scholarly journals Drug Flux Across RPE Cell Models: The Hunt for An Appropriate Outer Blood–Retinal Barrier Model for Use in Early Drug Discovery

Pharmaceutics ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 176 ◽  
Author(s):  
Laura Hellinen ◽  
Heidi Hongisto ◽  
Eva Ramsay ◽  
Kai Kaarniranta ◽  
Kati-Sisko Vellonen ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Retinal Pigment ◽  
Cell Monolayer ◽  
Barrier Properties ◽  
Cell Models ◽  
Blood Retinal Barrier ◽  
Rpe Cells ◽  
Cell Monolayers ◽  
Value Range ◽  
Drug Flux

The retinal pigment epithelial (RPE) cell monolayer forms the outer blood–retinal barrier and has a crucial role in ocular pharmacokinetics. Although several RPE cell models are available, there have been no systematic comparisons of their barrier properties with respect to drug permeability. We compared the barrier properties of several RPE secondary cell lines (ARPE19, ARPE19mel, and LEPI) and both primary (hfRPE) and stem-cell derived RPE (hESC-RPE) cells by investigating the permeability of nine drugs (aztreonam, ciprofloxacin, dexamethasone, fluconazole, ganciclovir, ketorolac, methotrexate, voriconazole, and quinidine) across cell monolayers. ARPE19, ARPE19mel, and hfRPE cells displayed a narrow Papp value range, with relatively high permeation rates (5.2–26 × 10−6 cm/s. In contrast, hESC-RPE and LEPI cells efficiently restricted the drug flux, and displayed even lower Papp values than those reported for bovine RPE-choroid, with the range of 0.4–32 cm−6/s (hESC-RPE cells) and 0.4–29 × 10−6 cm/s, (LEPI cells). Therefore, ARPE19, ARPE19mel, and hfRPE cells failed to form a tight barrier, whereas hESC-RPE and LEPI cells restricted the drug flux to a similar extent as bovine RPE-choroid. Therefore, LEPI and hESC-RPE cells are valuable tools in ocular drug discovery.

scholarly journals Oxidative stress-mediated TXNIP loss causes RPE dysfunction

2019 ◽  
Vol 51 (10) ◽  
pp. 1-13 ◽  
Author(s):  
Min Ji Cho ◽  
Sung-Jin Yoon ◽  
Wooil Kim ◽  
Jongjin Park ◽  
Jangwook Lee ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Function ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Common Factor ◽  
Age Related Macular Degeneration ◽  
Autophagic Flux ◽  
Blood Retinal Barrier ◽  
Rpe Cells ◽  
Age Related

Abstract The disruption of the retinal pigment epithelium (RPE), for example, through oxidative damage, is a common factor underlying age-related macular degeneration (AMD). Aberrant autophagy also contributes to AMD pathology, as autophagy maintains RPE homeostasis to ensure blood–retinal barrier (BRB) integrity and protect photoreceptors. Thioredoxin-interacting protein (TXNIP) promotes cellular oxidative stress by inhibiting thioredoxin reducing capacity and is in turn inversely regulated by reactive oxygen species levels; however, its role in oxidative stress-induced RPE cell dysfunction and the mechanistic link between TXNIP and autophagy are largely unknown. Here, we observed that TXNIP expression was rapidly downregulated in RPE cells under oxidative stress and that RPE cell proliferation was decreased. TXNIP knockdown demonstrated that the suppression of proliferation resulted from TXNIP depletion-induced autophagic flux, causing increased p53 activation via nuclear localization, which in turn enhanced AMPK phosphorylation and activation. Moreover, TXNIP downregulation further negatively impacted BRB integrity by disrupting RPE cell tight junctions and enhancing cell motility by phosphorylating, and thereby activating, Src kinase. Finally, we also revealed that TXNIP knockdown upregulated HIF-1α, leading to the enhanced secretion of VEGF from RPE cells and the stimulation of angiogenesis in cocultured human retinal microvascular endothelial cells. This suggests that the exposure of RPE cells to sustained oxidative stress may promote choroidal neovascularization, another AMD pathology. Together, these findings reveal three distinct mechanisms by which TXNIP downregulation disrupts RPE cell function and thereby exacerbates AMD pathogenesis. Accordingly, reinforcing or restoring BRB integrity by targeting TXNIP may serve as an effective therapeutic strategy for preventing or attenuating photoreceptor damage in AMD.

scholarly journals Avoiding the Pitfalls of siRNA Delivery to the Retinal Pigment Epithelium with Physiologically Relevant Cell Models

Pharmaceutics ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 667
Author(s):  
Eva Ramsay ◽  
Manuela Raviña ◽  
Sanjay Sarkhel ◽  
Sarah Hehir ◽  
Neil R. Cameron ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Cell Model ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Sirna Delivery ◽  
Target Cells ◽  
Cell Models ◽  
Rpe Cells ◽  
Age Related

Inflammation is involved in the pathogenesis of several age-related ocular diseases, such as macular degeneration (AMD), diabetic retinopathy, and glaucoma. The delivery of anti-inflammatory siRNA to the retinal pigment epithelium (RPE) may become a promising therapeutic option for the treatment of inflammation, if the efficient delivery of siRNA to target cells is accomplished. Unfortunately, so far, the siRNA delivery system selection performed in dividing RPE cells in vitro has been a poor predictor of the in vivo efficacy. Our study evaluates the silencing efficiency of polyplexes, lipoplexes, and lipidoid-siRNA complexes in dividing RPE cells as well as in physiologically relevant RPE cell models. We find that RPE cell differentiation alters their endocytic activity and causes a decrease in the uptake of siRNA complexes. In addition, we determine that melanosomal sequestration is another significant and previously unexplored barrier to gene silencing in pigmented cells. In summary, this study highlights the importance of choosing a physiologically relevant RPE cell model for the selection of siRNA delivery systems. Such cell models are expected to enable the identification of carriers with a high probability of success in vivo, and thus propel the development of siRNA therapeutics for ocular disease.

scholarly journals Ultrastructural localization of blood-retinal barrier breakdown in diabetic and galactosemic rats.

1990 ◽  
Vol 38 (9) ◽  
pp. 1341-1352 ◽  
Author(s):  
S A Vinores ◽  
R McGehee ◽  
A Lee ◽  
C Gadegbeku ◽  
P A Campochiaro
Keyword(s):  
Tight Junctions ◽  
Retinal Pigment ◽  
Cell Junctions ◽  
Capillary Endothelium ◽  
Intercellular Spaces ◽  
Blood Retinal Barrier ◽  
Cytoplasmic Staining ◽  
Rpe Cells ◽  
Perinuclear Cytoplasm ◽  
Brb Breakdown

Breakdown of the blood-retinal barrier (BRB) is an early event in diabetic and galactosemic rats, but the location and nature of the specific defect(s) are controversial. Using an electron microscopic immunocytochemical technique, the retinas of normal, diabetic, and galactosemic rats were immunostained for endogenous albumin. Normal rats showed little evidence of BRB breakdown at either the inner barrier (retinal vasculature) or the outer barrier (retinal pigment epithelium) (RPE). In diabetic and galactosemic rats, as was true in human diabetics, BRB breakdown occurred predominantly at the inner BRB, but in some cases at the outer barrier as well. Treatment with the aldose reductase inhibitor sorbinil largely prevented BRB failure in galactosemic rats. In the inner retina of diabetic and galactosemic rats, albumin was frequently demonstrated on the abluminal side of the retinal capillary endothelium (RCE) in intercellular spaces, basal laminae, pericytes, ganglion cells, astrocytes, and the perinuclear cytoplasm of cells in the inner nuclear layer. Albumin did not appear to cross RCE cell junctions; however, it was occasionally seen in RCE cytoplasm of galactosemic rats. In the outer retina, albumin was frequently detected in the subretinal space, in the intercellular space between photoreceptors, and in the perinuclear cytoplasm of photoreceptor cells, but was only infrequently found in the RPE cells constituting the barrier. Albumin derived from the choroidal vasculature did not appear to cross the tight junctions of the RPE. These findings suggest that specific sites of BRB compromise are infrequent but that once albumin has crossed the RCE or RPE it freely permeates the retinal tissue by filling intercellular spaces and permeating the membranes of cells not implicated in BRB formation. The diffuse cytoplasmic staining of some RCE and RPE cells suggests that the predominant means of BRB breakdown in diabetes and galactosemia involves increased focal permeability of the surface membranes of the RCE and RPE cells rather than defective tight junctions or vesicular transport.

scholarly journals Inhibiting TLR7 Expression in the Retinal Pigment Epithelium Suppresses Experimental Autoimmune Uveitis

2022 ◽  
Vol 12 ◽  
Author(s):  
Sheng-Min Lo ◽  
Yih-Shiou Hwang ◽  
Chao-Lin Liu ◽  
Chia-Ning Shen ◽  
Wei-Hsin Hong ◽  
...  
Keyword(s):  
T Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Experimental Autoimmune Uveitis ◽  
Blood Retinal Barrier ◽  
Autoreactive T Cells ◽  
Autoimmune Uveitis ◽  
Rpe Cells ◽  
Physical Defense ◽  
Experimental Autoimmune

Experimental autoimmune uveitis (EAU), a model of human uveitis, is an organ-specific, T cell-mediated autoimmune disease. Autoreactive T cells can penetrate the blood-retinal barrier, which is a physical defense composed of tight junction-linked retinal pigment epithelial (RPE) cells. RPE cells serve as antigen-presenting cells (APCs) in the eye since they express MHC class I and II and Toll-like receptors (TLRs). Although previous studies have shown that supplementation with TLR agonists exacerbates uveitis, little is known about how TLR signaling in the RPE contributes to the development of uveitis. In this study, we isolated the RPE from EAU mice, which were induced by active immunization (aEAU) or adoptive transfer of antigen-specific T cells (tEAU). The expression of TLRs on RPE was determined, and both aEAU and tEAU mice exhibited induced tlr7 expression. The TLR7 agonist R848 was shown to induce aggressive disease progression, along with significantly elevated levels of the uveopathogenic cytokine IL-17. Furthermore, not only IL-17 but also R848 appeared to enhance the inflammatory response and to impair the barrier function of the RPE, indicating that TLR7 signaling is involved in the pathogenesis of EAU by affecting the behaviors of the RPE and consequently allowing the infiltration of autoreactive T cells intraocularly. Finally, local application of shRNA against TLR7 delivered by recombinant AAV effectively inhibited disease severity and reduced IFN-γ and IL-17. Our findings highlight an immunomodulatory role of RPE TLR7 in EAU development and provide a potential therapeutic strategy for autoimmune uveitis.

scholarly journals Eucalyptol Inhibits Amyloid-β-Induced Barrier Dysfunction in Glucose-Exposed Retinal Pigment Epithelial Cells and Diabetic Eyes

Antioxidants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1000
Author(s):  
Dong Yeon Kim ◽  
Min-Kyung Kang ◽  
Eun-Jung Lee ◽  
Yun-Ho Kim ◽  
Hyeongjoo Oh ◽  
...  
Keyword(s):  
Tight Junction ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Amyloid Β ◽  
Diabetic Mouse ◽  
Retinal Pigment Epithelial Cells ◽  
Barrier Dysfunction ◽  
Blood Retinal Barrier ◽  
Rpe Cells ◽  
Pigment Epithelial

Hyperglycemia elicits tight junction disruption and blood-retinal barrier breakdown, resulting in diabetes-associated vison loss. Eucalyptol is a natural compound found in eucalyptus oil with diverse bioactivities. This study evaluated that eucalyptol ameliorated tight junctions and retinal barrier function in glucose/amyloid-β (Aβ)-exposed human retinal pigment epithelial (RPE) cells and in db/db mouse eyes. RPE cells were cultured in media containing 33 mM glucose or 5 μM Aβ for 4 days in the presence of 1–20 μM eucalyptol. The in vivo animal study employed db/db mice orally administrated with 10 mg/kg eucalyptol. Nontoxic eucalyptol inhibited the Aβ induction in glucose-loaded RPE cells and diabetic mouse eyes. Eucalyptol reversed the induction of tight junction-associated proteins of ZO-1, occludin-1 and matrix metalloproteinases in glucose- or Aβ-exposed RPE cells and in diabetic eyes, accompanying inhibition of RPE detachment from Bruch’s membrane. Adding eucalyptol to glucose- or Aβ-loaded RPE cells, and diabetic mouse eyes reciprocally reversed induction/activation of apoptosis-related bcl-2, bax, cytochrome C/Apaf-1 and caspases. Eucalyptol attenuated the generation of reactive oxygen species and the induction of receptor for advanced glycation end products in Aβ-exposed RPE cells and diabetic eyes. Eucalyptol may ameliorate RPE barrier dysfunction in diabetic eyes through counteracting Aβ-mediated oxidative stress-induced RPE cell apoptosis.

Improved Trans-endothelial Electrical Resistance Sensing using Microfluidic Low-Temperature Co-fired Ceramics

2013 ◽  
Vol 2013 (CICMT) ◽  
pp. 000162-000167
Author(s):  
William L. Mercke ◽  
Thomas Dziubla ◽  
Richard E. Eitel ◽  
Kimberly Anderson
Keyword(s):  
Endothelial Cell ◽  
Low Temperature ◽  
Electrical Resistance ◽  
Cell Monolayer ◽  
Three Dimensional ◽  
Barrier Properties ◽  
Ceramic Materials ◽  
Functional Change ◽  
Cell Monolayers ◽  
Lock In

Trans-endothelial Electrical Resistance (TEER) and cellular impedance measurements are widely used to evaluate the barrier properties and functional change of endothelial cell monolayers. In the current work, low temperature cofired ceramics (LTCC) are applied enabling the incorporation of TEER and impediametric measurements in an integrated microfluidic chip. LTCC materials are an ideal substrate for biomedical and cell-based microfluidics due to their biocompatibility and ability to combine complex three dimensional structures with optical, fluidic, and electrical functionality. Multilayer microfluidic ceramic devices incorporating gold measurement electrodes where prepared using standard LTCC manufacturing procedures. The sensitivity of the resulting LTCC devices were compared to systems currently on the market for TEER measurements. These results indicate the LTCC device is able to effectively detect the growth of an endothelial cell monolayer. Results further evaluate endothelial cell viability using electrical resistance and Live/Dead assay. Finally, the results from this study also display improved sensitivity through the optimization of the electrode geometry and use of a lock-in amplifier. These results provide a solid basis for using low temperature co-fired ceramic materials for microfluidic TEER devices.

scholarly journals High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression

2016 ◽  
Vol 311 (3) ◽  
pp. C418-C436 ◽  
Author(s):  
Mitra Farnoodian ◽  
Caroline Halbach ◽  
Cassidy Slinger ◽  
Bikash R. Pattnaik ◽  
Christine M. Sorenson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
High Glucose ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Blood Retinal Barrier ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Proliferation And Apoptosis ◽  
The Impact

Defects in the outer blood-retinal barrier have significant impact on the pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin. We recently reported a method for the culture of retinal pigment epithelial (RPE) cells from wild-type and transgenic mice. The RPE cells are responsible for maintaining the integrity of the outer blood-retinal barrier whose dysfunction during diabetes has a significant impact on vision. Here we determined the impact of high glucose on the function of RPE cells. We showed that high glucose conditions resulted in enhanced migration and increased the level of oxidative stress in RPE cells, but minimally impacted their rate of proliferation and apoptosis. High glucose also minimally affected the cell-matrix and cell-cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including pigment epithelium-derived factor (PEDF) were altered under high glucose conditions. Incubation of RPE cells with the antioxidant N-acetylcysteine under high glucose conditions restored normal migration and PEDF expression. These cells also exhibited increased nuclear localization of the antioxidant transcription factor Nrf2 and ZO-1, reduced levels of β-catenin and phagocytic activity, and minimal effect on production of vascular endothelial growth factor, inflammatory cytokines, and Akt, MAPK, and Src signaling pathways. Thus high glucose conditions promote RPE cell migration through increased oxidative stress and expression of PEDF without a significant effect on the rate of proliferation and apoptosis.

Processing Immunoperoxidase Labeled Cell Monolayers and Cryostat Sesctions For Electron Microscopy

1985 ◽  
Vol 43 ◽  
pp. 714-715
Author(s):  
K. Chien ◽  
R. Van de Velde ◽  
I.P. Shintaku ◽  
A.F. Sassoon
Keyword(s):  
Cell Monolayer ◽  
Cell Types ◽  
Surface Antigens ◽  
Immunoperoxidase Method ◽  
Reaction Step ◽  
Hot Plate ◽  
Air Drying ◽  
New Approach ◽  
Cell Monolayers ◽  
Glass Slides

Immunoelectron microscopy of neoplastic lymphoma cells is valuable for precise localization of surface antigens and identification of cell types. We have developed a new approach in which the immunohistochemical staining can be evaluated prior to embedding for EM and desired area subsequently selected for ultrathin sectioning.A freshly prepared lymphoma cell suspension is spun onto polylysine hydrobromide- coated glass slides by cytocentrifugation and immediately fixed without air drying in polylysine paraformaldehyde (PLP) fixative. After rinsing in PBS, slides are stained by a 3-step immunoperoxidase method. Cell monolayer is then fixed in buffered 3% glutaraldehyde prior to DAB reaction. After the DAB reaction step, wet monolayers can be examined under LM for presence of brown reaction product and selected monolayers then processed by routine methods for EM and embedded with the Chien Re-embedding Mold. After the polymerization, the epoxy blocks are easily separated from the glass slides by heatingon a 100°C hot plate for 20 seconds.

scholarly journals A human model of Batten disease shows role of CLN3 in phagocytosis at the photoreceptor–RPE interface

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Cynthia Tang ◽  
Jimin Han ◽  
Sonal Dalvi ◽  
Kannan Manian ◽  
Lauren Winschel ◽  
...  
Keyword(s):  
Photoreceptor Cell ◽  
Vision Loss ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cell Loss ◽  
Human Model ◽  
Lysosomal Storage ◽  
Rpe Cells ◽  
Cln3 Disease

AbstractMutations in CLN3 lead to photoreceptor cell loss in CLN3 disease, a lysosomal storage disorder characterized by childhood-onset vision loss, neurological impairment, and premature death. However, how CLN3 mutations cause photoreceptor cell death is not known. Here, we show that CLN3 is required for phagocytosis of photoreceptor outer segment (POS) by retinal pigment epithelium (RPE) cells, a cellular process essential for photoreceptor survival. Specifically, a proportion of CLN3 in human, mouse, and iPSC-RPE cells localized to RPE microvilli, the site of POS phagocytosis. Furthermore, patient-derived CLN3 disease iPSC-RPE cells showed decreased RPE microvilli density and reduced POS binding and ingestion. Notably, POS phagocytosis defect in CLN3 disease iPSC-RPE cells could be rescued by wild-type CLN3 gene supplementation. Altogether, these results illustrate a novel role of CLN3 in regulating POS phagocytosis and suggest a contribution of primary RPE dysfunction for photoreceptor cell loss in CLN3 disease that can be targeted by gene therapy.

scholarly journals FHL-1 interacts with human RPE cells through the α5β1 integrin and confers protection against oxidative stress

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rawshan Choudhury ◽  
Nadhim Bayatti ◽  
Richard Scharff ◽  
Ewa Szula ◽  
Viranga Tilakaratna ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Fate ◽  
Retinal Pigment ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Bruch's Membrane ◽  
Bruch’S Membrane ◽  
Rpe Cells ◽  
Age Related

AbstractRetinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch’s membrane, are essential for RPE cell health and function, but the signals induced by Bruch’s membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch’s membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and β1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

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