect , Genetic Vectors , Genotype , Integrases/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , RNA, Messenger/metabolism , Recombination, Genetic , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
PURPOSE: To investigate the effect of mammalian target of rapamycin (mTOR) specific siRNA on proliferative vitreoretinopathy (PVR). METHODS: Cultured human retinal pigment epithelial (hRPE) cell line D407 was treated with three mTOR specific small interfering RNAs. Cell proliferation, attachment, spreading, and migration were performed. The impact of the mTOR specific siRNA on PVR was tested using a rabbit model in which PVR was induced by the injection of hRPE cells. RESULTS: Decreasing mTOR expression by about 82% using small interfering RNA resulted in a significant decrease in cell spreading and migration. Whereas retinal detachment occurred in 100% of the control group animals, co-injection of the mTOR specific siRNA substantially reduced the severity and incidence (50%) of retinal detachments. CONCLUSIONS: Gene therapy with mTOR specific siRNA attenuates PVR in a rabbit model of the disease. This may be a new approach to preventing PVR in humans.