RPE Cultures Research Articles

Retinal pigment epithelial cells can be cultured on fluocinolone acetonide treated nanofibrous scaffold

Engineered tissue currently lacks requisite capacity to sustain cell viability and functionality. Here we demonstrate that human RPE cell lines (ARPE-19) can be cultured on ultrathin suspended electrospun nanofibre scaffolds (ENS) composed of hydrophobic polymer polyacrylonitrile (PAN) and a water-soluble aliphatic diamine, without (untreated) or with (treated) fluocinolone acetonide (FA). Cells survived and retained their characteristic morphology for up to 150 days with FA-treated ENS and manifested a morphological epithelial phenotype with expression of biomarkers critical for maintaining retinal physiological characteristics. This novel technique for producing culture substrates provides suitable hydrophilicity and a protective environment for prolonged RPE culture and has immense potential for subretinal transplantation. The findings indicate that FA-treated ENS is an excellent matrix for retaining the differentiated and epithelial phenotype.

Oxidation of DJ-1 Cysteines in Retinal Pigment Epithelium Function.

The retina and RPE cells are regularly exposed to chronic oxidative stress as a tissue with high metabolic demand and ROS generation. DJ-1 is a multifunctional protein in the retina and RPE that has been shown to protect cells from oxidative stress in several cell types robustly. Oxidation of DJ-1 cysteine (C) residues is important for its function under oxidative conditions. The present study was conducted to analyze the impact of DJ-1 expression changes and oxidation of its C residues on RPE function. Monolayers of the ARPE-19 cell line and primary human fetal RPE (hfRPE) cultures were infected with replication-deficient adenoviruses to investigate the effects of increased levels of DJ-1 in these monolayers. Adenoviruses carried the full-length human DJ-1 cDNA (hDJ) and mutant constructs of DJ-1, which had all or each of its three C residues individually mutated to serine (S). Alternatively, endogenous DJ-1 levels were decreased by transfection and transduction with shPARK7 lentivirus. These monolayers were then assayed under baseline and low oxidative stress conditions. The results were analyzed by immunofluorescence, Western blot, RT-PCR, mitochondrial membrane potential, and viability assays. We determined that decreased levels of endogenous DJ-1 levels resulted in increased levels of ROS. Furthermore, we observed morphological changes in the mitochondria structure of all the RPE monolayers transduced with all the DJ-1 constructs. The mitochondrial membrane potential of ARPE-19 monolayers overexpressing all DJ-1 constructs displayed a significant decrease, while hfRPE monolayers only displayed a significant decrease in their ΔΨm when overexpressing the C2S mutation. Viability significantly decreased in ARPE-19 cells transduced with the C53S construct. Our data suggest that the oxidation of C53 is crucial for regulating endogenous levels of ROS and viability in RPE cells.

Amniotic Membrane Enhances the Characteristics and Function of Stem Cell-Derived Retinal Pigment Epithelium Sheets by Inhibiting the Epithelial–Mesenchymal Transition

Human pluripotent stem cell-derived retinal pigment epithelium (iRPE) is an attractive cell source for disease modeling and cell replacement therapy of retinal disorders with RPE defects. However, there are still challenges to develop appropriate culture conditions close to in vivo microenvironment to generate iRPE sheets, which mimic more faithfully the characteristics and functions of the human RPE cells. Here, we developed a simple, novel platform to construct authentic iRPE sheets using human amniotic membrane (hAM) as a natural scaffold. The decellularized hAM (dAM) provided a Bruch's membrane (BM)-like bioscaffold, supported the iRPE growth and enhanced the epithelial features, polarity distribution and functional features of iRPE cells. Importantly, RNA-seq analysis was performed to compare the transcriptomes of iRPE cells cultured on different substrates, which revealed the potential mechanism that dAM supported and promoted iRPE growth was the inhibition of epithelial-mesenchymal transition (EMT). The tissue-engineered iRPE sheets survived and kept monolayer when transplanted into the subretinal space of rabbits. All together, our results indicate that the dAM imitating the natural BM allows for engineering authentic human RPE sheets, which will provide valuable biomaterials for disease modeling, drug screening and cell replacement therapy of retinal degenerative diseases. Statement of significanceEngineered RPE sheets have a great advantage over RPE cell suspension for transplantation as they support RPE growth in an intact monolayer which RPE functions are dependent on. The substrates for RPE culture play a critical role to maintain the physiological functions of the RPE in stem cell therapies for patients with retinal degeneration. In this study, we constructed engineered iRPE sheets on the decellularized human amniotic membrane scaffolds, which contributed to enhancing epithelial features, polarity distribution and functional features of iRPE. dAM exhibited the ability of anti-epithelial mesenchymal transition to support iRPE growth. Furthermore, the results of transplantation in vivo demonstrated the feasibility of iRPE sheets in retina regenerative therapy. Engineering RPE sheets on dAM is a promising strategy to facilitate the development of iRPE replacement therapy and retinal disease modeling.

Quantification of mitophagy using mKeima-mito in cultured human primary retinal pigment epithelial cells

The retinal pigment epithelium is a pigmented monolayer of cells that help maintain a healthy retina. Loss of this essential cell layer is implicated in a number of visual disorders, including age-related macular degeneration (AMD). Utilizing primary RPE cultures to investigate disease is an important step in understanding disease mechanisms. However, the use of primary RPE cultures presents a number of challenges, including the limited number of cells available and the presence of auto-fluorescent pigment that interferes with quantifying fluorescent probes. Additionally, primary RPE are difficult to transfect with exogenous nucleic acids traditionally used for fluorescent imaging. To overcome these challenges, we used an adeno-associated viral (AAV) vector to express a pH sensitive fluorescent protein, mKeima, fused to the mitochondrial targeting sequence of cytochrome oxidase subunit 8A (mKeima-mito). mKeima-mito allows for quantification of mitochondrial autophagy (mitophagy) in live-cell time-lapse imaging experiments. We also developed an image analysis pipeline to selectively quantify mKeima-mito while removing the signal of auto-fluorescent pigment from the dataset by utilizing information from the mKeima fluorescent channels. These techniques are demonstrated in primary RPE cultures expressing mKeima-mito treated with 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP), an uncoupler that depolarizes the mitochondrial membrane and leads to mitochondrial fragmentation and mitophagy. The techniques outlined provide a roadmap for investigating disease mechanisms or the effect of treatments utilizing fluorescent probes in an important cell culture model.

Complement C5 is not critical for the formation of sub-RPE deposits in Efemp1 mutant mice

The complement system plays a role in the formation of sub-retinal pigment epithelial (RPE) deposits in early stages of age-related macular degeneration (AMD). But the specific mechanisms that connect complement activation and deposit formation in AMD patients are unknown, which limits the development of efficient therapies to reduce or stop disease progression. We have previously demonstrated that C3 blockage prevents the formation of sub-RPE deposits in a mouse model of EFEMP1-associated macular degeneration. In this study, we have used double mutant Efemp1R345W/R345W:C5-/- mice to investigate the role of C5 in the formation of sub-RPE deposits in vivo and in vitro. The data revealed that the genetic ablation of C5 does not eliminate the formation of sub-RPE deposits. Contrarily, the absence of C5 in RPE cultures promotes complement dysregulation that results in increased activation of C3, which likely contributes to deposit formation even in the absence of EFEMP1-R345W mutant protein. The results also suggest that genetic ablation of C5 alters the extracellular matrix turnover through an effect on matrix metalloproteinases in RPE cell cultures. These results confirm that C3 rather than C5 could be an effective therapeutic target to treat early AMD.

Highly Differentiated Human Fetal RPE Cultures Are Resistant to the Accumulation and Toxicity of Lipofuscin-Like Material.

PurposeThe accumulation of undigestible autofluorescent material (UAM), termed lipofuscin in vivo, is a hallmark of aged RPE. Lipofuscin derives, in part, from the incomplete degradation of phagocytized photoreceptor outer segments (OS). Whether this accumulated waste is toxic is unclear. We therefore investigated the effects of UAM in highly differentiated human fetal RPE (hfRPE) cultures.MethodsUnmodified and photo-oxidized OS were fed daily to confluent cultures of ARPE-19 RPE or hfRPE. The emission spectrum, composition, and morphology of resulting UAM were measured and compared to in vivo lipofuscin. Effects of UAM on multiple RPE phenotypes were assessed.ResultsCompared to ARPE-19, hfRPE were markedly less susceptible to UAM buildup. Accumulated UAM in hfRPE initially resembled the morphology of lipofuscin from AMD eyes, but compacted and shifted spectrum over time to resemble lipofuscin from healthy aged human RPE. UAM accumulation mildly reduced transepithelial electrical resistance, ketogenesis, certain RPE differentiation markers, and phagocytosis efficiency, while inducing senescence and rare, focal pockets of epithelial-mesenchymal transition. However, it had no effects on mitochondrial oxygen consumption rate, certain other RPE differentiation markers, secretion of drusen components or polarity markers, nor cell death.ConclusionshfRPE demonstrates a remarkable resistance to UAM accumulation, suggesting mechanisms for efficient OS processing that may be lost in other RPE culture models. Furthermore, while UAM alters hfRPE phenotype, the effects are modest, consistent with conflicting reports in the literature on the toxicity of lipofuscin. Our results suggest that healthy RPE may adequately adapt to and tolerate lipofuscin accumulation.

Intravenous injection of l-aspartic acid β-hydroxamate attenuates choroidal neovascularization via anti-VEGF and anti-inflammation

Choroidal neovascularization (CNV) is a hallmark of exudative age-related macular degeneration (exAMD) and a major cause of visual loss in AMD. Despite the widespread use of anti-VEGF therapy, serious adverse effects arise from repeated intravitreal injection of anti-VEGF antibodies, which warrant alternative strategy. We report herein that in a CNV murine model created by krypton red laser, intravenous injection of a serine racemase inhibitor, l-Aspartic acid β-hydroxamate (L-ABH), significantly reduced CNV at the dose 6 mg/kg on the first day before and followed by 3 mg/kg on the third day after laser injury. The CNV volumes were analyzed with isolectin GS-IB4 staining on choroidal/RPE flat mounts on the seventh day after laser injury. Injection of L-ABH did not produce negative effects on retinal function and visual behavior. To dissect the mechanism in vitro, pretreatment with L-ABH in primary RPE cultures significantly reduced production of vascular endothelial growth factor (VEGF) and macrophage chemotactic protein 1 (MCP-1) by TNFα-primed RPEs. Consistent with these observations, L-ABH pretreatment significantly attenuated macrophage migration mediated by TNFα-primed RPE. Collectively, intravenous injection of L-ABH significantly reduced CNV volumes via reducing production of VEGF and MCP-1 by inflammation-primed RPEs.

Perspective on AMD Pathobiology: A Bioenergetic Crisis in the RPE.

AMD is the leading cause of blindness in developed countries. The dry form of AMD, also known as atrophic AMD, is characterized by the death of RPE and photoreceptors. Currently, there are no treatments for this form of the disease due in part to our incomplete understanding of the mechanism causing AMD. Strong experimental evidence from studies of human donors with AMD supports the emerging hypothesis that defects in RPE mitochondria drive AMD pathology. These studies, using different experimental methods, have shown disrupted RPE mitochondrial architecture and decreased mitochondrial number and mass, altered content of multiple mitochondrial proteins, increased mitochondrial DNA damage that correlates with disease severity, and defects in bioenergetics for primary RPE cultures from AMD donors. Herein, we discuss a model of metabolic uncoupling that alters bioenergetics in the diseased retina and drives AMD pathology. These data provide the rationale for targeting the mitochondria in the RPE as the most efficacious intervention strategy if administered early, before vision loss and cell death.

Stem Cell-Derived Retinal Pigment Epithelial Layer Model from Adult Human Globes Donated for Corneal Transplants.

An adult human retinal pigment epithelial layer (ahRPE) model derived from stem cells isolated from native RPE monolayers (ahRPE-SCs) exhibits key physiological characteristics of native tissue and therefore provides the means to create a human "disease in a dish" model to study RPE diseases. Traditionally, RPE lines are established from whole globes dedicated to research. Here we describe a new technique for establishing primary RPE lines from the posterior poles of globes used for corneal transplants. Since tissues from corneal transplants are derived from younger and healthier donors than those used for research, we have hypothesized that RPE cells isolated from corneal transplantation globes will result in improved primary RPE line establishment. Our new procedure increases the rate of establishing successful RPE cultures and improves the total cell number yield. Use of this advanced methodology can provide a new source of high-quality primary RPE line cultures. © 2018 by John Wiley & Sons, Inc.

Human Adult Retinal Pigment Epithelial Stem Cell–Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue

We tested what native features have been preserved with a new culture protocol for adult human RPE. We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used. Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 ± 0.24 mV (mean ± SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 ± 9.9 Ω·cm2 (mean ± SEM, n = 31). Application of 100 μM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 ± 0.53 μL·cm2·h-1 (mean ± SEM, n = 6) and TEP by 0.33 ± 0.048 mV (mean ± SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 ± 146 pg/mL/d, compared to an apical secretion of 1548 ± 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 ± 280 ng/mL/d compared to a basolateral secretion of 864 ± 132 ng/mL/d (n = 14, P < 0.01). The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.

Infection of Human Retinal Pigment Epithelium with Chlamydia trachomatis.

PurposeLittle is known about the susceptibility of posterior segment tissues, particularly the human retinal pigment epithelium (hRPE), to Chlamydia trachomatis. The purpose of the study was to investigate the possibility of infecting the hRPE with Chlamydia trachomatis, and to examine the infectivity of different Chlamydia trachomatis clinical isolates for hRPE cells and the hRPE cell response to the infection.MethodsCultured hRPE and McCoy cells were inoculated with eight Chlamydia trachomatis (serovar E) clinical isolates at multiplicity of infection (MOI) of 2.0 or 0.3. To detect Chlamydia trachomatis, samples were stained immunohistochemically with anti-major outer membrane protein antibodies at 24h, 48h, and 72h postinoculation (PI). The changes in the expression of signaling molecules and proteins of cytoskeleton and extracellular matrix in hRPE cells were examined immunohistochemically.ResultsAll eight clinical isolates demonstrated ability to infect hRPE cells. At equal MOI of 0.3, the infectivity of Chlamydia trachomatis clinical isolates for RPE culture was found to be at least as high as that for McCoy cell culture. At 24h PI, the percentage of inclusion-containing cells varied from 1.5 ± 0.52 to 14.6 ± 3.3% in hRPE cell culture infected at MOI of 2.0 against 0.37 ± 0.34 to 8.9 ± 0.2% in McCoy cell culture infected at MOI of 0.3. Collagen type I, collagen type IV, basic fibroblast growth factor, transforming growth factor-beta and interleukin–8 expression at 48h PI were maximally increased, by 2.1-, 1.3-, 1.5-, 1.5- and 1.6-fold, respectively, in the Chlamydia trachomatis-infected compared with control hRPE cell culture specimens (P < 0.05).ConclusionsThis study, for the first time, proved the possibility of infecting hRPE cultured cells with Chlamydia trachomatis, which leads to proproliferative and proinflammatory changes in the expression of signaling molecules and extracellular matrix components.

An improved method for the isolation and culture of retinal pigment epithelial cells from adult rats.

Since adult rats are used in pre-clinical studies, and due to the necessity of investigating the side-effects of drugs on RPE cells in vitro, there is a great need for primary RPE cells from these animals. The aim of this study was to develop a reproducible and quantifiable method of isolation, culture, and maintenance of adult rat RPE cells. Moreover, potential differences between RPE cells from albino versus pigmented rats were also investigated. A total of 180 pigmented rats and 340 albino rats aged 6-14 weeks were used. RPE cells were isolated and cultured for several weeks by using three different methods: 1) growing directly on flat mounts, 2) after enzymatic isolation, and 3) after they spontaneously detached from the flat mounts and continued to grow on the plastic. Yield, cell survival, and morphological characteristics were investigated using light and electron microscopy as well as immunohistochemistry. After 0 weeks, the yield of the first method was 30,000 cells/eye; after 2 weeks 18,000 cells/eye; and after 4 weeks 11,000 cells/eye. The yield of RPE cells was very low after enzymatic isolation in method 2 (0 weeks, 13.000 cells/eye; 2 weeks, 30,000 cells/eye; 4 weeks 38,000 cells/eye), whereas it was higher when the RPE cells spontaneously detached from the flat mounts and then continued to grow on the plastic in method three. (0 weeks, 30,000 cells/eye; 2 weeks, 314,000 cells/eye; 4 weeks, 659,000 cells/eye). The second method often showed contamination with fibroblasts, whereas the two other methods showed pure RPE cultures. The RPE cells were able to proliferate when using the second and the third method, but not when they were cultivated directly on the flat mounts (first method). The qualitative and quantitative best method for isolating adult rat RPE cells is the culture of RPE cells which spontaneously detach from flat mounts. No differences were observed between albino and pigmented RPE cells.

Subcellular distribution and activity of mechanistic target of rapamycin in aged retinal pigment epithelium.

Inhibiting mechanistic target of rapamycin (mTOR) by pharmacological or genetic approaches can extend lifespan in mammals. The kinase activity of mTOR is controlled by upstream regulatory proteins and its subcellular localization. The purpose of this study was to characterize age-related alterations and functional consequences of mTOR signaling in the postmitotic RPE cells. Activity of mTOR complex 1 (mTORC1) was monitored by measuring phosphorylation status of its downstream effector protein S6, in either cultured human RPE cells or RPE explants prepared from mice at different ages. Subcellular distribution of mTOR was investigated by immunofluorescent staining of RPE culture or flatmount. The signaling of mTORC1 was modulated by either overexpression of a small guanosine triphosphatase, Ras homolog enriched in brain (Rheb), or disruption of the Ragulator complex with small interference RNA targeting p18. The effects of mTOR pathway on degradation of phagocytosed photoreceptor outer segments (POS) were determined by measuring the turnover rate of rhodopsin. Aged RPE cells had more lysosome-associated mTOR and had increased response to amino acid stimulation. The lysosome distribution was essential for mTORC1 function, as disruption of the Ragulator complex abolished mTORC1 activation by amino acids. Increased mTORC1 activity caused decreased rate of degradation of internalized POS in the RPE. Aging changes the subcellular localization and function of mTOR in the RPE. Increased mTORC1 inhibits POS degradation and may further exacerbate lysosome dysfunction of aged RPE.

Functional Analysis of Serially Expanded Human iPS Cell-Derived RPE Cultures

To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures. Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE). RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1-P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein-coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels. Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.

Longterm cultures of the aged human RPE do not maintain epithelial morphology and high transepithelial resistance

Dear Editor, Visual rehabilitation achieved with peripheral RPE autografts in exudative AMD is only modest. Moreover, complication rates using the popular RPE–choroid patch technique remain relatively high. Tissue-engineered transplants might improve the outcome of RPE transplantation. A culturing step could place cells on a substrate prior to transplantation, thereby optimizing delivery and long-term function [1]. This would require sufficient cellular plasticity (i.e., response to environmental stimuli) to achieve a well-differentiated, functional graft. While culture plasticity was confirmed for fetal cells or RPE cell lines [2, 3], only sparse information is available for aged RPE [4]. We studied the influence of culture conditions on select differentiation characteristics in long-term primary RPE cultures from ten human donors above age 55, and compared them to a 23-year-old and two fetal donors (20 and 21 weeks of gestation respectively). The main outcome measures at 6 weeks post-confluence (PC) were morphology and transepithelial resistance (TER), an electrophysiological method for assessing tight junctions build-up. Eyes were preserved within 6 hours of death. Adult primary RPE cultures (aRPE) were initiated from: (1) mechanically scraped RPE, or (2) collagenase pretreated (1 mg/ml for 40 minutes, CLS I, Worthington Biochemical Corp, Lakewood, NJ, USA) and mechanically scraped RPE, or (3) fetal (fRPE) and adult cultures started from floater passages [2]. Cells were grown on permeable polyester inserts (Transwells, Corning Inc., Corning, NY, USA) in modified Hu & Bok media [2], containing 2% calf serum, basic FGF (5 ng/ml), L-alanyl-L-glutamine (2 mM), Albumax (200 μg/ml), and antibiotics/antimycotics. RPE culture purity was confirmed by a uniform pancytokeratin staining. Experimental matrices analyzed the influence of above harvesting methods, seeding density, laminin coating (2 μg/cm), and media composition (2% vs 10% serum until confluence, or calcium switch at confluence, or 5 mM vs 25 mM glucose) on above outcome measures. Morphology was monitored weekly in all and transepithelial resistance (TER) in all conditions, except the laminin matrix (i.e., seven of ten aged donors). Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, FL, USA, April 30–May 4 2006.

Constitutive and oxidative-stress-induced expression of VEGF in the RPE are differently regulated by different Mitogen-activated protein kinases

Vascular endothelial growth factor (VEGF) is a fundamental factor for angiogenesis. It plays important roles in pathological conditions (e.g. the development of wet AMD), but also in the healthy organism) e.g. in maintaining the vasculature and supporting the retina). Recent therapies to treat the wet AMD focus on neutralizing VEGF indiscriminately. VEGF is constitutively expressed in the retina, but its expression is upregulated by various (noxious) stimuli, e.g. oxidative stress or hypoxia. Discrimination between constitutive expression of VEGF and its pathological upregulation might provide the possibility of focusing on inhibiting the pathological expression only. Here, we focused on the influence of different mitogen-activated protein kinase (MAPK) (p38, Erk, JNK) on the secretion and expression of VEGF, with or without being challenged by oxidative stress. VEGF secretion was measured using a perfusion organ culture model; expression was examined in primary RPE culture and Western blotting. Constitutive VEGF expression and secretion can be diminished by inhibiting p38, while inhibiting Erk or JNK does not show a significant effect. When challenged with oxidative stress (250 microM t-butylhydroperoxide), VEGF expression and secretion increases and the influence of the MAPK changes: While p38 still accounts for about 30% of the secretion, Erk shows a similar influence. Inhibiting JNK presents conflicting results. In organ culture, inhibiting JNK significantly increases VEGF secretion after stimulation with 250 microM tBH, while with regard to VEGF expression in RPE cell culture, this effect could not be seen. Constitutive and oxidative stress induced VEGF secretion, and expression is differently regulated, which might offer an opportunity to selectively inhibit pathological VEGF expression only.

Expression, Localization, and Function of Junctional Adhesion Molecule-C (JAM-C) in Human Retinal Pigment Epithelium

To determine the localization of JAM-C in human RPE and characterize its functions. Immunofluorescence, Western blot, and PCR was used to identify the localization and expression of JAM-C, ZO-1, N-cadherin, and ezrin in cultures of human fetal RPE (hfRPE) with or without si-RNA mediated JAM-C knockdown and in adult native RPE wholemounts. A transepithelial migration assay was used to study the migration of leukocytes through the hfRPE monolayer. JAM-C localized at the tight junctions of cultured hfRPE cells and adult native RPE. During initial junction formation JAM-C was recruited to the primordial cell-cell contacts and after JAM-C knockdown, the organization of N-cadherin and ZO-1 at those contacts was disrupted. JAM-C knockdown caused a delay in the hfRPE cell polarization, as shown by reduced apical staining of ezrin. JAM-C inhibition significantly decreased the chemokine-induced transmigration of granulocytes but not monocytes through the hfRPE monolayer. JAM-C localizes specifically in the tight junctions of hfRPE and adult native RPE. It is important for tight junction formation in hfRPE, possibly by regulating the recruitment of N-cadherin and ZO-1 at the cell-cell contacts, and has a role in the polarization of hfRPE cells. Finally, JAM-C promotes the basal-to-apical transmigration of granulocytes but not monocytes through the hfRPE monolayer.

Complement factor H and factor B expression in RPE cells

Abstract Purpose Age‐related macular degeneration (AMD) is the leading cause of untreatable blindness in the developed world. The pathogenesis of AMD is not fully understood. Recent evidence suggests that local inflammation in particular complement activation plays an important role. We aim to understand how complement activation is regulated at retina/choroidal interface. Methods The expression and distribution of complement factor H (CFH) and factor B (CFB) in mouse ocular tissues were examined by immunohistochemistry. Regulation of CFH and CFB gene expression by various cytokines or photoreceptor outer segments (POS) was investigated in vitro in cultured RPE cells. Changes in CFH or CFB gene expression after treatment were evaluated by RT‐PCR. Results In normal mouse eyes, CFH was detected in corneal epithelial cells, ciliary body, RPE cells, Bruch’s membrane and choroidal vessels. There is no significant change in either the expression level or the distribution pattern of CFH in ocular tissues of different ages of mice. CFB was exclusively detected in RPE cells in normal mice. The expression of CFB in RPE cells increases with age. In vitro in RPE cultures, the expression of CFH was negatively regulated by cytokine TNF‐alpha and IL‐6, whereas the expression of CFB was positively regulated by TNF‐alpha and IFN‐gamma. Short‐term incubation of RPE cells with POS did not alter the expression of CFH or CFB, whereas long‐term incubation of RPE cells with POS significantly down‐regulated CFH expression but up‐regulated CFB expression. Conclusion Complement regulatory factors CFH and CFB are produced locally in the retina/choroidal interface by RPE cells. The production of CFH and CFB in RPE cells is regulated differently by various cytokines and oxidized POS.

Expression of ZnT and ZIP Zinc Transporters in the Human RPE and Their Regulation by Neurotrophic Factors

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. 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. 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. 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.

Therapeutic effect of microencapsulated porcine retinal pigmented epithelial cells transplantation on rat model of Parkinson’s disease

To investigate the therapeutic effect of microencapsulated porcine retinal pigmented epithelial cells (RPE-M) transplantation on rat model of Parkinson's disease (PD). Primary porcine RPE cells were harvested by enzyme digestion and expanded in culture medium. Determine the levels of dopamine (DA) and homovanillic acid (HVA) by high performance liquid chromatography electrochemical (HPLC) assay, and the levels of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) were detected by ELISA. Alginate-polylysine-alginate (APA) microencapsulated cells were produced by using a high voltage electrostatic system. PD rat model was established by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB). After that, the RPE-M was transplanted into the corpus striatum of PD rat, and then the rotation test scores were recorded and biochemical changes of the corpus striatum were tested. The levels of DA, HVA, BDNF and GDNF secreted by RPE were stable in the RPE culture supernatant and were not changed by the microencapsulation. Eighty-three percent rats developed PD by unilateral lesion of 6-OHDA in the MFB. The RPE-M transplantation had therapeutic effect on 33% PD rats. Porcine RPE cells grow actively in vitro and could secrete DA, HVA, BDNF, and GDNF constantly, which does not be affected by the passage culture and the APA miroencapsulation. RPE-M transplantation of may be a curative therapy for PD.