Primary Human Retinal Pigment Epithelial Cells Research Articles

Human retinal pigment epithelial cells dying through autophagy are engulfed by professional and non‐professional phagocytes

AbstractPurpose Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional and non‐professional phagocytes can result in debris formation and development of age‐related macular degeneration (AMD). We aimed to study the clearance of autophagic dying RPE cells serving as a model for dry and wet type of AMD, respectively.Methods Autophagic cell death was induced by serum deprivation and H2O2 co‐treatment in ARPE‐19 and primary human RPE (hRPE) cells. Annexin‐V FITC/PI labelling was used to determine the cell death rate, while autophagy detection was achieved by Western blot quantification of LC3 II/LC3 I ratio. The clearance of autophagic dying cells by non‐professional and professional phagocytes was quantified using flow cytometry.Results An increasing percentage of dying RPE cells was observed in a time‐ and concentration dependent manner upon H2O2 treatment. Paralelly, an induction of autophagy could be detected within 2hrs of H2O2 treatment. Phagocytosis studies found autophagic dying cells to be efficiently and increasingly engulfed by both professional and non‐professional phagocytes over time.Conclusion The clearance of autophagic dying RPE cells can be used as a model for studying both dry and wet type of AMD in vitro, as well as for testing future pharmacological agents for treating this disease.

Cytokine gene expression and protein production in West Nile virus‐infected retinal pigment epithelium

AbstractPurpose To investigate if West Nile virus (WNV) infection of human retinal pigment epithelium (RPE) cells induces differential expression of mRNA and proteins for cytokines important for leucocyte recruitment (e.g. CCL5) and integrity of the outer blood retinal barrier (BRB) (e.g. TNF).Methods Primary human RPE cells (P3‐5) were cultured until a complete monolayer was observed, and then infected with 1, 5 or 10 plaque‐forming units (p.f.u.) of WNV (Sarafend) for 24, 48 and 72hrs. Supernatants were collected for Luminex‐based cytokine assay. RNA was prepared from cells and processed for real‐time (RT)‐PCR. Pro‐inflammatory, leucocyte recruitment and viral defense cytokines and chemokines were studied.Results CCL5, IL8, IL6 (pro‐inflammatory) and IFNB1 (anti‐viral) showed increases in mRNA expression from 24 h post‐infection (p.i.) and protein levels from 48h p.i. However in WNV‐infected RPE, protein levels of VEGF and TNF were similar to mock‐infected cells, despite significant increases in mRNA (TNF 24 h p.i., p <0.05; VEGF 48 h, p<0.01). Interestingly, CCL2 mRNA and protein showed no change in expression post‐WNV infection at 24, 48 and 72hConclusion We previously observed that WNV induced transepithelial electrical resistance (TEER) changes in human RPE (outer BRB) after 48 hours, although the mechanisms(s) remain unclear. Studies in mice show that both TNF and CCL2 are important for blood‐brain barrier integrity and leucocyte recruitment following WNV infection. Surprisingly we find little or no effect on TNF or CCL2 expression in WNV‐infected RPE, suggesting alternate mechanisms operate in RPE post‐viral infection. Funding‐ NFMRI and NHMRC

The Retinal Pigment Epithelium Utilizes Fatty Acids for Ketogenesis: IMPLICATIONS FOR METABOLIC COUPLING WITH THE OUTER RETINA

Every day, shortly after light onset, photoreceptor cells shed approximately a tenth of their outer segment. The adjacent retinal pigment epithelial (RPE) cells phagocytize and digest shed photoreceptor outer segment, which provides a rich source of fatty acids that could be utilized as an energy substrate. From a microarray analysis, we found that RPE cells express particularly high levels of the mitochondrial HMG-CoA synthase 2 (Hmgcs2) compared with all other tissues (except the liver and colon), leading to the hypothesis that RPE cells, like hepatocytes, can produce β-hydroxybutyrate (β-HB) from fatty acids. Using primary human fetal RPE (hfRPE) cells cultured on Transwell filters with separate apical and basal chambers, we demonstrate that hfRPE cells can metabolize palmitate, a saturated fatty acid that constitutes .15% of all lipids in the photoreceptor outer segment, to produce β-HB. Importantly, we found that hfRPE cells preferentially release β-HB into the apical chamber and that this process is mediated primarily by monocarboxylate transporter isoform 1 (MCT1). Using a GC-MS analysis of (13)C-labeled metabolites, we showed that retinal cells can take up and metabolize (13)C-labeled β-HB into various TCA cycle intermediates and amino acids. Collectively, our data support a novel mechanism of RPE-retina metabolic coupling in which RPE cells metabolize fatty acids to produce β-HB, which is transported to the retina for use as a metabolic substrate.

Human Complement Factor H and Factor H-Like Protein 1 Are Expressed in Human Retinal Pigment Epithelial Cells

Background: A common haplotype in the gene for the regulator of the alternative pathway of complement activation factor H has been linked to individual predisposition to age- related macular degeneration (AMD). Methods: In this study, retinal pigment epithelial (RPE) cells, i.e. immortalized ARPE-19 as well as primary human RPE cells, were investigated for expression of factor H and FHL-1 by immunohistochemistry and in situ hybridization analysis. Results: Factor H and the alternative spliced product FHL-1 are expressed in RPE cells, i.e. in immortalized ARPE-19 and primary human RPE cells. Factor H and FHL-1 expression was induced in a dose-dependent manner in ARPE-19 cells upon treatment with the inflammatory marker interleukin-6 (IL-6). In situ hybridization experiments confirmed an elevated expression rate of the factor H gene in IL-6-treated ARPE-19 cells. AMD is characterized by complement-associated inflammatory processes in the retina. Thus, local synthesis of complement regulators affects the protection of retinal cells and may be involved in the pathogenesis at the RPE-choroid interface.

Constitutive Expression of HCA2 in Human Retina and Primary Human Retinal Pigment Epithelial Cells

Purpose/Aim: HCA2, a receptor of β-hydroxybutyrate and niacin, has recently been described in mouse retina and immortalized human retinal pigment epithelial (RPE) cell lines. As HCA2 might be a pharmacologic target, e.g. in diabetic retinopathy, we studied its expression in human retina and primary human RPE cells.Materials and methods: Paraffin sections of human retina and primary human RPE cells were obtained from human donor eyes. Expression of HCA2 in human retina was investigated by immunohistochemistry of paraffin sections and by RT-PCR. HCA2 expression in primary human RPE cells was examined by immunocytochemistry and by Western-blot analysis.Results: Positive immunohistochemical staining for HCA2 was found in paraffin sections of human retina, and positive immunocytochemical staining for HCA2 in primary human RPE cells. RT-PCR analysis detected mRNA expression of HCA2 in human retina. The expression of HCA2 protein was found in primary human RPE cells.Conclusions: Based on these results, HCA2 appears to be constitutively expressed in human retina and in primary human RPE cells. Although its functional role is still unknown, HCA2 may be potentially involved in the pathogenesis of various retinopathies and may offer a new therapeutic target.

Biological Effects of Cigarette Smoke in Cultured Human Retinal Pigment Epithelial Cells

The goal of the present study was to determine whether treatment with cigarette smoke extract (CSE) induces cell loss, cellular senescence, and extracellular matrix (ECM) synthesis in primary human retinal pigment epithelial (RPE) cells. Primary cultured human RPE cells were exposed to 2, 4, 8, and 12% of CSE concentration for 24 hours. Cell loss was detected by cell viability assay. Lipid peroxidation was assessed by loss of cis-parinaric acid (PNA) fluorescence. Senescence-associated ß-galactosidase (SA-ß-Gal) activity was detected by histochemical staining. Expression of apolipoprotein J (Apo J), connective tissue growth factor (CTGF), fibronectin, and laminin were examined by real-time PCR, western blot, or ELISA experiments. The results showed that exposure of cells to 12% of CSE concentration induced cell death, while treatment of cells with 2, 4, and 8% CSE increased lipid peroxidation. Exposure to 8% of CSE markedly increased the number of SA-ß-Gal positive cells to up to 82%, and the mRNA expression of Apo J, CTGF, and fibronectin by approximately 3–4 fold. Treatment with 8% of CSE also increased the protein expression of Apo J and CTGF and the secretion of fibronectin and laminin. Thus, treatment with CSE can induce cell loss, senescent changes, and ECM synthesis in primary human RPE cells. It may be speculated that cigarette smoke could be involved in cellular events in RPE cells as seen in age-related macular degeneration.

Indocyanine green increases light‐induced oxidative stress, senescence, and matrix metalloproteinases 1 and 3 in human RPE cells

Indocyanine green (ICG) is a commonly used vital dye for macular surgery. Recent reports implicate that its use might be associated with less favourable results regarding postoperative visual outcome and damage of retinal cells, and atrophic degeneration of the retinal pigment epithelium (RPE) has been described. This study investigates the effects of ICG on light-induced senescence of RPE cells. Primary human RPE cells were either pre-incubated with ICG in concentrations of 0.005% and 0.05% or not and then exposed to white light. After 10 min of irradiation viability, induction of intracellular reactive oxygen species (ROS) and senescence-associated β-galactosidase activity (SA β-Gal) were determined. Expression and secretion of matrix metalloproteinases (MMPs) 1 and 3 and their mRNA were determined by RT-PCR and ELISA. Light exposure decreased RPE cell viability by 46%. Treatment with 0.005% and 0.05% ICG alone decreased RPE cell viability by 7% and 21%. In addition, expression of ROS, SA β-Gal, and MMP-1 and 3 was significantly increased. When 0.005% and 0.05% ICG treatments were combined with light exposure, viability decreased by 69% and 82% compared to the untreated control. Effects on the expression of ROS, SA β-Gal, and MMP-1 and 3 were, depending on the ICG dose, significantly increased when cells were pre-incubated with ICG and then illuminated. In this study, pretreatment with ICG significantly increased light-induced oxidative stress and senescence. This might indicate a potential, supplementary mechanism that could explain RPE alterations and reduced functional results after ICG-assisted internal limiting membrane peeling.

Ataxin-1 Poly(Q)-induced Proteotoxic Stress and Apoptosis Are Attenuated in Neural Cells by Docosahexaenoic Acid-derived Neuroprotectin D1

Neurodegenerative diseases share two common features: enhanced oxidative stress and cellular inability to scavenge structurally damaged abnormal proteins. Pathogenesis of polyglutamine (poly(Q)) diseases involves increased protein misfolding, along with ubiquitin and chaperon protein-containing nuclear aggregates. In spinocerebellar ataxia, the brain and retina undergo degeneration. Neuroprotectin D1 (NPD1) is made on-demand in the nervous system and retinal pigment epithelial (RPE) cells in response to oxidative stress, which activates prosurvival signaling via regulation of gene expression and other processes. We hypothesized that protein misfolding-induced proteotoxic stress triggers NPD1 synthesis. We used ARPE-19 cells as a cellular model to assess stress due to ataxin-1 82Q protein expression and determine whether NPD1 prevents apoptosis. Ectopic ataxin-1 expression induced RPE cell apoptosis, which was abrogated by 100 nm docosahexaenoic acid, 10 ng/ml pigment epithelium-derived factor, or NPD1. Similarly, NPD1 was protective in neurons and primary human RPE cells. Furthermore, when ataxin-1 82Q was expressed in 15-lipoxygenase-1-deficient cells, apoptosis was greatly enhanced, and only NPD1 (50 nm) rescued cells from death. NPD1 reduced misfolded ataxin-1-induced accumulation of proapoptotic Bax in the cytoplasm, suggesting that NPD1 acts by preventing proapoptotic signaling pathways from occurring. Finally, NPD1 signaling interfered with ataxin-1/capicua repression of gene expression and decreased phosphorylated ataxin-1 in an Akt-independent manner, suggesting that NPD1 signaling modulates formation or stabilization of ataxin-1 complexes. These data suggest that 1) NPD1 synthesis is an early response induced by proteotoxic stress due to abnormally folded ataxin-1, and 2) NPD1 promotes cell survival through modulating stabilization of ataxin-1 functional complexes and pro-/antiapoptotic and inflammatory pathways.

Microphthalmia-associated Transcription Factor (MITF) Promotes Differentiation of Human Retinal Pigment Epithelium (RPE) by Regulating microRNAs-204/211 Expression

The retinal pigment epithelium (RPE) plays a fundamental role in maintaining visual function and dedifferentiation of RPE contributes to the pathophysiology of several ocular diseases. To identify microRNAs (miRNAs) that may be involved in RPE differentiation, we compared the miRNA expression profiles of differentiated primary human fetal RPE (hfRPE) cells to dedifferentiated hfRPE cells. We found that miR-204/211, the two most highly expressed miRNAs in the RPE, were significantly down-regulated in dedifferentiated hfRPE cells. Importantly, transfection of pre-miR-204/211 into hfRPE cells promoted differentiation whereas adding miR-204/211 inhibitors led to their dedifferentiation. Microphthalmia-associated transcription factor (MITF) is a key regulator of RPE differentiation that was also down-regulated in dedifferentiated hfRPE cells. MITF knockdown decreased miR-204/211 expression and caused hfRPE dedifferentiation. Significantly, co-transfection of MITF siRNA with pre-miR-204/211 rescued RPE phenotype. Collectively, our data show that miR-204/211 promote RPE differentiation, suggesting that miR-204/211-based therapeutics may be effective treatments for diseases that involve RPE dedifferentiation such as proliferative vitreoretinopathy.

Axitinib modulates hypoxia-induced blood–retina barrier permeability and expression of growth factors

This study investigates the effects of the multikinase inhibitor axitinib on the expression of vascular endothelial growth factor (VEGF) receptors 1/2 (VEGFR-1/2) and platelet-derived growth factor (PDGF) receptor beta (PDGFR-β), hypoxia-induced increased tissue permeability, occludin, zonula occludens protein 1 (ZO-1), VEGF-A, and PDGF expression of human retinal pigment epithelial (RPE) cells and human umbilical vein endothelial cells (HUVECs). Primary human RPE cells and HUVECs were exposed to hypoxia and axitinib. Viability of cells, tissue permeability, and expression of occludin, ZO-1, VEGF, PDGF, VEGFR-1/2 and PDGFR-β, and their mRNAs, were investigated by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, and immunohistochemistry. Treatment with axitinib reduced expression of VEGFR-1/2 and PDGFR-β. Hypoxia decreased cell viability, occludin, and ZO-1 expression and increased tissue permeability, expression, and secretion of VEGF and PDGF. Axitinib significantly reduced hypoxia-induced effects on HUVEC and RPE cells. Our in vitro results suggest that axitinib may have promising properties as a potential treatment for diabetic macular edema.

Axitinib modulates hypoxia‐induced blood–retina barrier permeability and expression of growth factors

AbstractPurpose Hypoxia‐induced breakdown of the inner and outer blood–retina barrier and increased expression of growth factor are closely associated with the development and progression of diabetic macular edema. This study was performed to investigate the effects of the multikinase inhibitor axitinib on hypoxia‐induced increased tissue permeability, vascular endothelial growth factor A (VEGF), and platelet‐derived growth factor (PDGF) expression of human retinal pigment epithelial (RPE) cells and human umbilical vein endothelial cells (HUVECs). In addition, the effects of axitinib on the expression of VEGF receptors 1/2 (VEGFR‐1/2) and PDGF receptor beta (PDGFR‐β) were explored.Methods Primary human RPE cells and HUVECs were treated with axitinib (0.5 µg/mL). Viability of cells and expression of VEGFR‐1/2 and PDGFR‐β, and their mRNAs, were investigated by reverse transcription‐polymerase chain reaction (RT‐PCR) and immunohistochemistry. Cells were exposed to hypoxia. Viability, tissue permeability, expression, and secretion of VEGF and PDGF were determined by RT‐PCR and enzyme‐linked immunosorbent assays.Results Treatment with axitinib reduced expression of VEGFR‐1/2 and PDGFR‐β. Hypoxia decreased cell viability and increased tissue permeability, expression, and secretion of VEGF and PDGF. Axitinib significantly reduced hypoxia‐induced overexpression and secretion of the growth factors and tissue permeability.Conclusion Our in vitro results suggest that axitinib may have promising properties as a potential treatment for diabetic macular edema.

Lectin from Agaricus Bisporus Suppresses Akt Phosphorylation and Arrests Cell Cycle Progression in Primary Human Retinal Pigment Epithelial Cells

Anomalous retinal pigment epithelial (RPE) cells have been implicated in the development of retinal diseases. Lectin from the edible mushroom Agaricus bisporus (ABL) was found to inhibit growth of RPE cells. To elucidate the mechanism through which ABL inhibits RPE cell proliferation, we investigated the changes in cell proliferation-related signaling pathways and cell cycle distribution patterns. Primary human RPE cells were grown with or without the lectin (ABL) supplement (20ug or 90ug/ml) for three days. Phosphorylation statuses of Akt, Jnk and p38 as well as p53 expression level were investigated by Western blotting. Cellular distributions in various cell cycle phases were investigated using flow cytometry. After ABL treatment (90ug/ml), Akt was found to be hypo-phosphorylated while the expression levels of p53, phosphorylated-Jnk and phosphorylated-p38 were not altered. The amount of cells present at S phase was reduced. Our results showed that ABL hypo-phosphorylated Akt and this observation is in line with the finding that ABL could attenuate cell proliferation. As the level of p53 was not significantly altered by ABL, this suggested that the mechanism in which ABL arrested cell proliferation was independent of Akt-mediated MDM2 activation but was possibly mediated by altering G1 to S phase transition.

Zytoprotektive und antiangiogene Wirkung des Multikinaseinhibitors Sorafenib im retinalen Pigmentepithel

Cumulative light exposure is significantly associated with progression of age-related macular degeneration (AMD). Inhibition of vascular endothelial growth factor A (VEGF) is the main target of current antiangiogenic treatment strategies for AMD. Previous reports indicated that sorafenib, an oral multikinase inhibitor, might have beneficial effects on exudative AMD. This study investigates the effects of sorafenib on light-induced overexpression of VEGF and its receptors VEGFR1 and 2 in human retinal pigment epithelial (RPE) cells. The effects of sorafenib on VEGFR1 and 2 expression of primary human RPE cells was investigated by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and western blotting. In addition, RPE cells were exposed to white light and incubated with sorafenib. Viability, expression of VEGF and its mRNA were determined by RT-PCR, immunohistochemistry, western blotting, and enzyme-linked immunosorbent assays. Sorafenib reduced VEGFR1 and 2 expression of RPE cells. Light exposure decreased cell viability and increased expression and secretion of VEGF. These light-induced effects were significantly reduced when cells were treated with sorafenib at a dose of 1 µg/ml. The results show that sorafenib has promising properties as a potential antiangiogenic treatment for AMD.

Inhibitory Effect of Epigallocatechin Gallate (EGCG), Resveratrol, and Curcumin on Proliferation of Human Retinal Pigment Epithelial Cells In Vitro

Purpose: To investigate potential inhibitory effects of three polyphenolic agents, epigallocatechin gallate (EGCG; from green tea), resveratrol (from red wine), and curcumin (from turmeric), on the proliferation of human retinal pigment epithelial (RPE) cells and to elucidate unwanted effects.Methods: ARPE19 cells and primary human RPE cells were cultured in the presence of various concentrations of EGCG, resveratrol, or curcumin, and compared with controls. The number of viable cells was determined after 24, 48, and 72 hr by flow cytometrical enumeration. Furthermore, cell division was measured by dye dilution assay using carboxyfluorescein succinimidyl ester (CFSE), cell death by Hoechst 33258 staining, and apoptosis by staining for active caspase 3/7 and 8.Results: The three drugs inhibited the increase of RPE cell numbers at all time points, with resveratrol being the most efficient and curcumin being the least efficient. EGCG inhibited cell proliferation with intermediate efficiency, and showed little induction of cell death. Resveratrol almost completely suppressed cell proliferation, and induced RPE cell necrosis and caspase 3/7- and caspase 8-dependent apoptosis. Curcumin inhibited RPE cell increase exclusively by inducing caspase 3/7-dependent but caspase 8-independent cell death and necrosis.Conclusions: All three polyphenols tested reduced the absolute number of cells, but had different effects on cell proliferation, apoptosis, and necrosis. Resveratrol was most potent and EGCG induced the least cell death. These polyphenols may aid treatment of proliferative vitreoretinopathy (PVR).

Rescue and repair during photoreceptor cell renewal mediated by docosahexaenoic acid-derived neuroprotectin D1

Retinal degenerative diseases result in retinal pigment epithelial (RPE) and photoreceptor cell loss. These cells are continuously exposed to the environment (light) and to potentially pro-oxidative conditions, as the retina's oxygen consumption is very high. There is also a high flux of docosahexaenoic acid (DHA), a PUFA that moves through the blood stream toward photoreceptors and between them and RPE cells. Photoreceptor outer segment shedding and phagocytosis intermittently renews photoreceptor membranes. DHA is converted through 15-lipoxygenase-1 into neuroprotectin D1 (NPD1), a potent mediator that evokes counteracting cell-protective, anti-inflammatory, pro-survival repair signaling, including the induction of anti-apoptotic proteins and inhibition of pro-apoptotic proteins. Thus, NPD1 triggers activation of signaling pathway/s that modulate/s pro-apoptotic signals, promoting cell survival. This review provides an overview of DHA in photoreceptors and describes the ability of RPE cells to synthesize NPD1 from DHA. It also describes the role of neurotrophins as agonists of NPD1 synthesis and how photoreceptor phagocytosis induces refractoriness to oxidative stress in RPE cells, with concomitant NPD1 synthesis.

Lipid peroxidation products reduce lysosomal protease activities in human retinal pigment epithelial cells via two different mechanisms of action

In age-related macular degeneration (AMD), reduced lysosomal capacity may contribute to lipofuscinogenesis and progressive dysfunction of the retinal pigment epithelium (RPE). We previously demonstrated that lipid peroxidation-related protein modifications inhibit lysosomal degradation of photoreceptor outer segment (POS) proteins in RPE cells. Herein, we investigate the effects of lipid peroxidation products on activities of key RPE lysosomal proteases. In lysosomes isolated from primary human RPE cells, lipid peroxidation products 4-hydroxynonenal (HNE) and malondialdehyde (MDA) exerted a dose-dependent inhibitory effect on cysteine proteases cathepsin B and L, with biologically relevant concentrations of 1 muM resulting in a reduction of enzyme activities by 88-94%. This effect was confirmed in cultured RPE cells. Using mass spectrometry, covalent HNE and MDA adducts were detected in the active center region of inactivated cathepsins. POS previously modified with HNE and MDA likewise caused a dose-dependent reduction of cathepsin B and L activities in isolated lysosomes and, in addition, inhibited the aspartic protease cathepsin D. Our results indicate that lipid peroxidation products in vitro interfere with RPE lysosomal protease activities by two different mechanisms of action: (i) HNE and MDA directly inactivate lysosomal cysteine proteases by covalent binding to the active center; (ii) HNE- and MDA-mediated protein modifications convert proteolytic substrates into competitive inhibitors of lysosomal proteases. Via these mechanisms, lipid peroxidation products may induce lysosomal dysfunction and lipofuscinogenesis in the aging RPE and thus contribute to the pathogenesis of AMD.

Lipid peroxidation products reduce lysosomal protease activities in human retinal pigment epithelial cells via two different mechanisms of action

In age-related macular degeneration (AMD), reduced lysosomal capacity may contribute to lipofuscinogenesis and progressive dysfunction of the retinal pigment epithelium (RPE). We previously demonstrated that lipid peroxidation-related protein modifications inhibit lysosomal degradation of photoreceptor outer segment (POS) proteins in RPE cells. Herein, we investigate the effects of lipid peroxidation products on activities of key RPE lysosomal proteases. In lysosomes isolated from primary human RPE cells, lipid peroxidation products 4-hydroxynonenal (HNE) and malondialdehyde (MDA) exerted a dose-dependent inhibitory effect on cysteine proteases cathepsin B and L, with biologically relevant concentrations of 1 μM resulting in a reduction of enzyme activities by 88–94%. This effect was confirmed in cultured RPE cells. Using mass spectrometry, covalent HNE and MDA adducts were detected in the active center region of inactivated cathepsins. POS previously modified with HNE and MDA likewise caused a dose-dependent reduction of cathepsin B and L activities in isolated lysosomes and, in addition, inhibited the aspartic protease cathepsin D. Our results indicate that lipid peroxidation products in vitro interfere with RPE lysosomal protease activities by two different mechanisms of action: (i) HNE and MDA directly inactivate lysosomal cysteine proteases by covalent binding to the active center; (ii) HNE- and MDA-mediated protein modifications convert proteolytic substrates into competitive inhibitors of lysosomal proteases. Via these mechanisms, lipid peroxidation products may induce lysosomal dysfunction and lipofuscinogenesis in the aging RPE and thus contribute to the pathogenesis of AMD.

Molecular regulation of cigarette smoke induced-oxidative stress in human retinal pigment epithelial cells: implications for age-related macular degeneration

Cigarette smoke is the most important environmental risk factor for developing age-related macular degeneration (AMD). Damage to the retinal pigment epithelium (RPE) caused by cigarette smoke may underlie the etiology of AMD. This study investigated the molecular and cellular effects of cigarette smoke exposure on human RPE cells. ARPE-19 or primary human RPE cells were exposed to cigarette smoke extract (CSE) or hydroquinone (HQ), a component of cigarette smoke. The effect of this exposure on key aspects of RPE vitality including viability, cell size, mitochondrial membrane potential (DeltaPsi(m)), superoxide production, 4-hydroxy-2-nonenal (4-HNE), vascular endothelial growth factor (VEGF), and heme oxygenase-1 (HO-1) expression was determined. Exposure of RPE cells to CSE or HQ caused oxidative damage and apoptosis, characterized by a reduction in cell size and nuclear condensation. Evidence of oxidative damage also included increased lipid peroxidation (4-HNE) and mitochondrial superoxide production, as well as a decrease in intracellular glutathione (GSH). Exogenous administration of antioxidants (GSH and N-acetyl-cysteine) prevented oxidative damage to the RPE cells caused by CSE. Cigarette smoke also induced expression of VEGF, HO-1, and the transcription factor nuclear factor erythroid-derived 2, like 2 (NRF2). However, NRF2 was only modestly involved in CSE-induced HO-1 expression, as shown by the NRF2 small interfering RNA studies. These new findings demonstrate that cigarette smoke is a potent inducer of oxidative damage and cell death in human RPE cells. These data support the hypothesis that cigarette smoke contributes to AMD pathogenesis by causing oxidative damage and cell death to RPE cells.

Biological effects of native and oxidized low-density lipoproteins in cultured human retinal pigment epithelial cells

Age-related macular degeneration (AMD) and artherosclerosis share common characteristics in their pathogenesis. In this study, we investigated the effects of lipoproteins like native (n)-LDL, oxidized (ox)-LDL and high-density lipoprotein (HDL) on advanced senescence, extracellular matrix accumulation, cell loss, and transforming growth factor-beta2 (TGF-β2) expression in cultured human retinal pigment epithelial (RPE) cells. Primary human RPE cells were incubated with 10–100μg/ml n-LDL, ox-LDL, and HDL for 24h. For determination of advanced senescence, beta-galactosidase staining was used. The induction of fibronectin (Fn), laminin alpha 1 (Laa1), and collagen type IV alpha 2 (Col4a2) mRNA was quantified by real-time PCR. Cell loss was investigated by live dead assay. Expression of TGF-β2 was analyzed by real-time PCR and ELISA assays. Ox-LDL accelerated dose-dependently the onset of RPE senescence, whereas LDL and HDL had no effect. LDL and ox-LDL led to induced expression of Fn, Laa1 and Col4a2, whereas HDL had no influence. Incubation of RPE cells with 100μg/ml ox-LDL induced marked cell death compared to untreated control cells. Expression of TGF-β2 was dose-dependently increased by LDL and ox-LDL.LDL and ox-LDL induced cellular changes in RPE cells in vitro, which may resemble pathogenic events of AMD. These results may provide further information about the effects of LDL and ox-LDL in the human RPE and their potential role in the pathogenesis of AMD.

Proteomic and Transcriptomic Analyses of Retinal Pigment Epithelial Cells Exposed to REF-1/TFPI-2

The authors previously reported a growth-promoting factor, REF-1/TFPI-2, that is specific to retinal pigment epithelial (RPE) cells. The purpose of this study was to determine the genes and proteins of human RPE cells that are altered by exposure to TFPI-2. Human primary RPE cells were cultured with or without TFPI-2. Cell extracts and isolated RNA were subjected to proteomic and transcriptomic analyses, respectively. Proteins were separated by two-dimensional gel electrophoresis followed by gel staining and ion spray tandem mass spectrometry analyses. Transcriptomic analysis was performed using a DNA microarray to detect 27,868 gene expressions. Proteomic analysis revealed c-Myc binding proteins and ribosomal proteins L11 preferentially induced by TFPI-2 in human RPE cells. Transcriptomic analysis detected 10,773 of 33,096 probes in the TFPI-2 treated samples, whereas only 2186 probes were detected in the nontreated samples. Among the genes up-regulated by TFPI-2 at the protein level were c-myc, Mdm2, transcription factor E2F3, retinoblastoma binding protein, and the p21 gene, which is associated with the c-myc binding protein and ribosomal protein L11. The mechanisms by which TFPI-2 promotes the proliferation of RPE cells may be associated with augmented c-myc synthesis and the activation of E2F in the retinoblastoma protein (Rb)/E2F pathway at the G1 phase of the RPE cells. Activation of ribosomal protein L11 and the Mdm2 complex of the p53 pathway may be counterbalanced by the hyperproliferative conditions.