Rat Retinal Pigment Epithelial Cells Research Articles

Retinal pigment epithelial cell culture

Objective: Retinal pigment epithelial cells (RPEs), as the main cellular component of the preretinal membrane, whose excessive proliferation can lead to the development of proliferative vitreoretinopathy as interaction with other cellular components and intercellular substance. Here, we developed an effective method to culture RPEs so as to provide effective cell source for later experimental usage. Methods: The harvested of eye cup that retains the retinal pigment epithelium was gently washed three times with phosphate buffered saline (PBS), and was added with 0.02 mg/ml dispase enzyme, which was cut into small pieces and then placed in the incubator for digestion for about half an hour. Subsequently, gently blowing was performed multiple times to detach the RPEs from the retina to collect the RPEs suspension. After implantation into the culture dish, when the cells were grown and covered with 80-90% of the bottom of the bottle, digestion of the cells is carried out with 0.25% trypsin. After removing them from the wall, the digested cell suspension was collected, followed by addition of Dulbecco's modified Eagle medium/Nutrient Mixture F-12 (DMEM/F12) to stop digestion. The mixture was subject to centrifuge at 1000 r/min for 8 min, with the supernatant discarded, and then the cells located in bottom were passaged at 1:3 ratios. During the process, the morphology and viability were observed and cell identification were also performed. Results: Primary cultured cells began to adhere to the wall at 4 h, characterized with a flat and polygonal morphology, rich in pigment particles. On 3 days, there was a significant increase in cell numbers accompanied by cluster formation, resulting in a stabilized cobblestone-like appearance. By 5 days, cells elongated and formed dense clusters. Growth curve analysis revealed robust proliferation at 3 and 5 days. Immunofluorescence staining confirmed that over 90% of the cells were positive for RPE-65, indicating successful isolation and culture of pure rat RPEs. Conclusions: The RPEs cultured by the above method are in good growth and it can be used for further experimental studies.

Characterization of LysoTracker Red uptake by in vitro model cells of the outer blood-retinal barrier: Implication of lysosomal trapping with cytoplasmic vacuolation and cytotoxicity

Lysosomal trapping, a physicochemical process in which lipophilic cationic compounds are sequestered in lysosomes, can affect drug disposition and cytotoxicity. To better understand lysosomal trapping at the outer blood-retinal barrier (BRB), we investigated the distribution of LysoTracker Red (LTR), a probe compound for lysosomal trapping, in conditionally immortalized rat retinal pigment epithelial (RPE-J) cells. LTR uptake by RPE-J cells was dependent on temperature and attenuated by ammonium chloride and protonophore, which decreased the pH gradient between the lysosome and cytoplasm, suggesting lysosomal trapping of LTR in RPE-J cells. The involvement of lysosomal trapping in response to cationic drugs, including neuroprotectants such as desipramine and memantine, was also suggested by an inhibition study of LTR uptake. Chloroquine, which is known to show ocular toxicity, induced cytoplasmic vacuolization in RPE-J cells with a half-maximal effective concentration of 1.35 μM. This value was 59 times lower than the median lethal concentration (= 79.1 μM) of chloroquine, suggesting that vacuolization was not a direct trigger of cell death. These results are helpful for understanding the lysosomal trapping of cationic drugs, which is associated with drug disposition and cytotoxicity in the outer BRB.

A Multifunctional Hybrid Nanocarrier for Non-Invasive siRNA Delivery to the Retina.

Drug therapy for retinal diseases (e.g., age-related macular degeneration, the leading cause of blindness) is generally performed by invasive intravitreal injection because of poor drug delivery caused by the blood-retinal barrier (BRB). This study aimed to develop a nanocarrier for the non-invasive delivery of small interfering RNA (siRNA) to the posterior segment of the eye (i.e., the retina) by eyedrops. To this end, we prepared a hybrid nanocarrier based on a multifunctional peptide and liposomes, and the composition was optimized. A cytoplasm-responsive stearylated peptide (STR-CH2R4H2C) was used as the multifunctional peptide because of its superior ability to enhance the complexation, cell permeation, and intracellular dynamics of siRNA. By adding STR-CH2R4H2C to the surface of liposomes, intracellular uptake increased regardless of the liposome surface charge. The STR-CH2R4H2C-modified cationic nanocarrier demonstrated significant siRNA transfection efficiency with no cytotoxicity, enhanced siRNA release from endosomes, and effectively suppressed vascular endothelial growth factor expression in rat retinal pigment epithelium cells. The 2.0 mol% STR-CH2R4H2C-modified cationic nanocarrier enhanced intraocular migration into the retina after instillation into rat eyes.

Synthetic torpor protects rats from exposure to accelerated heavy ions

Hibernation or torpor is considered a possible tool to protect astronauts from the deleterious effects of space radiation that contains high-energy heavy ions. We induced synthetic torpor in rats by injecting adenosine 5′-monophosphate monohydrate (5′-AMP) i.p. and maintaining in low ambient temperature room (+ 16 °C) for 6 h immediately after total body irradiation (TBI) with accelerated carbon ions (C-ions). The 5′-AMP treatment in combination with low ambient temperature reduced skin temperature and increased survival following 8 Gy C-ion irradiation compared to saline-injected animals. Analysis of the histology of the brain, liver and lungs showed that 5′-AMP treatment following 2 Gy TBI reduced activated microglia, Iba1 positive cells in the brain, apoptotic cells in the liver, and damage to the lungs, suggesting that synthetic torpor spares tissues from energetic ion radiation. The application of 5′-AMP in combination with either hypoxia or low temperature environment for six hours following irradiation of rat retinal pigment epithelial cells delays DNA repair and suppresses the radiation-induced mitotic catastrophe compared to control cells. We conclude that synthetic torpor protects animals from cosmic ray-simulated radiation and the mechanism involves both hypothermia and hypoxia.

Hypoxic and osmotic expression of Kir2.1 potassium channels in retinal pigment epithelial cells: Contribution to vascular endothelial growth factor expression

Retinal pigment epithelial (RPE) cells express different subtypes of inwardly rectifying potassium (Kir) channels. We investigated whether human and rat RPE cells express genes of strongly rectifying Kir2 channels. We also determined the hypoxic and hyperosmotic regulation of Kir2.1 gene expression in cultured human RPE cells and the effects of siRNA-mediated knockdown of Kir2.1 on VEGFA expression, VEGF secretion, proliferation, and viability of the cells. Extracellular hyperosmolarity was induced by addition of NaCl or sucrose. Hypoxia and chemical hypoxia were produced by cell culture in 0.25% O2 and addition of CoCl2, respectively. Gene expression levels were evaluated by real-time RT-PCR. Rat RPE cells contained Kir2.1, Kir2.2, Kir2.3, and Kir2.4 gene transcripts while human RPE cells contained Kir2.1, Kir2.2, and Kir2.4 transcripts. Immunocytochemical data may suggest that Kir2.1 protein in cultured human cells is expressed in both perinuclear and plasma membranes. Kir2.1 gene expression and Kir2.1 protein level in human cells increased under hypoxic and hyperosmotic conditions. The expression of the Kir2.1 gene was mediated in part by diverse intracellular signal transduction pathways and transcription factor activities under both conditions; the hyperosmotic, but not the CoCl2-induced Kir2.1 gene expression was dependent on intracellular calcium signaling. Autocrine/paracrine activation of purinergic receptors contributed to Kir2.1 gene expression under hyperosmotic (P2Y1, P2Y2, P2X7) and CoCl2-induced conditions (P2Y2, P2X7). Exogenous VEGF, TGF-β1, and blood serum decreased Kir2.1 gene expression. Inhibition of VEGF receptor-2 increased the Kir2.1 gene expression under control conditions and in CoCl2-simulated hypoxia, and decreased it under high NaCl conditions. Knockdown of Kir2.1 by siRNA inhibited the CoCl2-induced and hyperosmotic transcription of the VEGFA gene and caused a delayed decrease of the constitutive VEGFA gene expression while VEGF protein secretion was not altered. Kir2.1 knockdown stimulated RPE cell proliferation under control and hyperosmotic conditions without affecting cell viability. The data indicate that Kir2.1 channel activity is required for the expression of the VEGFA gene and inhibits the proliferation of RPE cells. Under control and hypoxic conditions, the extracellular VEGF level may regulate the production of VEGF via its inhibitory effect on the Kir2.1 gene transcription; this feedback loop may prevent overproduction of VEGF.

PEDF-Mediated Mitophagy Triggers the Visual Cycle by Enhancing Mitochondrial Functions in a H2O2-Injured Rat Model.

Retinal degenerative diseases result from oxidative stress and mitochondrial dysfunction, leading to the loss of visual acuity. Damaged retinal pigment epithelial (RPE) and photoreceptor cells undergo mitophagy. Pigment epithelium-derived factor (PEDF) protects from oxidative stress in RPE and improves mitochondrial functions. Overexpression of PEDF in placenta-derived mesenchymal stem cells (PD-MSCs; PD-MSCsPEDF) provides therapeutic effects in retinal degenerative diseases. Here, we investigated whether PD-MSCsPEDF restored the visual cycle through a mitophagic mechanism in RPE cells in hydrogen peroxide (H2O2)-injured rat retinas. Compared with naïve PD-MSCs, PD-MSCsPEDF augmented mitochondrial biogenesis and translation markers as well as mitochondrial respiratory states. In the H2O2-injured rat model, intravitreal administration of PD-MSCsPEDF restored total retinal layer thickness compared to that of naïve PD-MSCs. In particular, PTEN-induced kinase 1 (PINK1), which is the major mitophagy marker, exhibited increased expression in retinal layers and RPE cells after PD-MSCPEDF transplantation. Similarly, expression of the visual cycle enzyme retinol dehydrogenase 11 (RDH11) showed the same patterns as PINK1 levels, resulting in improved visual activity. Taken together, these findings suggest that PD-MSCsPEDF facilitate mitophagy and restore the loss of visual cycles in H2O2-injured rat retinas and RPE cells. These data indicate a new strategy for next-generation MSC-based treatment of retinal degenerative diseases.

MicroRNA-126 inhibits pathological retinal neovascularization via suppressing vascular endothelial growth factor expression in a rat model of retinopathy of prematurity

Vascular endothelial growth factor (VEGF) is the principal growth factor responsible for the retinal neovascularization in the pathogenesis of retinopathy of prematurity (ROP). Current therapies for ROP include laser ablation and intravitreal anti-VEGF injection. However, these treatments either destroy the peripheral retina or associate with problems of persistent peripheral avascular retina or later recurrence of ROP. In the present study we investigated a new therapeutic approach by exploring the potential role of a specific microRNA, miR-126, in regulating VEGFA expression and retinal neovascularization in a rat oxygen-induced retinopathy (OIR) model. We demonstrated that miR-126 mimic and plasmid effectively suppresses VEGFA mRNA expression in both human and rat retinal pigment epithelium cell lines, quantified with qRT-PCR. Animal experiments on rat OIR model revealed that intravitreal injection of miR-126 plasmid efficiently downregulated VEGFA expression in the intraocular fluid and retinal tissues measured by ELISA, and significantly suppressed retinal neovascularization, which was confirmed by calculating sizes of neovascularization areas on fluorescence microscopic images of flat mounted retina stained with Alexa Fluor 594-conjugated isolectin B4 to visualize blood vessels. Together, these results showed that intravitreal injection of miR-126 plasmid could inhibit retinal neovascularization by down-regulating VEGFA expression, suggesting a potential therapeutic effect for ROP.

Development of Cell Delivery System for the Subretinal Space Using Biodegradable Nanosheets

Age-related macular degeneration (AMD) is a major ophthalmic disease that causes visual impairment and blindness, particularly in elderly people. The developing choroidal vessels invade Bruch’s membrane and disrupt the monolayer of retinal pigment epithelial (RPE) cells. Subsequently, degeneration of photoreceptors occurs. Hence, subretinal transplantation of RPE cells to the site of degeneration would offer an ideal treatment providing the transplanted cells could generate a monolayer, reconstruct the RPE–photoreceptor interface, and inhibit further development of choroidal vessels. Although transplantation of autologous peripheral RPE cells has been tested by injection of cell suspensions using a syringe needle, limited visual improvement result due to the low viability of the injected cells, and their restricted distribution and integration into the subretinal tissue.Recently, we investigated the use of biodegradable polymeric nanosheets as cell scaffolds [1,2]. Because this system enabled the easy manipulation of the cell/nanosheet constructs with a syringe needle, it raises the possibility of transplanting cell sheets into a narrow space, such as the subretinal space, in a minimally-invasive manner. So far, poly(lactic-co-glycolic acid) (PLGA) was used as the material for the nanosheets. However, this biodegradable polymer is relatively inflammatory because it produces lactic and glycolic acids as degradation intermediates and these acid dissociation constants are about 3.8. In this study, we fabricated biodegradable nanosheets using polycaprolactone (PCL) and collagen as low inflammatory materials and investigated a minimally invasive and effective method for cell transplantation into the subretinal space (Fig.1(a)).Micropatterned nanosheets consisting of PCL, collagen and Nile red were prepared by a combination of spin-coating and micro-contact printing technique (Fig.1(b)). The diameter and thickness of the nanosheets used for the following experiments were 200 μm and 200 nm, respectively. The cell/nanosheet constructs were prepared by culturing rat retinal pigment epithelial (RPE-J) cells on the micropatterned nanosheets in a monolayer. A hand-made glass capillary needle was as thick as a 27G medical needle, and the tip angle was 30 degrees. Also, it was coated with 2-methayloxyethyl phosphorylcholine (MPC) to prevent cells from attaching the inner surface of the needle. The cell/nanosheet constructs and a sodium hyaluronate solution with the medium were loaded into the capillary needle. Subsequently, the cell/nanosheet was injected into the subretinal space through the sclera of rat. Several days after implantation, the rats were euthanized, and the eyeballs were enucleated.The micropatterned nanosheet was shown in Fig.1(c). The nanosheets had sufficient strength and flexibility to be handled in water. The shape or diameter of the nanosheets could be varied by adjusting the microstructure of the PDMS stamp. Also, the thickness of the nanosheets could be controlled by changing the concentration of the solution. As shown in Fig.1b, the cells formed a monolayer on the nanosheet of PCL/collagen. Finally, we attempted to deliver the RPE-J cell/nanosheet constructs to the subretinal space of a rat eye in vivo. The hand-made glass capillary needle shape allowed the needle insertion along the retina through the sclera. The fluorescence of PCL/collagen nanosheet and cells could be observed in the eyeballs under excitation light. Also, as shown in Fig.1(e), it was confirmed that the cell/nanosheet construct was successively delivered into the subretinal space.We fabricated biodegradable nanosheets using PCL and collagen as low inflammatory materials. Also, we developed a minimally invasive and effective method for cell transplantation into the subretinal space using capillary needles and confirmed that cell/nanosheet was successfully delivered. This method holds great promise for transplantation of monolayer cells and for the development of local cell delivery systems. In order to further improve this method, we plan to evaluate the inflammatory properties of the nanosheets and the efficacy of the delivered cells.

Uptake Study in Lysosome-Enriched Fraction: Critical Involvement of Lysosomal Trapping in Quinacrine Uptake but Not Fluorescence-Labeled Verapamil Transport at Blood-Retinal Barrier.

Lysosomal trapping at the blood–retinal barrier (BRB) was investigated through quinacrine and fluorescence-labeled verapamil (EFV) uptake. Quinacrine uptake by conditionally immortalized rat retinal capillary endothelial (TR-iBRB2) cells suggested saturable and non-saturable transport processes in the inner BRB. The reduction of quinacrine uptake by bafilomycin A1 suggested quinacrine distribution to the acidic intracellular compartments of the inner BRB, and this notion was also supported in confocal microscopy. In the study using the lysosome-enriched fraction of TR-iBRB2 cells, quinacrine uptake was inhibited by bafilomycin A1, suggesting the lysosomal trapping of quinacrine in the inner BRB. Pyrilamine, clonidine, and nicotine had no effect on quinacrine uptake, suggesting the minor role of lysosomal trapping in their transport across the inner BRB. Bafilomycin A1 had no effect on EFV uptake, and lysosomal trapping driven by the acidic interior pH was suggested as a minor mechanism for EFV transport in the inner BRB. The minor contribution of lysosomal trapping was supported by the difference in inhibitory profiles between EFV and quinacrine uptakes. Similar findings were observed in the outer BRB study with the fraction of conditionally immortalized rat retinal pigment epithelial (RPE-J) cells. These results suggest the usefulness of lysosome-enriched fractions in studying lysosomal trapping at the BRB.

Astragaloside IV protects retinal pigment epithelial cells from apoptosis by upregulating miR‑128 expression in diabetic rats.

The present study aimed to investigate the protective effects exerted by astragaloside-IV (AIV) on retinal pigment epithelial (RPE) cells of rats with diabetes mellitus (DM), and to explore the underlying molecular mechanisms. For this purpose, a rat model of DM was established by injecting rats with an intraperitoneal injection of streptozotocin. AIV was then intragastrically administered. An electroretinogram (ERG) was used to assess retinopathy and TUNEL staining was used to detect the level of apoptosis of RPE cells. Western blot analysis was used to determine protein expression in RPE cells in vitro and in vivo. AIV was found to be able to significantly increase body weight and decrease blood glucose levels in rats with DM in a dose-dependent manner. Compared with the rats with DM, the rat rod cell response a wave, b wave, maximum response b wave, photopic (photo)-ERG b wave and oscillatory potential (OP) p4 wave latency significantly decreased and the amplitude of OP Os1 wave increased significantly in the rats with DM treated with AIV for 11 weeks. In addition, AIV significantly decreased the apoptotic levels of RPE cells from rats with DM and significantly decreased the protein expression levels of Bax/Bcl-2, Fas/FasL, active caspase-3, active caspase-8, active caspase-9, homeobox B3 (HOXB3), p-phosphoinositide 3-kinase (PI3K)/PI3K, p-AKT/AKT and p-p70S6K1/p70S6K1, whereas it significantly increased miR-128 expression in the RPE cells from rats with DM. In vitro, AIV significantly inhibited the high glucose (HG)-induced apoptosis of RPE cells by increasing miR-128 expression and Bcl-2 and FasL protein expression in vivo. On the whole, the findings of the present study demonstrate that AIV treatment protects RPE cells of diabetic rats from apoptosis, and that these effects may be associated with the upregulation of miR-128 expression.

Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System Reduces Neovascularisation in Rats

Background: Age-related macular degeneration (AMD) is a progressive retinal disorder, characterised by imbalanced pro- and anti-angiogenic signals, leading vision loss. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy on primary cells with stable expression of human pigment epithelium derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Methods: Rat retinal pigment epithelial cells (RPEs) and iris pigment epithelial cells (IPEs) were co-transfected with pFAR4-ITRs CMV PEDF BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats and transfected primary cells (tRPE and tIPE) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinase 2 and 9 (MMP-2/9) and microglial/macrophage markers were measured and evaluated. Findings: The animals injected with tRPE/IPE cells significantly showed a reduction in the leakage area at 14 days while at 7 days only tIPE cells showed a significant reduction. The CNV area at 7 days was reduced with tRPE/IPE cells. There was a significant increase in PEDF and PEDF/VEGF ratio with tRPE cells along with a reduction in the MMP-2 activity. Activated microglial fluorescence intensity increased in all groups analyzed. Interpretation: Our data demonstrate that a single administration of SB100X-engineered RPE/IPE cells release enough hPEDF to favour an anti-angiogenic environment that ultimately reduces CNV suggest that ex vivo non-viral gene therapy could be an effective and safe therapeutic option for angiogenic retinal diseases. Funding: This work was supported by the European Union’s Seventh Framework Programme for research, technological development and demonstration, grant agreement no. 305134 and Fundacion Jesus Gangoiti Barrera. LGG received a predoctoral grant from the Asociacion de Amigos de la Universidad de Navarra. Declaration of Interest: A. Garcia-Layana is consultant in Thea, Allergan, Bayer, Novartis and Roche Laboratories. Z. Izsvak and Z. Ivics are co-inventors on several patents on Sleeping Beauty transposon technology. Ethical Approval: The study was performed according to the Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Ophthalmic and Vision Research and approved by the Ethics Committee for Animal Research of the University of Navarra (protocol approval number 023-13).

MiRNA-1273g-3p Involvement in Development of Diabetic Retinopathy by Modulating the Autophagy-Lysosome Pathway.

BackgroundDiabetic retinopathy (DR) is one of the most common and serious complications of diabetes mellitus (DM). The autophagy-lysosome pathway (ALP) is one of the main intracellular self-digestive degradation systems. Lysosomal impairment and autophagic dysfunction are early events in the pathogenesis of DR, suggesting autophagy might be a novel therapeutic strategy for DR treatment.Material/MethodsIn our study, we screened a differentially expressed miRNA, miR-1273g-3p, in streptozotocin (STZ)-injected DR rat retinal pigment epithelial (RPE) cells. miR-1273g-3p inhibitor and mimic were employed to treat RPE cells to assess the role of miR-1273g-3p. QRT-PCR and Western blot analysis were performed to examine the function of miR-1273g-3p on ALP-related and DR-related proteins.ResultsmiR-1273g-3p was highly expressed in STZ-induced DM RPE cells. miR-1273g-3p mimic promoted the expression of DR-related MMP-2, MMP-9, and TNF-α proteins, and ALP-related LC3, cathepsin B, and cathepsin L factors, but miR-1273g-3p inhibitor suppressed the levels of these factors.ConclusionsmiR-1273g-3p is involved in the progression of DR by modulating the autophagy-lysosome pathway. These findings provided new evidence of the close relationship between DR and ALP, and reveal a new target for DR therapy.

Optimization of ultrastructure observation of human embryonic stem cell-derived retinal pigment epithelial cells

Background Normal ultrastructure is the anatomical basis of retinal pigment epithelial(RPE)cells to perform normal physiological function.At present the precipitation method is often used to detect the ultrastructure of RPE cells with transmission electron microscopy(TEM). Objective The aim of this study was to explore a simple and feasible approach to examine the ultrastructure of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells. Methods hESCs were induced and differentiated into RPE cells by the spontaneous differentiation method, and the expressions of microphthalmia associated transcription factor MITF and paired-box gene 6 (PAX6), specific protein of RPE cells, in the cells were detected by immunofluorescence assay.hESC-RPE cells were inoculated into Transwell filter, and the ultrastructure of the cell sheet was examined under the TEM.Then the ultrastructure of the cell sheet specimens was compared with those of hESC-RPE cells from cell precipitation and RPE cell specimens of 90-day-old Long Evans rats. Results MITF and PAX6 were positively expressed in hESC-RPE cells.The normal ultrastructure were visible in the RPE cells of rats under the TEM, including apical microvilli, polarized melanin granules, cellular nucleus, basement membrane and intercellular junctions, and the ultrastructure of hESC-RPE cell sheet on Transwell was similar to the RPE cells in rats.However, only scatter melanin granules, nonpolar nucleus and scanty microvilli were observed under the TEM in the hESC-RPE cells by cell precipitation method. Conclusions Without digestion process, hESC-RPE cell sheet on Transwell can retain the normal ultrastructure of hESC-RPE cells under the TEM, with a more simple and reliable advantage. Key words: Retinal pigment epithelium/cytology; Embryonic stem cells; Microscopy, electron, transmission; Cell differentiation/physiology; Cells, cultured; Rats; Retinal pigment epithelial sheets, human

A Method for the Isolation and Culture of Adult Rat Retinal Pigment Epithelial (RPE) Cells to Study Retinal Diseases.

Diseases such as age-related macular degeneration (AMD) affect the retinal pigment epithelium (RPE) and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 min yielded 4 × 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

Hyperglycemia-induced GLP-1R downregulation causes RPE cell apoptosis

Glucagon-like peptide-1 receptor (GLP-1R) is closely associated with the onset of diabetes and its complications. However, its roles in diabetic retinopathy are unknown. Retinal pigment epithelial (RPE) cells are a crucial component of the outer blood–retina barrier and their death is related to the progression of diabetic retinopathy. Thus, we examined the pathophysiological role of GLP-1R in RPE cell apoptosis. We found that GLP-1R expression was lower in the isolated neuroretina and RPE cells of streptozotocin-treated rats than in vehicle-treated rats. High-glucose treatment also decreased GLP-1R expression in a human RPE cell line (ARPE-19 cells). GLP-1R was silenced in ARPE-19 cells, in order to elucidate the pathophysiological roles of GLP-1R. This increased intracellular reactive oxygen species (ROS) generation and activated p53-mediated Bax promoter and endoplasmic reticulum (ER) stress signaling. We also found that GLP-1R knockdown-mediated p53 expression was regulated by ER stress. Interestingly, antioxidant treatment and peroxiredoxin 1 (Prx1) overexpression attenuated GLP-1R knockdown-induced ER stress signaling and p53 expression. Finally, to confirm that GLP-1R activation has protective effects, ARPE-19 cells were treated with exendin-4, a synthetic GLP-1R agonist. This attenuated high-glucose-induced ROS generation, ER stress signaling, and p53 expression. Collectively, these results indicated that hyperglycemia decreases GLP-1R expression in RPE cells. Such a decrease generates intracellular ROS, which increases ER stress-mediated p53 expression, and subsequently causes apoptosis by increasing Bax promoter activity. Our data suggested that regulation of GLP-1R expression is a promising approach for the treatment of diabetic retinopathy.

Enhanced downregulation of transforming growth factor‑β2 in rat retinal pigment epithelium cells by adeno‑associated virus‑mediated ribonucleic acid interference combined with ultrasound or microbubbles.

The present study was designed to determine the efficiency and safety of ultrasound (US) and/or US contrast agent microbubbles (MBs) in the delivery of type 2 recombinant adeno-associated virus‑delivered transforming growth factor‑β2 short hairpin ribonucleic acid encoding the enhanced green fluorescent protein gene (rAAV2‑TGFβ2 shRNA‑EGFP) and the downregulation of TGFβ2 in rat retinal pigment epithelium (RPE‑J) cells. The effects of US and/or MBs on the delivery of rAAV2‑EGFP and rAAV2‑TGFβ2 shRNA‑EGFP were evaluated by fluorescence microscopy and flow cytometry. The potential toxicity of cell viability under various US or MB conditions was assessed by CellTiter 96® AQueous One solution cell proliferation assay. The level of TGFβ2 mRNA in RPE‑J cells under various conditions was estimated by reverse transcription‑quantitative polymerase chain reaction analysis. The results obtained demonstrated that low-intensity US (0.5 W/cm2 and 30 sec) or SonoVue (MB:cell ratio, 40:1) increased the delivery efficiency of rAAV2‑EGFP and rAAV2‑TGFβ2 shRNA‑EGFP to RPE‑J cells, whereas the combination of US with MBs did not further increase but instead decreased rAAV transfection. Under the optimal conditions of rAAV delivery, enhanced TGFβ2 gene silencing with a combination of US or SonoVue with rAAV2‑TGFβ2 shRNA resulted in a significant decrease in mRNA levels compared with rAAV2‑TGFβ2 shRNA alone. US or SonoVue was used safely to enhance the delivery of rAAV2‑TGFβ2 shRNA to RPE‑J cells. A combination of the biological (rAAV2‑TGFβ2 shRNA) and physical (US or SonoVue) approaches downregulated the mRNA level of TGFβ2 more effectively.

Use of antibiotic‐resistance‐free plasmids for non‐viral transfection of pigment epithelium‐derived factor in rat primary cells

AbstractPurpose We aim to assess the pigment epithelium‐derived factor (PEDF) production in cells after transfection with plasmids free of antibiotic resistance markers (pFAR4) in a non‐viral Sleeping Beauty transposon system (SB100X).Methods ARPE‐19 cell line and primary rat iris and retinal pigment epithelial (IPE and RPE) cells were transfected with SB100X transposase and PEDF transposon encoding pFAR4 plasmids (1:16 ratio). Control cells were transfected with SB100X and pFAR4‐Venus plasmids. Supernatants were taken every 24 hours for kinetics. The effect of transfecting different amount of cells was also analyzed. PEDF production was tested by Western blot and ELISA.Results A cumulative PEDF secretion over time in ARPE‐19 and primary cells was shown, reaching almost 3‐fold higher PEDF production in ARPE‐19 cells. Control cells did not express recombinant PEDF, but showed fluorescence. The transfection of 10,000 cells clearly showed higher PEDF expression than cells that were transfected as a group of 70,000 and subsequently divided.Conclusion Rat primary cells are efficiently transfected and produced PEDF as it happens in ARPE‐19 cell line. Merging of the SB100X and pFAR4 technologies led to increased PEDF expression and improved safety by avoiding the transfer and potential integration of antibiotic resistance genes. This study demonstrates the feasibility of our approach based on ex vivo transfection of a low number of primary autologous pigment epithelial cells.

Epigallocatechin Gallate (EGCG) Prevents H2O2-Induced Oxidative Stress in Primary Rat Retinal Pigment Epithelial Cells

Purpose: To determine whether the green tea polyphenol epigallocatechin gallate (EGCG) could prevent H2O2-induced oxidative stress in primary rat retinal pigment epithelial cells.Methods: Primary cultures of retinal pigment epithelium (RPE) cells were established from Long–Evans newborn rats. RPE cells were pretreated with various concentrations of EGCG for 24 h before being exposed to hydrogen peroxide (H2O2) for 2 h to induce oxidative stress. Cell metabolic activity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death was quantified by flow cytometry using propidium iodide (PI).Results: Treatment of RPE cells with EGCG alone does not affect the cell viability up to 50 µM. Exposure of RPE cells to 600 µM H2O2 caused a significant decrease in cell viability; whereas pretreatment with 10, 25, and 50 µM EGCG significantly reduced this decrease in a dose-dependent manner. The proportion of PI-positive cells increased significantly in cultures treated with H2O2 alone; whereas pretreatment of RPE cells with 50 µM EGCG significantly reduced H2O2-induced RPE cell death.Conclusions: Our study shows that EGCG pretreatment can protect primary rat RPE cells from H2O2-induced death. This suggests potential effect of EGCG in the prevention of retinal diseases associated with H2O2-induced oxidative stress.

Localization of organic anion transporting polypeptide (Oatp) 1a4 and Oatp1c1 at the rat blood-retinal barrier.

BackgroundOrganic anion transporting polypeptide (Oatp) transporters at the blood–brain barrier (BBB) and the blood-retinal barrier (BRB), which consists of retinal capillary endothelial cells and retinal pigment epithelial cells, are major determinants of the control of anionic drugs into the brain and retina. Although Oatp1a4 (Slco1a4) and Oatp1c1 (Slco1c1) are known to be expressed in the abluminal and luminal membrane of the rat BBB and Oatp1a4 is known to be expressed at the BRB, the expression and localization of Oatp1c1 at the BRB and subcellular localization of Oatp1a4 at the BRB have received little attention. Therefore, the purpose of present study was to determine the cellular and subcellular localization of Oatp1a4 and 1c1 at the BRB.MethodsWe used guinea pig polyclonal antibodies to Oatp1a4 and 1c1 for immunoblot and immunohistochemical analysis to determine their cellular and subcellular distributions in the rat retina. We compared these distributions with those of the glucose transporter 1 (GLUT1/Slc2a1). Whole brain, brain capillary fractions and kidney were used as control.ResultsOatp1a4 and 1c1 immunoreactivities were detected in the rat retinal capillaries and co-localized with GLUT1, suggesting that both proteins are located on the abluminal and luminal membrane of the retinal capillary endothelial cells. Oatp1a4 and 1c1 immunoreactivities were preferentially detected on the apical and basolateral membrane of rat retinal pigment epithelial cells, respectively, suggesting that Oatp1a4 and 1c1 are localized on the apical membrane and the basolateral membrane of the retinal pigment epithelial cells, respectively.ConclusionOatp1a4 and 1c1 are present at the BRB and contribute to the transcellular transport of amphipathic organic anions across the BRB.

P41 Hydrogen sulfide: Endogenous production and regulatory role in ocular tissues

Hydrogen sulfide (H 2 S), a colorless gas characterized by its pungent odor, has been portrayed for decades as a toxic pollutant. Evidence of endogenous production of H 2 S in mammalian tissues, and its potential biological role stimulated our interest in the pharmacological significance of this gaseous molecule in ocular tissues. In the present study, we tested the hypothesis that H 2 S is endogenously produced in mammalian ocular tissues, and may produce pharmacological effects in retinal pigment epithelial cells. Methods Endogenous levels of H 2 S were measured in bovine ocular tissues, using a well established colorimetric assay. In addition, basal level of H 2 S in bovine retina was examined in response to the H 2 S substrate, l -cysteine as well as the H 2 S biosynthetic enzymes inhibitors, propargylglycine (PAG) and aminooxyacetic acid (AOA); or activator, S-adenosyl- l -methionine (SAM). Using a H 2 S-releasing compound, sodium hydrosulfide (NaHS), we also determined the effects of H 2 S on cyclic AMP (cAMP) production in rat retinal pigment epithelial cells (RPE-J) by Enzyme Immuno Assay. The observed effect of H 2 S on cAMP was studied in the presence or absence of forskolin, an adenylate cyclase activator; glibenclamide, an ATP-sensitive potassium channel (K ATP ) antagonist; or flubiprofen and indomethacin, which are cyclooxygenase (COX) inhibitors. Results H 2 S is endogenously produced in ocular tissues with highest level detected in cornea (19 ± 2.85 nmoles/mg protein) and retina (17 ± 2.1 nmoles/mg protein). Interestingly, the inhibitors of cystathionine γ -lyase (CSE) and cystathionine β -synthase (CBS) (1 mM PAG and 1 mM AOA, respectively) significantly decreased H 2 S production in the bovine retina by 56.8 and 42%. On the other hand, activator of CBS (SAM, 100 μ M), and H 2 S substrate ( l -cysteine, 10–300 μM) increased ( p 2 S in bovine retina. In RPE-J cells, NaHS (1 nM–100 μM) produced a concentration-dependent increase ( p p ATP channel antagonist, glibenclamide (100 μM) inhibited the NaHS-induced increase of cAMP production in RPE-J cells. Conclusions Our results indicate that H 2 S is endogenously produced in various tissues in the bovine eye. Endogenous level of H 2 S is increased in the presence of l -cysteine or SAM, and decreased by PAG or AOA, inhibitors of enzymes that synthesize H 2 S in neural retina. Moreover, H 2 S can increase cAMP production in RPE-J cells, and removal of the apparent inhibitory effect of prostaglandins unmasks an excitatory effect of H 2 S on cAMP production. In addition to the adenylyl cylcase pathway, K ATP channels are involved in mediating the observed effects of H 2 S on cAMP production. The fact that H 2 S is endogenously produced in ocular tissues and exhibits a regulatory role in cyclic nucleotide production in retina, supports a physiological and/or pharmacological role for this gaseous molecule in the eye.