Cell-Based Therapies for Glaucoma.

Glaucomatous optic neuropathy (GON) is the major cause of irreversible visual loss worldwide and can result from a range of disease etiologies. The defining features of GON are retinal ganglion cell (RGC) degeneration and characteristic cupping of the optic nerve head (ONH) due to tissue remodeling, while intraocular pressure remains the only modifiable GON risk factor currently targeted by approved clinical treatment strategies. Efforts to understand the mechanisms that allow species such as the zebrafish to regenerate their retinal cells have greatly increased our understanding of regenerative signaling pathways. However, proper integration within the retina and projection to the brain by the newly regenerated neuronal cells remain major hurdles. Meanwhile, a range of methods for in vitro differentiation have been developed to derive retinal cells from a variety of cell sources, including embryonic and induced pluripotent stem cells. More recently, there has been growing interest in the implantation of glial cells as well as cell-derived products, including neurotrophins, microRNA, and extracellular vesicles, to provide functional support to vulnerable structures such as RGC axons and the ONH. These approaches offer the advantage of not relying upon the replacement of degenerated cells and potentially targeting earlier stages of disease pathogenesis. In order to translate these techniques into clinical practice, appropriate cell sourcing, robust differentiation protocols, and accurate implantation methods are crucial to the success of cell-based therapy in glaucoma. Translational Relevance: Cell-based therapies for glaucoma currently under active development include the induction of endogenous regeneration, implantation of exogenously derived retinal cells, and utilization of cell-derived products to provide functional support.

Transcriptomics of CD29+/CD44+ cells isolated from hPSC retinal organoids reveals a single cell population with retinal progenitor and Müller glia characteristics.

Müller glia play very important and diverse roles in retinal homeostasis and disease. Although much is known of the physiological and morphological properties of mammalian Müller glia, there is still the need to further understand the profile of these cells during human retinal development. Using human embryonic stem cell-derived retinal organoids, we investigated the transcriptomic profiles of CD29+/CD44+ cells isolated from early and late stages of organoid development. Data showed that these cells express classic markers of retinal progenitors and Müller glia, including NFIX, RAX, PAX6, VSX2, HES1, WNT2B, SOX, NR2F1/2, ASCL1 and VIM, as early as days 10-20 after initiation of retinal differentiation. Expression of genes upregulated in CD29+/CD44+ cells isolated at later stages of organoid development (days 50-90), including NEUROG1, VSX2 and ASCL1 were gradually increased as retinal organoid maturation progressed. Based on the current observations that CD24+/CD44+ cells share the characteristics of early and late-stage retinal progenitors as well as of mature Müller glia, we propose that these cells constitute a single cell population that upon exposure to developmental cues regulates its gene expression to adapt to functions exerted by Müller glia in the postnatal and mature retina.

MicroRNA profile of extracellular vesicles released by Müller glial cells.

Introduction: As with any other radial glia in the central nervous system, Müller glia derive from the same neuroepithelial precursors, perform similar functions, and exhibit neurogenic properties as radial glia in the brain. Müller glial cells retain progenitor-like characteristics in the adult human eye and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Recently, it has been demonstrated that intracellular communication is possible via the secretion of nano-sized membrane-bound extracellular vesicles (EV), which contain bioactive molecules like microRNA (miRNA) and proteins that induce phenotypic changes when internalised by recipient cells. Methods: We conducted high-throughput sequencing to profile the microRNA signature of EV populations secreted by Müller glia in culture and used bioinformatics tools to evaluate their potential role in the neuroprotective signalling attributed to these cells. Results: Sequencing of miRNA within Müller EV suggested enrichment with species associated with stem cells such as miR-21 and miR-16, as well as with miRNA previously found to play a role in diverse Müller cell functions in the retina: miR-9, miR-125b, and the let-7 family. A total of 51 miRNAs were found to be differentially enriched in EV compared to the whole cells from which EV originated. Bioinformatics analyses also indicated that preferential enrichment of species was demonstrated to regulate genes involved in cell proliferation and survival, including PTEN, the master inhibitor of the PI3K/AKT pathway. Discussion: The results suggest that the release by Müller cells of miRNA-enriched EV abundant in species that regulate anti-apoptotic signalling networks is likely to represent a significant proportion of the neuroprotective effect observed after the transplantation of these cells into animal models of retinal ganglion cell (RGC) depletion. Future studies will seek to evaluate the modulation of putative genes as well as the activation of these pathways in in vitro and in vivo models following the internalisation of Müller-EV by target retinal neurons.

Effects of fluoroquinolones and tetracyclines on mitochondria of human retinal MIO-M1 cells.

Our goal was to explore the detrimental impacts of ciprofloxacin (CPFX) and tetracycline (TETRA) on human retinal Müller (MIO-M1) cells in vitro. Cells were exposed to 30, 60 and 120 µg/ml of CPFX and TETRA. The cellular metabolism was measured with the MTT assay. The JC-1 and CM-H2DCFDA assays were used to evaluate the levels of mitochondrial membrane potential (MMP) and ROS (reactive oxygen species), respectively. Mitochondrial DNA (mtDNA) copy number, along with gene expression levels associated with apoptotic (BAX, BCL2-L13, BCL2, CASP-3 and CASP-9), inflammatory (IL-6, IL-1ß, TGF-α, TGF-ß1 and TGF-ß2) and antioxidant pathways (SOD2, SOD3, GPX3 and NOX4) were analyzed via Quantitative Real-Time PCR (qRT-PCR). Bioenergetic profiles were measured using the Seahorse® XF Flux Analyzer. Cells exposed 24 h to 120 µg/ml TETRA demonstrated higher cellular metabolism compared to vehicle-treated cells. At each time points, (i) all TETRA concentrations reduced MMP levels and (ii) ROS levels were reduced by TETRA 120 µg/ml treatment. TETRA caused (i) higher expression of CASP-3, CASP-9, TGF-α, IL-1B, GPX3 and SOD3 but (ii) decreased levels of TGF-B2 and SOD2. ATP production and spare respiratory capacity declined with TETRA treatment. Cellular metabolism was reduced with CPFX 120 µg/ml in all cultures and 60 µg/ml after 72 h. The CPFX 120 µg/ml reduced MMP in all cultures and ROS levels (72 h). CPFX treatment (i) increased expression of CASP-3, CASP-9, and BCL2-L13, (ii) elevated the basal oxygen consumption rate, and (iii) lowered the mtDNA copy numbers and expression levels of TGF-B2, IL-6 and IL-1B compared to vehicle-control cells. We conclude that clinically relevant dosages of bactericidal and bacteriostatic antibiotics can have negative effects on the cellular metabolism and mitochondrial membrane potential of the retinal MIO-M1 cells in vitro. It is noteworthy to mention that apoptotic and inflammatory pathways in exposed cells were affected significantly This is the first study showing the negative impact of fluoroquinolones and tetracyclines on mitochondrial behavior of human retinal MIO-M1 cells.

Strain Specific Responses in a Microbead Rat Model of Experimental Glaucoma.

PURPOSE: A major challenge in glaucoma research is the lack of reproducible animal models of RGC and optic nerve damage, the characteristic features of this condition. We therefore examined the glaucomatous responses of two different rat strains, the Brown Norway (BN) and Lister Hooded (LH) rats, to high intraocular pressure (IOP) induced by injection of magnetic beads into the anterior chamber. METHODS: Magnetic microsphere suspensions (20 µl of 5-20 mg/ml) were injected into the anterior chamber of BN (n = 9) or LH (N = 15) rats. Animals from each strain were divided into three groups, each receiving a different dose of microspheres. IOP was measured over 4 weeks using a rebound tonometer. Retinal ganglion cell (RGC) damage and function were assessed using scotopic electroretinograms (ERGs), retinal flatmounts and optic nerve histology. ANOVA and Student's t-tests were used to analyse the data. RESULTS: A significant elevation in IOP was observed in BN rats receiving injections of 20 mg (37.18 ± 12.28 mmHg) or 10 mg microspheres/ml (36.95 ± 13.63 mmHg) when compared with controls (19.63 ± 4.29 mmHg) (p < .001) over 2 weeks. This correlated with a significant impairment of RGC function, as determined by scotopic ERG (p < .001), reduction in axon number (p < .05) and lower RGC density (P < .05) in animals receiving 20 mg or 10 mg microspheres/ml as compared with controls. LH rats receiving similar microsphere doses showed reduced scotopic ERG function (p < .001) after 2 weeks. No changes in IOP was seen in this strain, although a reduction in axon density was observed in optic nerve cross-sections (p < .05). Initial changes in IOP and ERG responses observed in BN rats remained unchanged for a duration of 7 weeks. In LH animals, ERG responses were decreased at 1-2 weeks and returned to control levels after 5 weeks. CONCLUSIONS: Although this model was easily reproducible in BN rats, the phenotype of injury observed in LH rats was very different from that observed in BN animals. We suggest that differences in the glaucomatous response observed in these two strains may be ascribed to anatomical and physiological differences and merits further investigation.

Galectins and their involvement in ocular disease and development.

Galectins are carbohydrate binding proteins with high affinity to ß-galactoside containing glycoconjugates. Understanding of the functions of galectins has grown steadily over the past decade, as a result of substantial advancements in the field of glycobiology. Galectins have been shown to be versatile molecules that participate in a range of important biological systems, including inflammation, neovascularisation and fibrosis. These processes are of particular importance in ocular tissues, where a major theme of recent research has been to divert diseases away from pathways which result in loss of function into pathways of repair and regeneration. This review summarises our current understanding of galectins in the context important ocular diseases, followed by an update on current clinical studies and future directions.

Insights into the design of spray systems for cell therapies for retinal disease using computational modelling.

Chronic eye diseases are the main cause of vision loss among adults. Among these, retinal degenerative diseases affect millions of people globally, causing permanent loss of cells and organ dysfunction. Despite recent progress in developing stem cell therapies for retinal diseases, methods for delivery remain an area of intense research. Aerosol technology is a promising technique with the potential to spray cells evenly and directly across the retinal surface, promoting cell attachment and survival. Here we implement mathematical modelling of the spraying process to develop organ-specific spraying parameters in this therapeutic scenario. Firstly, we characterise the rheological parameters for a typical hydrogel used for spraying cells. These parameters are then integrated into a 3D computational model of an adult human eye under realistic surgical conditions. Simulation results provide quantitative relationships between the volume flow rate of the cell-laden hydrogel, external pressure needed for aerosolization, angle of the spraying, and properties of the cell delivery. An experimental assessment is also carried out to explore the impact of spraying under the regimes identified by the computational model on cell viability. This is the first stage towards using computational models to inform the design of spray systems to deliver cell therapies onto the human retina.

Potential of Müller Glia for Retina Neuroprotection.

Müller glia constitute the main glial cells of the retina. They are spatially distributed along this tissue, facilitating their close membrane interactions with all retinal neurons. Müller glia are characterized by their active metabolic functions, which are neuroprotective in nature. Although they can become reactive under pathological conditions, leading to their production of inflammatory and neurotoxic factors, their main metabolic functions confer neuroprotection to the retina, resulting in the promotion of neural cell repair and survival. In addition to their protective metabolic features, Müller glia release several neurotrophic factors and antioxidants into the retinal microenvironment, which are taken up by retinal neurons for their survival. This review summarizes the Müller glial neuroprotective mechanisms and describes advances made on the clinical application of these factors for the treatment of retinal degenerative diseases. It also discusses prospects for the use of these cells as a vehicle to deliver neuroprotective factors into the retina.

Phenotypic and Functional Characterization of Müller Glia Isolated from Induced Pluripotent Stem Cell-Derived Retinal Organoids: Improvement of Retinal Ganglion Cell Function upon Transplantation.

Glaucoma is one of the leading causes of blindness, and there is an ongoing need for new therapies. Recent studies indicate that cell transplantation using Müller glia may be beneficial, but there is a need for novel sources of cells to provide therapeutic benefit. In this study, we have isolated Müller glia from retinal organoids formed by human induced pluripotent stem cells (hiPSCs) in vitro and have shown their ability to partially restore visual function in rats depleted of retinal ganglion cells by NMDA. Based on the present results, we suggest that Müller glia derived from retinal organoids formed by hiPSC may provide an attractive source of cells for human retinal therapies, to prevent and treat vision loss caused by retinal degenerative conditions. Stem Cells Translational Medicine 2019;8:775&784.

Comparative proteomic analysis of normal and gliotic PVR retina and contribution of Müller glia to this profile.

Müller glia are responsible for the neural retina regeneration observed in fish and amphibians throughout life. Despite the presence of these cells in the adult human retina, there is no evidence of regeneration occurring in humans following disease or injury. It may be possible that factors present in the degenerated retina could prevent human Müller glia from proliferating and neurally differentiating within the diseased retina. On this basis, investigations into the proteomic profile of these cells and the abundance of key proteins associated to Müller glia in the gliotic PVR retina, may assist in the identification of factors with the potential to control Müller proliferation and neural differentiation in vivo. Label free mass spectrometry identified 1527 proteins in Müller glial cell preparations, 1631 proteins in normal retina and 1074 in gliotic PVR retina. Compared to normal retina, 28 proteins were upregulated and 196 proteins downregulated by 2-fold or more in the gliotic PVR retina. As determined by comparative proteomic analyses, of the proteins highly upregulated in the gliotic PVR retina, the most highly abundant proteins in Müller cell lysates included vimentin, GFAP, polyubiquitin and HSP90a. The observations that proteins highly upregulated in the gliotic retina constitute major proteins expressed by Müller glia provide the basis for further studies into mechanisms that regulate their production. In addition investigations aimed at controlling the expression of these proteins may aid in the identification of factors that could potentially promote endogenous regeneration of the adult human retina after disease or injury.

Comparison of proteomic profiles in the zebrafish retina during experimental degeneration and regeneration.

Zebrafish spontaneously regenerate the retina after injury. Although the gene expression profile has been extensively studied in this species during regeneration, this does not reflect protein function. To further understand the regenerative process in the zebrafish, we compared the proteomic profile of the retina during injury and upon regeneration. Using two-dimensional difference gel electrophoresis (2D-DIGE) and label-free quantitative proteomics (quadrupole time of flight LC-MS/MS), we analysed the retina of adult longfin wildtype zebrafish at 0, 3 and 18 days after Ouabain injection. Gene ontology analysis indicates reduced metabolic processing, and increase in fibrin clot formation, with significant upregulation of fibrinogen gamma polypeptide, apolipoproteins A-Ib and A-II, galectin-1, and vitellogenin-6 during degeneration when compared to normal retina. In addition, cytoskeleton and membrane transport proteins were considerably altered during regeneration, with the highest fold upregulation observed for tubulin beta 2 A, histone H2B and brain type fatty acid binding protein. Key proteins identified in this study may play an important role in the regeneration of the zebrafish retina and investigations on the potential regulation of these proteins may lead to the design of protocols to promote endogenous regeneration of the mammalian retina following retinal degenerative disease.

Characteristics and vitreoretinal management of retinal detachment in eyes with Boston keratoprosthesis.

PURPOSE: To review the incidence and features of vitreoretinal complications of a permanent Boston keratoprosthesis and to report the use and outcomes of 23-gauge vitrectomy to manage vitreoretinal pathology. DESIGN: Retrospective non-comparative, interventional case series. SUBJECT, PARTICIPANTS: 27 eyes of 27 patients managed with a Boston keratoprosthesis at Moorfields Eye Hospital over a 3-year period. METHODS: All eyes that underwent pars plana vitrectomy (PPV) and had at least 6 months follow-up were analysed with a specific focus on the anatomical and histological characteristics of retinal detachment and outcomes of surgery. MAIN OUTCOME MEASURES: Anatomical success and characteristics of retinal detachment over the follow-up period. RESULTS: 27 patients underwent Boston keratoprosthesis implantation over the study period. Of these, six (22%) required PPV for retinal detachment which demonstrated a specific pattern of serous elevation with subsequent severe anterior proliferative vitreoretinopathy (PVR). The mean follow-up period was 9 months (range 6-14 months). At final follow-up, visual acuity ranged from perception of light to 6/18, and five of six cases had attached retinae under the silicone oil. Histological analysis of a subretinal membrane demonstrated a predominantly glial/retinal pigment epithelium fibrocellular tissue, consistent with PVR. CONCLUSIONS: The study showed that retinal detachment complicated by PVR, as demonstrated by the clinical and histological characteristics of this condition, is common in patients undergoing Boston keratoprosthesis. We also showed that 23-gauge vitrectomy can be effectively performed in patients with a permanent prosthesis. Visual acuity often remains poor, despite successful anatomical results.

Allogeneic Transplantation of Müller-Derived Retinal Ganglion Cells Improves Retinal Function in a Feline Model of Ganglion Cell Depletion.

Human Müller glia with stem cell characteristics (hMGSCs) have been shown to improve retinal function upon transplantation into rat models of retinal ganglion cell (RGC) depletion. However, their translational potential may depend upon successful engraftment and improvement of retinal function in experimental models with anatomical and functional features resembling those of the human eye. We investigated the effect of allogeneic transplantation of feline Müller glia with the ability to differentiate into cells expressing RGC markers, following ablation of RGCs by N-methyl-d-aspartate (NMDA). Unlike previous observations in the rat, transplantation of hMGSC-derived RGCs into the feline vitreous formed aggregates and elicited a severe inflammatory response without improving visual function. In contrast, allogeneic transplantation of feline MGSC (fMGSC)-derived RGCs into the vitrectomized eye improved the scotopic threshold response (STR) of the electroretinogram (ERG). Despite causing functional improvement, the cells did not attach onto the retina and formed aggregates on peripheral vitreous remnants, suggesting that vitreous may constitute a barrier for cell attachment onto the retina. This was confirmed by observations that cellular scaffolds of compressed collagen and enriched preparations of fMGSC-derived RGCs facilitated cell attachment. Although cells did not migrate into the RGC layer or the optic nerve, they significantly improved the STR and the photopic negative response of the ERG, indicative of increased RGC function. These results suggest that MGSCs have a neuroprotective ability that promotes partial recovery of impaired RGC function and indicate that cell attachment onto the retina may be necessary for transplanted cells to confer neuroprotection to the retina. Significance: Müller glia with stem cell characteristics are present in the adult human retina, but they do not have regenerative ability. These cells, however, have potential for development of cell therapies to treat retinal disease. Using a feline model of retinal ganglion cell (RGC) depletion, cell grafting methods to improve RGC function have been developed. Using cellular scaffolds, allogeneic transplantation of Müller glia-derived RGC promoted cell attachment onto the retina and enhanced retinal function, as judged by improvement of the photopic negative and scotopic threshold responses of the electroretinogram. The results suggest that the improvement of RGC function observed may be ascribed to the neuroprotective ability of these cells and indicate that attachment of the transplanted cells onto the retina is required to promote effective neuroprotection.

Brimonidine Can Prevent In Vitro Hydroquinone Damage on Retinal Pigment Epithelium Cells and Retinal Müller Cells.

PURPOSE: Brimonidine is a selective alpha-2 adrenergic agonist used to reduce intraocular pressure and it has been shown to have some neuroprotective effects. Hydroquinone (HQ) is a toxicant present in cigarette smoke, and other sources. In this study, we investigated the cyto-protective effects in vitro of Brimonidine on human retinal pigment epithelium cells (ARPE-19) and human retinal Müller cells (MIO-M1) that had been treated with HQ. METHODS: Cells were pretreated for 6 h with different doses of Brimonidine tartrate 0.1% (1/2×, 1×, 5×, 10×), followed by a 24-h exposure to 100 µM of HQ, while the Brimonidine was still present. Assays were used to measure cell viability, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) production, and lactate dehydrogenase (LDH) release. RESULTS: Brimonidine increased the cell viability at all concentrations studied in both cell lines studied. ΔΨm also improved at all Brimonidine doses in ARPE-19 cells and in the 5× and 10× dosages MIO-M1 cells. The ROS levels decreased at 1×, 5×, and 10× doses of Brimonidine in ARPE-19 but only at 10× on MIO-M1 cells. The 10×-Brimonidine ARPE-19 cells had decreased LDH release, but no LDH changes were observed on MIO-M1 cells. CONCLUSION: HQ-induced toxicity is mediated through mitochondrial damaging, oxidative stress-related and necrosis-related pathways; Brimonidine significantly prevented the mitochondrial damaging and oxidative stress-related effects but had little effect on blocking the necrosis component of HQ-toxicity. Brimonidine protective effects differ between the different retinal cell types and high concentrations of Brimonidine (10×) have minimal damaging effects on human retinal cells.

Short Communication: Contribution of Voltage-Gated Sodium Channels to the Rabbit Cone Electroretinograms.

PURPOSE: Although the rabbit eye is of a similar size to the human eye, our limited understanding of the differences in retinal physiology to other species hinders its use in retinal research. The role of voltage-gated sodium channels (Nav) in the propagation of excitatory potentials along bipolar cells remains unclear, as conflicting data have been reported in the rabbit. The present study assesses the relative contributions of Nav to the scotopic and photopic flash ERGs as well as the wavelength-dependence of Nav blockade on the rabbit flicker ERG. MATERIALS AND METHODS: Tetrodotoxin (TTX, 1 µM) was injected into the vitreous cavity of Chinchilla bastard rabbits. Scotopic ERGs were evoked by white flashes ranging from 10(-5) to 10 cds m(-2), photopic ERGs on a background of 25 cdm(-2) using flash intensities of 0.032-25 cds m(-2). Flicker ERGs (3-50 Hz) were elicited by blue, green and yellow stimuli at 2.34 cds m(-2) on a white background of 30 cdm(-2). RESULTS: The a- and b-waves of the scotopic ERG were unaffected by intravitreal injection of the Nav blocker TTX. In contrast, the b-wave, but not the a-wave, of the photopic ERG was selectively blocked by TTX. The reduction by TTX of the flicker ERG was greater for blue than for green and yellow stimuli. DISCUSSION: The data suggest that Nav selectively contribute to the generation of the photopic b-wave in the rabbit, indicating that they play an important role in the propagation of excitatory signals on bipolar cells in the cone, but not rod pathways. Importantly, the present study resolves conflicting previous reports into the role of Nav in the retinal function of the rabbit.

Downregulation of the Canonical WNT Signaling Pathway by TGFß1 Inhibits Photoreceptor Differentiation of Adult Human Müller Glia with Stem Cell Characteristics.

Müller glia are responsible for the retina regeneration observed in zebrafish. Although the human retina harbors Müller glia with stem cell characteristics, there is no evidence that they regenerate the retina after disease or injury. Transforming growth factor-ß (TGFß) and Wnt signaling regulate retinal neurogenesis and inflammation, but their roles in the neural differentiation of human Müller stem cells (hMSC) are not known. We examined hMSC lines in vitro for the expression of various Wnt signaling components and for their modulation by TGFß1, as well as the effect of this cytokine on the photoreceptor differentiation of these cells. Culture of hMSC with a combination of factors that induce photoreceptor differentiation of hMSC (FGF2, taurine, retinoic acid, and insulin-like growth factor type1; FTRI), markedly upregulated the expression of components of the canonical Wnt signaling pathway, including WNT2B, DKK1, and active ß-CATENIN. Although FTRI did not modify mRNA expression of WNT5B, a component of the noncanonical/planar cell polarity Wnt pathway, it upregulated its secretion. Furthermore, TGFß1 not only decreased WNT2B expression, but also inhibited FTRI-induced photoreceptor differentiation of hMSC, as determined by expression of the photoreceptor markers NR2E3, RHODOPSIN, and RECOVERIN. Inhibition of TGFß1 signaling by an ALK5 inhibitor prevented TGFß1-induced changes in the expression of the two Wnt ligands examined. More importantly, inhibition of the canonical WNT signaling by XAV-939 prevented FTRI-induced photoreceptor differentiation. These observations suggest that TGFß may play a key role in preventing neural differentiation of hMSC and may constitute a potential target for induction of endogenous regeneration of the human retina.

Amyloid ß Peptide Induces Apoptosis Through P2X7 Cell Death Receptor in Retinal Cells: Modulation by Marine Omega-3 Fatty Acid DHA and EPA.

Retinal Müller glial cells have already been implicated in age-related macular degeneration (AMD). AMD is characterized by accumulation of toxic amyloid-ß peptide (Aß); the question we raise is as follows: is P2X7 receptor, known to play an important role in several degenerative diseases, involved in Aß toxicity on Müller cells? Retinal Müller glial cells were incubated with Aß for 48 h. Cell viability was assessed using the alamarBlue assay and cytotoxicity using the lactate dehydrogenase (LDH) release assay. P2X7 receptor expression was highlighted by immunolabeling observed on confocal microscopy and its activation was evaluated by YO-PRO-1 assay. Hoechst 33342 was used to evaluate chromatin condensation, and caspases 8 and 3 activation was assessed using AMC assays. Lipid formulation rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) used in Age-Related Eye Disease Study 2 was incubated on cells for 15 min prior to Aß incubation. For the first time, we showed that Aß induced caspase-independent apoptosis through P2X7 receptor activation on our retinal model. DHA and EPA are polyunsaturated fatty acids recommended in food supplement to prevent AMD. We therefore modulated Aß cytotoxicity using a lipid formulation rich in DHA and EPA to have a better understanding of the results observed in clinical studies. We showed that fish oil rich in EPA and DHA, in combination with a potent P2X7 receptor antagonist, represents an efficient modulator of Aß toxicity and that P2X7 could be an interesting therapeutic target to prevent AMD.

Effects of light on retinal pigment epithelial cells, neurosensory retinal cells and Müller cells treated with Brilliant Blue G.

BACKGROUND: The aim of this study is to evaluate the safety profile of Brilliant Blue G (BBG) with and without exposure to light (L) on three different retinal cell lines. METHOD: ARPE-19, R28 and MIO-M1 cells were treated with BBG: 0.125 mg/mL (0.5x clinical concentration), 0.25 mg/mL (1x) or 0.5 mg/mL (2x) with or without surgical illumination of halogen light exposure for 10 min, 15 min or 30 min. Cells were further cultured after 24 h and then analysed for cell viability, late stages of apoptosis and mitochondrial damage associated with early apoptosis using assays that measure trypan blue dye exclusion, increases in caspase-3/7 activity or changes in mitochondrial membrane potential (ΔΨm), respectively. RESULT: All three cell lines that were exposed to BBG in the presence or absence of light exposure for 30 min were found to have cell viability and caspase-3/7 activity levels similar to the untreated cultures. The mitochondrial membrane potential (ΔΨm) was decreased significantly at the 2x + L dose and 2x dose in all three retinal cell lines compared to their respective untreated control cells. At the lower doses of BBG, with or without exposure to light, the ΔΨm values were similar to the untreated control cultures. CONCLUSION: Exposure to BBG dye concentrations that are used clinically (0.125 mg/mL and 0.25 mg/mL) in the presence up to 30 min of surgically equivalent light intensity is safe for retinal cells.

IL-1ß induces IL-6 production in retinal Müller cells predominantly through the activation of p38 MAPK/NF-κB signaling pathway.

IL-6 plays an important role in various inflammatory ocular diseases, including diabetic retinopathy. Müller cells are the major source of inflammatory mediators, including IL-6, in the retina. However, the mechanism of regulating IL-6 production in these cells remains unclear. Examination of signaling pathways in human retinal Müller cells (MIO-M1 cell line) cultured with IL-1ß, TNF-α, IL-6, IL-8, VEGF, IFN-γ, glucose or mannitol showed that IL-1ß was the most potent stimulator of IL-6 production. In addition, IL-1 ß also increased NF-κB p50 protein level and phosphorylation of p38 MAPK, ERK1/2 and c-Jun. Induction of IL-6 production by IL-1ß was significantly reduced by addition of p38 MAPK (SB203580), MEK1/2 (U0126) or NF-κB (BAY11-7082) inhibitors, with the highest effect being observed with SB203580. To explore the specific elements in IL-6 promoter responsible for IL-1ß-induction of IL-6 expression, a series of plasmids bearing various IL-6 promoter mutations were transiently expressed in MIO-MI cells cultured in the presence or absence of IL-1ß (10ng/ml) and/or SB203580 (10µM). Results showed that IL-6 promoter activity of the parent pIL-6-Luc651 was significantly enhanced by IL-1ß, but the level was significantly attenuated by SB203580. Furthermore, the IL-6 promoter activity was also reduced upon deletion of NF-κB, AP-1 or C/EBP binding sites, with NF-κB deletion being the greatest. These results are the first demonstration that IL-1ß induces IL-6 production in Müller cells by activation of IL-6 promoter activity predominantly through the p38 MAPK/NF-κB pathway.