Retinal Pigment Epithelial Cell Layer Research Articles

Elovl4b knockout zebrafish as a model for ocular very-long-chain PUFA deficiency

Very-long-chain PUFAs (VLC-PUFAs) are a group of lipids with chain lengths >24 carbons, and the ELOVL4 (elongation of very-long-chain FA-4) enzyme is responsible for vertebrate VLC-PUFA biosynthesis. Studies on the role of VLC-PUFAs in vision have been hindered because of the need for adequate animal models to capture the global loss of VLC-PUFAs. Since homozygous Elovl4 ablation is lethal in neonatal mice because of catastrophic drying from the loss of their protective skin barrier, we established a zebrafish (Danio rerio) model of Elovl4 ablation. We generated Elovl4b KO zebrafish by creating a 56-bp deletion mutation in exon 2 of the Elovl4b gene using CRISPR-Cas9. We used GC-MS and LC-MS/MS to analyze the VLC-PUFA and lipid profiles from wild-type and Elovl4b KO fish eyes. We also performed histology and visual-behavioral tests. We found that heterozygous and homozygous Elovl4b KO zebrafish eyes had altered lipid profiles and a significantly lower C30 to C36 VLC-PUFA abundance than wild-type fish. Moreover, Elovl4b+/− and Elovl4b−/− KO larvae had significantly lower motor activity in response to light-dark cycles than their age-matched controls. Elovl4b−/− adult fish showed no obvious differences in gross retinal morphology and lamination compared with wild type, except for the presence of lipid droplets within the retinal pigment epithelial cell layer of Elovl4b−/− fish. Our data indicate that the loss of Elovl4b in zebrafish changes ocular lipid profiles and leads to visual abnormalities and subtle retinal changes. These findings highlight the use of zebrafish as a model for VLC-PUFA depletion and ELOVL4-related dysfunction.

4D-DIA quantitative proteomics revealed the core mechanism of diabetic retinopathy after berberine treatment

ObjectiveTo reveal the core mechanism of berberine (BBR) in the treatment of diabetic retinopathy (DR), by using Four-dimensional independent data acquisition (4D-DIA) proteomics combined bioinformatics analysis with experimental validation. Methods: DR injury model was established by injecting streptozotocin intraperitoneally. At 8 weeks after BBR administration, optical coherence tomography (OTC) photos and Hematoxylin-eosin staining from retina in each group were performed, then the retina was collected for 4D-DIA quantitative proteomics detection. Moreover, difference protein analysis, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, protein-protein interaction (PPI) network, as well as molecular docking was performed, respectively. In the part of experiment, Western blot (WB) and immunofluorescent staining was used to confirm the change and distribution of carbonic anhydrase 1 (CA1), one of the most important molecules from quantitative PCR detection. Lastly, RNA knockdown was used to determine the crucial role of CA1 in retinal pigment epithelial cells (RPEs) administrated with berberine. ResultsOCT detection showed that the outer nucleus, inner layer and outer accessory layer of RPEs were thinned in DR group, compared with in sham one, while they were thickened after berberine administration, when compared with in DR group. 10 proteins were screened out by using proteomic analysis and Venny cross plot, in which, denn domain containing 1A (DENND1A) and UTP6 small subunit processome component (UTP6) was down-regulated, while ATPase copper transporting alpha (ATP7A), periplakin (PPL), osteoglycin (OGN), nse1 Homolog (NSMCE1), membrane metalloendopeptidase (MME), lim domain only 4 (LMO4), CA1 and fibronectin 1 (FN1) was up-regulated in DR group, and the BBR treatment can effectively reverse their expressions. PPI results showed that 10 proteins shared interactions with each other, but only ATP7A, FN1 and OGN exhibited directly associated with each other. Moreover, we enlarged the linked relation up to 15 genes in network, based on 10 proteins found from proteomics detection, so as to perform deep GO and KEGG analysis. As a result, the most important biological process is involving rRNA processing; the most important cell component is small subunit processor; the most important molecular function is Phospholipid binding; the KEGG pathway was Ribosome biogenesis in eukaryotes. Moreover, molecular docking showed that LMO4, ATP7A, PPL, NSMCE1, MME, CA1 could form a stable molecular binding pattern with BBR. Of these, the mRNA expression of CA1, PPL and ATP7A and the protein level of CA1 was increased in DR, and decreased in BBR group. Lastly, CA1 RNA knockdown confirmed the crucial role of CA1 in RPE administered with BBR. ConclusionThe present findings confirmed the role of BBR in DR treatment and explained associated molecular network mechanism, in which, CA1 could be considered as a crucial candidate in the protection of RPEs with berberine treatment.

Myopic macular Bruch’s membrane defects

PurposeTo examine histologic characteristics of macular Bruchś membrane defects (BMD) in axially elongated eyes. DesignHistomorphometric study. MethodsUsing light microscopy, we examined enucleated human globes for BMDs. ResultsIn 247 eyes, BMDs were detected in 15 (6.1%) eyes (axial length:27.0–36.0 mm), in 10 of them in the macular region. Prevalence and size of BMDs (mean:1.93 ± 1.62 mm; range:0.22mm–6.24 mm) correlated with longer axial length (OR:1.52; 95%CI:1.19,1.94; P = 0.001) and higher prevalence of scleral staphylomas (OR:16.3; 95%CI:2.67,99.3; P < 0.001). The BMDs were smaller than corresponding gaps in the retinal pigment epithelium (RPE) (1.93 ± 1.62 mm versus 2.61 mm ± 1.73 mm; P = 0.003), and larger than corresponding gaps in the inner nuclear layer (0.43 ± 0.76 mm; P = 0.008) and inner limiting membrane bridges (0.13 ± 0.33 mm; P = 0.001). Choriocapillaris thickness, BM thickness and RPE cell density did not vary (all P > 0.05) between the BDM border and adjacent areas. In the BMD, choriocapillaris and RPE were absent. The sclera was thinner in the BDM area than in adjacent areas (0.28 ± 0.19 mm versus 0.36 ± 0.13 mm; P = 0.006). ConclusionsBMDs as hallmarks of myopic macular degeneration are characterized by longer gaps in the RPE and smaller gaps in the outer nuclear layer and inner nuclear layer, by localized scleral thinning, and by a spatial association with scleral staphylomas. Thickness of the choriocapillaris and density of the RPE cell layer, both absent within the BDMs, do not vary between the BMD border and adjacent regions. The results suggest an association between BDMs and absolute scotomas, stretching of the adjacent retinal nerve fiver layer, and an axial elongation-associated stretching effect on BM as etiology of the BDMs.

Myopic macular Bruch's membrane defects

AbstractPurpose: To examine peculiarities of macular Bruch's membrane defects (BMD) in axially elongated eyes.Methods: Using light microscopy, enucleated human globes were histomorphometrically.Results: The study included 247 eyes (axial length: 20.0–37.0 mm). BMDs were detected in 15 (6.1%) eyes (axial length: 30.6 ± 2.4 mm) (1.9 ± 0.9 BMD/eye with BMDs), located in the macular region in 10 eyes. Mean BMD size was 1.93 ± 1.62 mm (range: 0.22 mm–6.24 mm). Prevalence and size of BMDs were associated with longer axial length (OR: 1.52; p = 0.001) and higher prevalence of scleral staphyloma (OR: 16.3; p &lt; 0.001). BMD width was smaller than the defect width in the overlying retinal pigment epithelium (RPE) layer (p = 0.003) and larger than the length of a defect in the overlying inner nuclear layer (0.43 ± 0.76 mm; p = 0.008) and the width of an inner limiting membrane bridge (0.13 ± 0.33 mm; p = 0.001). Choriocapillaris thickness (8.8 ± 5.0 μm vs. 7.9 ± 3.2 μm; p = 0.94), BM thickness (3.1 ± 0.6 μm versus 3.0 ± 0.5 μm; p = 0.43) and RPE cell density (31.6 ± 8.4 cells/480 μm vs. 27.4 ± 8.8 cells/480 μm; p = 0.051) did not differ significantly between the region at the BDM margin and neighbouring regions. The sclera was thinner in the BDM region than in the neighbouring regions (0.28 ± 0.19 mm vs. 0.36 ± 0.13 mm; p = 0.006).Conclusions: BMDs as hallmark of myopic macular degeneration are characterized by a larger defect in the overlying RPE layer, smaller defects in the overlying retinal outer nuclear layer and inner nuclear layer, localized scleral thinning and an association with scleral staphylomas. Thickness of the choriocapillaris and density of the RPE cell layer, both absent within the BDMs, do not differ between the BMD margin and neighbouring regions. The results suggest an association between BDMs and absolute scotomas, stretching of the overlying retinal nerve fiver layer, an axial elongation‐associated stretching effect as aetiology of the BDMs, and a biomechanical role BM may play.

Targeting the Complement Cascade for Treatment of Dry Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in the elderly population. AMD is characterized in its late form by neovascularization (wet type) or geographic atrophy of the retinal pigment epithelium cell layer (dry type). Regarding the latter type, there is growing evidence supporting an association between the pathophysiology of dry AMD and key proteins in the complement cascade. The complement cascade works as a central part of the innate immune system by defending against foreign pathogens and modified self-tissues. Through three distinct pathways, a series of plasma and membrane-associated serum proteins are activated upon identification of a foreign entity. Several of these proteins have been implicated in the development and progression of dry AMD. Potential therapeutic targets include C1q, C3, C5, complement factors (B, D, H, I), membrane attack complex, and properdin. In this review, we provide an understanding of the role of the complement system in dry AMD and discuss the emerging therapies in early phase clinical trials. The tentative hope is that these drugs may offer the potential to intervene at earlier stages in dry AMD pathogenesis, thereby preventing progression to late disease.

Histology of neovascular myopic macular degeneration

To assess the histological correlate of neovascular or exudative myopic macular degeneration (nMMD) in highly myopic human eyes, we examined histomorphometrically histologic sections of enucleated eyes of Caucasian patients. The study included 284 eyes (age: 61.9 ± 13.7 years; range: 24–89 years; axial length: 25.5 ± 3.1 mm; range: 20–37 mm). An nMMD was detected in 5 eyes (axial length: 29.6 ± 2.6 mm; range: 26.0–31.0 mm). All these eyes showed within or close to the nMMD a macular Bruch’s membrane (BM) defect, fibrous tissue with erythrocyte-filled blood vessels, and proliferations of irregularly pigmented and irregularly piled-up retinal pigment epithelium (RPE) cells each of which was connected with a curled-up, PAS (Periodic-Acid-Shiff)-positive membrane. The nMMD lesions were covered by proliferated RPE cells. RPE cells were not detected within the retina. In binary regression analysis, a higher nMMD prevalence was associated with a higher prevalence of macular BM defects (odds ratio: > 1000; P < 0.001), while the association with axial length was not significant (P = 0.43) in that model. After adjustment for the presence of macular BM defects, the nMMD prevalence was not associated with BM thickness (measured at the posterior pole, equator-posterior pole midpoint, equator and shortly posterior to the ora serrata) (P = 0.10; P = 0.87; P = 0.40; and P = 0.36, respectively), RPE cell layer thickness (P = 0.83; P = 0.79; P = 0.31; P = 0.38, resp.), RPE cell density (P = 0.56; P = 0.91; P = 0.47; P = 0.87, resp.), choriocapillaris thickness (P = 0.47; P = 0.93; P = 0.41; P = 0.75, resp.), and choriocapillaris density (P = 0.99; P = 0.94; P = 0.17; P = 0.97, resp.). The results suggest that nMMD is characterized by a fibrous pseudo-metaplasia of the RPE and is strongly associated with macular BM defects, without other detected histomorphometric differences in thickness or density of the RPE, BM, and choriocapillaris.

Complement System and Potential Therapeutics in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a complex multifactorial disease characterized in its late form by neovascularization (wet type) or geographic atrophy of the retinal pigment epithelium cell layer (dry type). The complement system is an intrinsic component of innate immunity. There has been growing evidence that the complement system plays an integral role in maintaining immune surveillance and homeostasis in AMD. Based on the association between the genotypes of complement variants and AMD occurrence and the presence of complement in drusen from AMD patients, the complement system has become a therapeutic target for AMD. However, the mechanism of complement disease propagation in AMD has not been fully understood. This concise review focuses on an overall understanding of the role of the complement system in AMD and its ongoing clinical trials. It provides further insights into a strategy for the treatment of AMD targeting the complement system.

Targeting of miR-33 ameliorates phenotypes linked to age-related macular degeneration

Abnormal cholesterol/lipid homeostasis is linked to neurodegenerative conditions such as age-related macular degeneration (AMD), which is a leading cause of blindness in the elderly. The most prevalent form, termed "dry" AMD, is characterized by pathological cholesterol accumulation beneath the retinal pigment epithelial (RPE) cell layer and inflammation-linked degeneration in the retina. We show here that the cholesterol-regulating microRNA miR-33 was elevated in the RPE of aging mice. Expression of the miR-33 target ATP-binding cassette transporter (ABCA1), a cholesterol efflux pump genetically linked to AMD, declined reciprocally in the RPE with age. In accord, miR-33 modulated ABCA1 expression and cholesterol efflux in human RPE cells. Subcutaneous delivery of miR-33 antisense oligonucleotides (ASO) to aging mice and non-human primates fed a Western-type high fat/cholesterol diet resulted in increased ABCA1 expression, decreased cholesterol accumulation, and reduced immune cell infiltration in the RPE cell layer, accompanied by decreased pathological changes to RPE morphology. These findings suggest that miR-33 targeting may decrease cholesterol deposition and ameliorate AMD initiation and progression.

HGF/Met Axis Protects Human Retinal Pigment Epithelial Cells from Cell Death

Demise of the retinal pigment epithelium (RPE) caused by etiologies such as age‐related macular degeneration is a major underlying factor leading to vision loss. Cell death can occur through two major pathways: apoptosis and necrosis/necroptosis. In the former, Fas/FasL death receptor signaling induces apoptosis, but its role in human RPE cell death is not thoroughly elucidated. In the latter, reactive oxygen species (ROS) can induce necrosis/necroptosis. Additionally, Hepatocyte Growth Factor (HGF) is a potent growth and survival factor for epithelial cells, but its functional role in RPE is not well characterized, especially its ability to protect RPE cells from death and to induce their growth. Accordingly, we hypothesized that HGF/Met protects human RPE cells from Fas‐promoted apoptosis and H2O2‐induced necrosis/necroptosis.We analyzed whether the HGF receptor (Met) is expressed in human and non‐human primate (NHP) eye tissue via western blot analysis and immunohistochemistry. To determine whether HGF/Met protects RPE cells against death induced by Fas/FasL and H2O2, we utilized gain‐ and loss‐of‐function models, cell survival assays and gene expression analysis (RNAseq). Human ARPE‐19 cell line was treated with various combinations of HGF, Met kinase inhibitor SU11274 (SU), FasL or H2O2. Cell growth and survival was determined via MTT assay. Met, Akt, and Erk activation were analyzed by western blot.Western blot analysis and immunohistochemistry confirmed Met activity and signaling in human and NHP eye tissue, including in the RPE cell layer. We found that HGF elicited Met stimulation and signaling culminating in activation of its downstream effectors Akt and Erk, proteins involved in two major pathways controlling cell survival and proliferation. We also uncovered that HGF inhibited FasL‐ and ROS‐induced killing of RPE. Conversely, inhibiting Met signaling rendered RPE susceptible to cell death induced by FasL and H2O2. Global gene expression analysis using an RNAseq approach revealed that HGF controls expression of numerous genes governing RPE cell growth, metabolism, and survival to name a few. In particular, HGF significantly upregulated 400 and repressed expression of 420 genes six hours post‐treatment; at 24 hours post‐treatment, HGF upregulated 291 and downregulated expression of 260 genes. Expression of 11,000 genes was unchanged.Our studies establish that HGF and Met play a major role in promoting the survival and homeostasis of RPE cells by inhibiting two major pathways of RPE cell death. Our findings suggest HGF/Met is a potential druggable target for therapeutic management of RPE‐related human ocular diseases.

MiR-194 suppresses epithelial-mesenchymal transition of retinal pigment epithelial cells by directly targeting ZEB1.

Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelial (RPE) cells is a critical step in the pathogenesis of proliferative vitreoretinopathy (PVR). Some microRNAs (miRNAs) participate in regulating RPE cell EMT as post-transcriptional regulators. However, the function of miR-194 in RPE cell EMT remains elusive. Here, the role of miR-194 in PVR was investigated. Retinal layers were obtained using laser capture microdissection (LCM). Gene expression at the mRNA and protein level in the tissues and cells was examined using quantitative reverse transcription (RT)-polymerase chain reaction and Western blotting, respectively. The related protein expression was observed by immunostaining. The effect of miR-194 on RPE cell EMT was examined by gel contraction, wound healing, and cell migration assays. RNAseq was performed in ARPE-19 with transfection of pSuper-scramble and pSuper-miR-194. The target gene of miR-194 was identified and confirmed via bioinformatics analysis and dual-luciferase reporter assay. ARPE-19 (adult retinal pigment epithelium-19) cells were treated with transforming growth factor (TGF)-β1 in the same fashion as the in vitro RPE cell EMT model. A PVR rat model was prepared by intravitreous injection of ARPE-19 cells with plasma-rich platelets. miR-194 was preferentially expressed in the RPE cell layer compared with the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer in rat retina. RNAseq analysis indicated that miR-194 overexpression was involved in RPE cell processes, including phagocytosis, ECM-receptor interaction, cell adhesion molecules, and focal adhesion. miR-194 overexpression significantly inhibited the TGF-β1-induced EMT phenotype of RPE cells in vitro. Zinc finger E-box binding homeobox 1 (ZEB1), a key transcription factor in EMT, was confirmed as the direct functional target of miR-194. Knockdown of ZEB1 attenuated TGF-β1-induced α-smooth muscle actin expression in ARPE-19 cells, and overexpression of miR-194 could significantly reduce the expression of some genes which were up-regulated by ZEB1. Exogenous miR-194 administration in vivo effectively suppressed PVR in the rat model, both functionally and structurally. Our findings demonstrate for the first time that miR-194 suppresses RPE cell EMT by functionally targeting ZEB1. The clinical application of miR-194 in patients with PVR merits further investigation.

RPE morphometry in geographic atrophy due to AMD

The geographic atrophy (GA) junctional zone includes changes in retinal pigment epithelium (RPE) and Bruch's membrane complex that are still difficult to interpret even with clinical high-resolution imaging technologies. We measured and evaluated degenerative RPE cell changes in histological sections of GA eyes. In this study seven GA eyes were evaluated by light microscopy. In three eye sections, zones of typical RPE alterations were graded (0= normal; 1= irregular cells but intact layer; 2= rounded, enlarged, and/or heaped cells; 3= migrating cells in retina; 4= RPE absent). In each graded zone, we measured and analyzed A)the total height of the RPE cell layer, B)the height of individual RPE cells and C)the height of basal deposits. From the outer macula towards the central RPE atrophic area the RPE passed almost steadily upward through grades of increased pathology. A)Zone2 exhibited highly variable total RPE height (16.9± 5.6 µm) with hypertrophic and heaped cells next to atrophic ones. In comparison, zone0 and zone1 showed less variability and aregular total RPE height (10.9± 2.6 µm). B)In zone2 the size of altered RPE cells varied widely (12.4± 5.2 µm, min.5.1, max. 27.5). All detected migrating RPE cells were hypertrophic. C)In zone0 basal deposits were found sporadically. With progressing RPE alterations, basal deposits became progressively continuous and thicker and reached aconsiderable height at the atrophic zone (9.5± 4.3 µm). Our measurements confirmed that degenerative RPE phenomena, particularly of degeneration grade2/3 close to the actual RPE atrophy zone, are often large enough to be visualized in detail with already available modern imaging technologies (e. g. SD-OCT).

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

Bone Marrow-Derived Cell Recruitment to the Neurosensory Retina and Retinal Pigment Epithelial Cell Layer Following Subthreshold Retinal Phototherapy.

PurposeWe investigated whether subthreshold retinal phototherapy (SRPT) was associated with recruitment of bone marrow (BM)–derived cells to the neurosensory retina (NSR) and RPE layer.MethodsGFP chimeric mice and wild-type (WT) mice were subjected to SRPT using a slit-lamp infrared laser. Duty cycles of 5%, 10%, 15%, and 20% (0.1 seconds, 250 mW, spot size 50 μm) with 30 applications were placed 50 to 100 μm from the optic disc. In adoptive transfer studies, GFP+ cells were given intravenously immediately after WT mice received SRPT. Immunohistochemistry was done for ionized calcium-binding adapter molecule-1 (IBA-1+), CD45, Griffonia simplicifolia lectin isolectin B4, GFP or cytokeratin). Expression of Ccl2, Il1b, Il6, Hspa1a, Hsp90aa1, Cryab, Hif1a, Cxcl12, and Cxcr4 mRNA and flow cytometry of the NSR and RPE-choroid were performed.ResultsWithin 12 to 24 hours of SRPT, monocytes were detected in the NSR and RPE-choroid. Detection of reparative progenitors in the RPE occurred at 2 weeks using flow cytometry. Recruitment of GFP+ cells to the RPE layer occurred in a duty cycle–dependent manner in chimeric mice and in mice undergoing adoptive transfer. Hspa1a, Hsp90aa1, and Cryab mRNAs increased in the NSR at 2 hours post laser; Hif1a, Cxcl12, Hspa1a increased at 4 hours in the RPE-choroid; and Ccl2, Il1b, Ifng, and Il6 increased at 12 to 24 hours in the RPE-choroid.ConclusionsSRPT induces monocyte recruitment to the RPE followed by hematopoietic progenitor cell homing at 2 weeks. Recruitment occurs in a duty cycle–dependent manner and potentially could contribute to the therapeutic efficacy of SRPT.

Quantitative study of zinc and metallothioneins in the human retina and RPE cells by mass spectrometry-based methodologies

The retina contains the highest concentration of zinc in the human eye and it is primarily associated with the retinal pigment epithelium (RPE). Metallothioneins (MTs) are the main cytosolic zinc-ion-binding proteins, exerting a tight control in the number of atoms of Zn-bound to the MTs related with their antioxidant and neuroprotective functions. In order to study the Zn-MT system in retina and RPE, we have implemented mass spectrometry (MS)-based technologies: two complementary element detection methodologies (HPLC- and laser ablation (LA)-ICP-MS) have been successfully employed to study metal content in the human eye as well as to perform speciation studies of Zn-MTs. First, Zn-elemental distribution was studied on cryogenic ocular sections by LA-ICP-MS. Quantitative images of Zn along RPE cell layer and the retina were obtained with a laser beam diameter of 25µm, showing a preferential distribution in the RPE. We carried out then the quantitative speciation of Zn, Fe, and Cu in the water-soluble protein fractions of RPE and retina to study their protein binding profile using HPLC-ICP-MS, where Zn is mainly associated to low molecular mass proteins (i.e., MTs). Finally, the effect of addition of different inductors, such as metal (i.e., 68ZnSO4), dexamethasone (DEX) and erythropoietin, was investigated in an in vitro cellular model of human RPE cells (HRPEsv), again using HPLC-ICP-MS in combination with stable isotopes and mathematical calculations based on isotope dilution and isotope pattern deconvolution. Exogenous Zn and DEX were found to increase MT proteins synthesis and exerted a stoichiometric transition in MT proteins in HRPEsv cells.

Targeting the cAMP and Transforming Growth Factor-β Pathway Increases Proliferation to Promote Re-Epithelialization of Human Stem Cell-Derived Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) cell integrity is critical to the maintenance of retinal function. Many retinopathies such as age-related macular degeneration (AMD) are caused by the degeneration or malfunction of the RPE cell layer. Replacement of diseased RPE with healthy, stem cell-derived RPE is a potential therapeutic strategy for treating AMD. Human embryonic stem cells (hESCs) differentiated into RPE progeny have the potential to provide an unlimited supply of cells for transplantation, but challenges around scalability and efficiency of the differentiation process still remain. Using hESC-derived RPE as a cellular model, we sought to understand mechanisms that could be modulated to increase RPE yield after differentiation. We show that RPE epithelialization is a density-dependent process, and cells seeded at low density fail to epithelialize. We demonstrate that activation of the cAMP pathway increases proliferation of dissociated RPE in culture, in part through inhibition of transforming growth factor-β (TGF-β) signaling. This results in enhanced uptake of epithelial identity, even in cultures seeded at low density. In line with these findings, targeted manipulation of the TGF-β pathway with small molecules produces an increase in efficiency of RPE re-epithelialization. Taken together, these data highlight mechanisms that promote epithelial fate acquisition in stem cell-derived RPE. Modulation of these pathways has the potential to favorably impact scalability and clinical translation of hESC-derived RPE as a cell therapy. Stem cell-derived retinal pigment epithelium (RPE) is currently being evaluated as a cell-replacement therapy for macular degeneration. This work shows that the process of generating RPE in vitro is regulated by the cAMP and transforming growth factor-β signaling pathway. Modulation of these pathways by small molecules, as identified by phenotypic screening, leads to an increased efficiency of generating RPE cells with a higher yield. This can have a potential impact on manufacturing transplantation-ready cells at large scale and is advantageous for clinical studies using this approach in the future.

Green Tea Polyphenol Epigallocatechin-3-Gallate Attenuates TNF-α-Induced Intercellular Adhesion Molecule-1 Expression and Monocyte Adhesion to Retinal Pigment Epithelial Cells

Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea (Camellia sinensis) and demonstrates anti-oxidant, anticancer and anti-inflammatory properties. EGCG has been shown to protect retinal pigment epithelium (RPE) against oxidative stress-induced cell death. The pathogenesis of diseases in the retina is usually initiated by local inflammation at the RPE cell layer, and inflammation is mostly associated with leukocyte migration and the secretion of pro-inflammatory cytokines. Whether EGCG can modulate the cytokine-induced inflammatory response of RPE, particularly leukocyte migration, has not been clearly elucidated, and was therefore the objective of this study. ARPE-19 cells were cultured with different concentrations of TNF-α in the presence or absence of EGCG to different time points. Intracellular reactive oxygen species (ROS) levels were determined. Intercellular adhesion molecule (ICAM)-1 and phosphor-NF-κB and IκB expression were determined by Western blot analysis. Phosphor-NF-κB nuclear translocation and monocyte-RPE adhesion were investigated using immunofluorescence confocal laser scanning microscopy. Scanning electron microscopy (SEM) was carried out to further determine the ultrastructure of monocyte-RPE adhesion. The results demonstrated that TNF-α modulated inflammatory effects in ARPE-19 by induction of ROS and up-regulation of ICAM-1 expression. Moreover, TNF-α-induced phosphor-NF-κB nuclear translocation, increased phosphor-NF-κB expression and IκB degradation, and increased the degree of monocyte-RPE adhesion. Pretreating the cells with EGCG ameliorated the inflammatory effects of TNF-α. The results indicated that EGCG significantly exerts anti-inflammatory effects in ARPE-19 cells, partly as a suppressor of TNF-α signaling and that the inhibition was mediated via the NF-κB pathway.

Senescent retinal pigment epithelial cells are more sensitive to vascular endothelial growth factor: implications for "wet" age-related macular degeneration.

Senescence of the retinal pigment epithelial (RPE) cell layer has been implicated in the occurrence of age-related macular degeneration (AMD). The present study examines whether the ability of vascular endothelial growth factor (VEGF) to decrease the barrier function of RPE cells is enhanced in senescent RPE cells, which could contribute to the pathology of "wet" AMD. Low or high population doubling level (PDL) range ARPE-19 human RPE cells were cultured in 6-well plates on membrane-containing inserts. After 2 weeks, the cells were treated with either VEGF or its vehicle and their transepithelial electrical resistance (TEER) was measured. One week later, the cells were stained for senescence-associated β-galactosidase (SABG) activity. VEGF was significantly more effective in reducing the TEER of the high PDL ARPE-19 cell layers than the low PDL layers (25% decrease vs. 6% decrease; t-test, P=0.0013). The low PDL cell layers had a modest uniform level of SABG staining. In contrast, the high PDL layers displayed darker and more mottled SABG staining indicative of the presence of senescent cells. The present results show that the ability of VEGF to reduce the barrier function of RPE cell layers is greater in high PDL layers, which display signs of senescence, than in low PDL layers. Senescence-induced changes in the responsiveness of RPE cell layers to VEGF could contribute to the pathology of AMD. Agents that strengthen the barrier properties of RPE cells or reduce their responsiveness to VEGF could be effective in treating "wet" AMD.

Enhancing RPE Cell-Based Therapy Outcomes for AMD: The Role of Bruch's Membrane.

Age-related macular degeneration (AMD) is the leading cause of legal blindness in older people in the developed world. The disease involves damage to the part of the retina responsible for central vision. Degeneration of retinal pigment epithelial (RPE) cells, photoreceptors, and choriocapillaris may contribute to visual loss. Over the past decades, scientists and clinicians have tried to replace lost RPE cells in patients with AMD using cells from different sources. In recent years, advances in generating RPE cells from stem cells have been made and clinical trials are currently evaluating the safety and efficiency of replacing the degenerated RPE cell layer with stem cell-derived RPE cells. However, the therapeutic success of transplantation of stem cell-derived RPE cells may be limited unless the transplanted cells can adhere and survive in the long term in the diseased eye. One hallmark of AMD is the altered extracellular environment of Bruch's membrane to which the grafted cells have to adhere. Here, we discuss recent approaches to overcome the inhibitory environment of the diseased eye and to enhance the survival rate of transplanted RPE cells. Our aim is to highlight novel approaches that may have the potential to improve the efficacy of RPE transplantation for AMD in the future.

THE VALUE OF RETINAL IMAGING WITH INFRARED SCANNING LASER OPHTHALMOSCOPY IN PATIENTS WITH STARGARDT DISEASE

To demonstrate the value of infrared scanning laser ophthalmoscopy (SLO) for determining structural retinal and choroidal changes in patients with Stargardt disease and its comparison to findings on short-wavelength fundus autofluorescence (SW-AF) imaging, spectral-domain optical coherence tomography, and microperimetry measurements. Forty-four eyes of 22 patients with Stargardt disease were studied using infrared-SLO, spectral-domain optical coherence tomography, macular microperimetry, SW-AF, electroretinography, and fundus photography. Although SW-AF imaging outlined the regions of retinal pigment epithelial (RPE) atrophy (hypofluorescence) and enhanced the visibility of more funduscopically apparent flecks (hyperfluorescence), infrared-SLO imaging outlined the regions of choroidal, and RPE, atrophic changes. Degenerative changes in photoreceptor and RPE cell layers, evident on spectral-domain optical coherence tomography imaging, were associated with either hyporeflective or hyperreflective images on infrared-SLO imaging, depending on whether both RPE and choroidal atrophy (hyperreflectance) or solely RPE atrophy (hyporeflectance) was present. Threshold elevations on microperimetry testing corresponded to both RPE and choroidal atrophy on infrared-SLO imaging and RPE atrophy on SW-AF. Although SW-AF identifies regions of RPE atrophy, infrared-SLO also identifies the involvement of the choroid that has important implications for the potential improvement in visual function from treatment. Thus, infrared-SLO imaging offers an additional advantage beyond that obtained with SW-AF.