Retinal Cell Cultures Research Articles

Immunocytochemical Profiling of Cultured Mouse Primary Retinal Cells

Primary retinal cell cultures and immunocytochemistry are important experimental platforms in ophthalmic research. Translation of retinal cells from their native environment to the in vitro milieu leads to cellular stress, jeopardizing their in vivo phenotype features. Moreover, the specificity and stability of many retinal immunochemical markers are poorly evaluated in retinal cell cultures. Hence, we here evaluated the expression profile of 17 retinal markers, that is, recoverin, rhodopsin, arrestin, Chx10, PKC, DCX, CRALBP, GS, vimentin, TPRV4, RBPMS, Brn3a, β-tubulin III, NeuN, MAP2, GFAP, and synaptophysin. At 7 and 18 days of culture, the marker expression profiles of mouse postnatal retinal cells were compared with their age-matched in vivo retinas. We demonstrate stable in vitro expression of all markers, except for arrestin and CRALBP. Differences in cellular expression and location of some markers were observed, both over time in culture and compared with the age-matched retina. We hypothesize that these differences are likely culture condition dependent. Taken together, we suggest a thorough evaluation of the antibodies in specific culture settings, before extrapolating the in vitro results to an in vivo setting. Moreover, the identification of specific cell types may require a combination of different genes expressed or markers with structural information.

Purified mouse retinal ganglion cell culture

To establish a culture system for purified mouse retinal ganglion cells (RGC) culture in order to lay a foundation for the in vitro study of RGC. It was a experiment study. Eight to twelve C57BL/6 mice on postnatal day 4 to 6 were used. The retinas were dissected and dissociated enzymatically to make a suspension of single cells. The retinal suspension was incubated in rat anti-mouse-macrophage antiserum for 5 minutes and incubated on a 100 mm anti-rat IgG panning plate at room temperature for 30 min twice. The nonadherent cells were removed with the suspension and placed on the Thy-1.2 panning plate at room temperature. After 45 min, plates were washed 6 - 10 times with phosphate-buffered saline and swirled moderately to dislodge nonadherent cells. Trypsin (0.125% solution) dissociation was used to remove adherent cells from the plate. Then cells were spun and resuspended in neurobasal medium containing some neurotrophic factors. The cell suspension was implanted in 24 well culture plates and cultured under 37°C in an incubator with 5% CO(2). Most of the cells adhered to the plate after 24 hours in culture and showed dendrites at different lengths. The dendrites grew longer with time. Most of the RGC can survive more than two weeks. Monoclonal antibody to the Thy-1.2 antigen can be used to purify the mice RGC.

Modified primary culture and identification of human retinal Müller cells

Background Retinal Muller cells are important gliocytes and the source of retinal stem cells.Researching the biological behavior of Muller cells is of important significance to the study on retinal physiopathological process and stem cell therapy of retinal diseases.To establish a stable culture method of Muller cells is a solid basis of relative basic research. Objective This study was to establish a simple and stable method of isolation and culture of human retinal Muller cells and provide sufficient and high-quality Muller cell source. Methods Human retinal Muller cells were isolated from healthy human donor eyes.The mixture solution of hyaluronidase (100 U) and 0.25% trypsin were used to digest chopped retinal tissue.The DMEM/F12 medium with 20% fetal bovine serum (FBS) was added to stop the digestion process.RPMI1640 medium with 20% FBS was used to culture the cell for 72 hours and then replaced the half medium.The cells were passaged by the RPMI1640 medium with 20% FBS.The morphology of the cells were examied under the optical microscope, and the expressions of glial fibrillary acidic protein (GFAP), a marker of gliocytes, and glutamine synthetase (GS), a special marker of retinal Muller cells, were detected by immunochemistry and immunofluorescence technology. Results Human retinal Muller cells were successfully isolated by enzyme mixture solution of hyaluronidase (100 U) and 0.25% trypsin.The cells were adherent to walls 24 hours after primary culture and completely merged 9-10 days after culture.The cells showed oval in shape with abundant cytoplasm, and a part of cells presented with cone-shaped bulge bilaterally and ectasia in the posterior containing large nuclei.After cells passage, the cells were enlarged and grew toward polygonal shape.The positive expression of GFAP was observed in more than 95% cells and strongly positive expression of GS was observed in more than 90% cells by immunohistochemstry and immunofluorescent staining. Conclusions Human retinal Muller cells can be successfully isolated by hyaluronidase combined with trypsin digestion.Abundent and pure human retinal Muller cells can be obtained by successively using RPMI1640 medium with 20% FBS and 10% FBS. Key words: Retina/cytology; Neuroglia; Cells, cultured; Humans; Muller cells

Elevated Glucose and Interleukin-1β Differentially Affect Retinal Microglial Cell Proliferation.

Diabetic retinopathy is considered a neurovascular disorder, hyperglycemia being considered the main risk factor for this pathology. Diabetic retinopathy also presents features of a low-grade chronic inflammatory disease, including increased levels of cytokines in the retina, such as interleukin-1 beta (IL-1β). However, how high glucose and IL-1β affect the different retinal cell types remains to be clarified. In retinal neural cell cultures, we found that IL-1β and IL-1RI are present in microglia, macroglia, and neurons. Exposure of retinal neural cell cultures to high glucose upregulated both mRNA and protein levels of IL-1β. High glucose decreased microglial and macroglial cell proliferation, whereas IL-1β increased their proliferation. Interestingly, under high glucose condition, although the number of microglial cells decreased, they showed a less ramified morphology, suggesting a more activated state, as supported by the upregulation of the levels of ED-1, a marker of microglia activation. In conclusion, IL-1β might play a key role in diabetic retinopathy, affecting microglial and macroglial cells and ultimately contributing to neural changes observed in diabetic patients. Particularly, since IL-1β has an important role in retinal microglia activation and proliferation under diabetes, limiting IL-1β-triggered inflammatory processes may provide a new therapeutic strategy to prevent the progression of diabetic retinopathy.

Development of an automated chip culture system with integrated on-line monitoring for maturation culture of retinal pigment epithelial cells

Development of an automated chip culture system with integrated on-line monitoring for maturation culture of retinal pigment epithelial cells

Development of an automated chip culture system with integrated on-line monitoring for maturation culture of retinal pigment epithelial cells

In cell manufacturing, the establishment of a fully automated, microfluidic, cell culture system that can be used for long-term cell cultures, as well as for process optimization is highly desirable. This study reports the development of a novel chip bioreactor system that can be used for automated long-term maturation cultures of retinal pigment epithelial (RPE) cells. The system consists of an incubation unit, a medium supply unit, a culture observation unit, and a control unit. In the incubation unit, the chip contains a closed culture vessel (2.5 mm diameter, working volume 9.1 μL), which can be set to 37 °C and 5% CO 2 , and uses a gas-permeable resin (poly- dimethylsiloxane) as the vessel wall. RPE cells were seeded at 5.0 × 10 4 cells/cm 2 and the medium was changed every day by introducing fresh medium using the medium supply unit. Culture solutions were stored either in the refrigerator or the freezer, and fresh medium was prepared before any medium change by warming to 37 °C and mixing. Automated culture was allowed to continue for 30 days to allow maturation of the RPE cells. This chip culture system allows for the long-term, bubble-free, culture of RPE cells, while also being able to observe cells in order to elucidate their cell morphology or show the presence of tight junctions. This culture system, along with an integrated on-line monitoring system, can therefore be applied to long-term cultures of RPE cells, and should contribute to process control in RPE cell manufacturing.

Melanocortin receptor agonists MCR1-5 protect photoreceptors from high-glucose damage and restore antioxidant enzymes in primary retinal cell culture.

Retinal photoreceptors are particularly vulnerable to local high‐glucose concentrations. Oxidative stress is a risk factor for diabetic retinopathy development. Melanocortin receptors represent a family of G‐protein‐coupled receptors classified in five subtypes and are expressed in retina. Our previous data indicate that subtypes 1 and 5 receptor agonists exert a protective role on experimental diabetic retinopathy. This study focuses on their role in primary retinal cell cultures in high‐glucose concentrations. After eye enucleation from wild‐type male C57BL/6 mice, retinal cells were isolated, plated in high‐glucose concentration and treated with melanocortin receptors 1 and 5 agonists and antagonists. Immunocytochemical and biochemical analysis showed that treatment with melanocortin receptors 1 and 5 agonists reduced anti‐inflammatory cytokines and chemokines and enhanced manganese superoxide dismutase and glutathione peroxidase levels, preserving photoreceptor integrity. According with these evidences, we propose a major role of melanocortin receptors 1 and 5 on primary retinal cell response against high glucose or oxidative insults.

Norbixin Protects Retinal Pigmented Epithelium Cells and Photoreceptors against A2E-Mediated Phototoxicity In Vitro and In Vivo.

The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9’-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9’-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial.

Potential of Small Molecule-Mediated Reprogramming of Rod Photoreceptors to Treat Retinitis Pigmentosa.

PurposeMutations in rod photoreceptor genes can cause retinitis pigmentosa (RP). Rod gene expression is regulated by the nuclear hormone receptor, Nr2e3. Genetic deletion of Nr2e3 reprograms rods into cells that resemble cone photoreceptors, and might therefore prevent their death from some forms of RP. There are no identified ligands for Nr2e3; however, reverse agonists might mimic the genetic rescue effect and may be therapeutically useful for the treatment of RP.MethodsWe screened for small molecule modulators of Nr2e3 using primary retinal cell cultures and characterized the most potent, which we have named photoregulin1 (PR1), in vitro and in vivo. We also tested the ability of PR1 to slow the progression of photoreceptor degeneration in two common mouse models of autosomal dominant RP, the RhoP23H and the Pde6brd1 mutations.ResultsIn developing retina, PR1 causes a decrease in rod gene expression and an increase in S opsin+ cones. Photoregulin1 continues to inhibit rod gene expression in adult mice. When applied to two mouse models of RP, PR1 slows the degeneration of photoreceptors.ConclusionsChemical compounds identified as modulators of Nr2e3 activity may be useful for the treatment of RP through their effects on expression of disease-causing mutant genes.

Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling.

Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue ‘Norgestrel’ is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel’s neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising treatment for RP, with dual actions as a neuroprotective and anti-inflammatory agent in the retina.

253 - N-Acetylcysteine Amide, a Thiol Antioxidant, Protects TBHP-Induced Oxidative Stress in Primary Human Retinal Pigment Epithelial Cells

Age-related macular degeneration (AMD) is a leading cause of blindness in Americans at ago 60 and older. AMD is a degenerative condition that begins in Bruch’s membrane and progresses to the retinal pigment epithelium and, ultimately, the overlying photoreceptors. While oxidative stress is implicated in the pathogenesis of AMD, dietary antioxidants have been shown to delay progression of AMD in clinical studies. We hypothesized that N-acetylcysteine amide (NACA), a thiol antioxidant, would impede progression of retinal degeneration. tert-Butyl hydroperoxide (tBHP) is a model hydroperoxidant used to induce oxidative stress in cell cultures. The goal of this work was to evaluate the efficacy of NACA in preventing tBHP-induced acute oxidative stress in primary human retinal pigment epithelial cells (hRPEpiC). Our data indicated that tBHP induced ROS generation and resulted in reduced cell viability, depletion of intracellular glutathione (GSH) levels and compromise of glutathione reductase (GR) activity. Pretreatment with NACA significantly reduced ROS generation, restored the level of GSH and GR activity, and recovered transepithelical electrical resistance (TEER). However, pretreatment of NACA did not decrease the number of dying cells, defined as early and late apoptotic cells from flow cytometry. Our data suggests that pretreatment with NACA reduces sublethal but not the lethal effect in primary cultures of human retinal pigment epithelial cells. NACA is a potent antioxidant that could be further evaluated as a potential treatment for dry AMD.

In vitro effects of bone marrow-derived mesenchymal stem cells on the biological behavior of lipopolysaccharide-activated retinal microglia

Background Retinal microglia(RMG) plays an important role in the pathogenesis of retinal degenerative diseases, while chemokine CX3CL1 participates in the regulation of steady-state of microglia.It has been determined that bone marrow-derived mesenchymal stem cells (BMSCs) have a remarkable role to modulate the immune response and protect the central nervous system through the release of soluble factors in a paracrine fashion and further affect the functional behavior of cells.However, whether BMSCs are able to interact with RMG and activate related signaling pathway for the maintaining of homeostasis in the retina is still unclear. Objective The aim of this study was to investigate the interaction between BMSCs and lipopolysaccharide (LPS)-activated RMG in vitro, and dissect the effects of CX3CL1/CX3CR1 signaling pathway on the biological behavior of BMSCs and RMG. Methods RMG was isolated from SD rats, cultured with mixed culture of retinal glial cells and purified by shaking.The cells were identified by detecting the expression of CD11b, Iba1 and glutamamine synthetase (GS) with indirect immunofluorescence assay.LPS (1 mg/ml, 2 μl) was added in the medium for 24 hours to stimulate RMG, and then the cells were divided into LPS control group, BMSCs group (cocultured with BMSCs for 24 hours) and CB-BMSCs group (cocultured with CX3CL1-blocking-BMSCs for 24 hours). The cells without LPS stimulation served as the blank control group.The functions of RMG, including the release content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), the proliferation, phagocytosis, and migration of RMG were examined. Results RMG was successfully isolated and harvested from SD rats by using mixed culture of retinal glial cells and purified by shaking.CD11b and Iba1 showed the positive expression with the green fluorescence in the cells and GS was absent.The contents of TNF-α in the cell supernatant were (2.55±0.97)ng/ml, (24.91±3.07)ng/ml, (20.38±2.97)ng/ml and (24.90±1.88)ng/ml in the blank control group, LPS control group, BMSCs group and CB-BMSCs group, respectively, showing a significant difference among the groups (F=119.90, P 0.05). The proliferative rate of RMG was lower in the BMSCs group than that in the LPS control group (P 0.05). The mean fluorescence intensity (MFI) and the number of migrated RMG were considerably different among the four groups (F=70.55, 15.49, both at P 0.05). Conclusions BMSCs could suppress the proliferation of LPS-activated RMG.Moreover, BMSCs might inhibit proinflammatory cytokines releasing, enhance phagocytosis and migration capabilities of RMG via CX3CL1/CX3CR1 signaling pathway. Key words: Microglia/pathology; Mesenchymal stem cells/metabolism; Chemokine CX3CL1/metabolism; Retinal degeneration; Inflammation; Cells, cultured; Rat, SD

The trophic effect of ouabain on retinal ganglion cells is mediated by IL-1β and TNF-α

Ouabain is a steroid hormone that binds to the enzyme Na+, K+ – ATPase and stimulates different intracellular pathways controlling growth, proliferation and cell survival. IL-1β and TNF-α are pleiotropic molecules, conventionally regarded as pro-inflammatory cytokines with well-known effects in the immune system. In addition, IL-1β and TNF-α also play important roles in the nervous system including neuroprotective effects. Previous data from our group showed that ouabain treatment is able to induce an increase in retinal ganglion cell survival kept in mixed retinal cell cultures. The aim of this work was to investigate if IL-1β and TNF-α could be mediating the trophic effect of ouabain on retinal ganglion cells. Our results show that the trophic effect of ouabain on retinal ganglion cell was inhibited by either anti-IL-1β or anti-TNF-α antibodies. In agreement, IL-1β or TNF-α increased the retinal ganglion cells survival in a dose-dependent manner. Accordingly, ouabain treatment induces a temporal release of TNF-α and IL-1β from retinal cell cultures. Interestingly, TNF-α and IL-1β regulate each other intracellular levels. Our results suggest that ouabain treatment triggers the activation of TNF-α and IL-1β signaling pathways leading to an increase in retinal ganglion cell survival.

Automated Measurement of Cobblestone Morphology for Characterizing Stem Cell Derived Retinal Pigment Epithelial Cell Cultures.

Assessing the morphologic properties of cells in microscopy images is an important task to evaluate cell health, identity, and purity. Typically, subjective visual assessments are accomplished by an experienced researcher. This subjective human step makes transfer of the evaluation process from the laboratory to the cell manufacturing facility difficult and time consuming. Automated image analysis can provide rapid, objective measurements of cultured cells, greatly aiding manufacturing, regulatory, and research goals. Automated algorithms for classifying images based on appearance characteristics typically either extract features from the image and use those features for classification or use the images directly as input to the classification algorithm. In this study we have developed both feature and nonfeature extraction methods for automatically measuring "cobblestone" structure in human retinal pigment epithelial (RPE) cell cultures. A new approach using image compression combined with a Kolmogorov complexity-based distance metric enables robust classification of microscopy images of RPE cell cultures. The automated measurements corroborate determinations made by experienced cell biologists. We have also developed an approach for using steerable wavelet filters for extracting features to characterize the individual cellular junctions. Two image analysis techniques enable robust and accurate characterization of the cobblestone morphology that is indicative of viable RPE cultures for therapeutic applications.

Simvastatin use associated with low intraocular ADP levels in patients with sight-threatening diabetic retinopathy

Dear Editor, Purinergic signalling controls vascular thrombosis, remodelling and inflammation [1], and may result in disrupted blood flow and vascular function in the diabetic retina [2]. High glucose conditions increase secretion and reduce degradation of extracellular ATP in retinal cell culture [3], with increased purine concentrations being reported in the vitreous of diabetic patients suffering from proliferative retinopathy [4]. Administration of simvastatin, a lipophilic HMG-CoA reductase inhibitor, did not only promote direct vasculoprotective effects in experimental in vitro and ex vivo models [5], but was also associated with low levels of vasoactive and tissue remodelling factors in the retinas of diabetic rats [6] and vitreous of diabetic patients [7]. Here, we evaluated the vitreous accumulation of extracellular purine nucleotides in a total of 52 eyes of 52 type I and type II adult diabetic patients admitted to primary vitrectomy for the management of sight-threatening forms of diabetic retinopathy. The patients included those without statin medication (n = 41) and those with preoperative simvastatin treatment (n = 11). No difference emerged in the baseline characteristics of the patients (age, gender, HbA1c, body mass index, physical status, blood pressure, duration of diabetes) and vitrectomized eyes (intraocular pressure, lens status, intravitreal anti-VEGF treatment, macular oedema, proliferative retinopathy, vitreous haemorrhage, fibrosis) between the study groups. Vitreous samples were subjected to protein concentration measurements of adenosine 5’-triphosphate (ATP), -5’-diphosphate (ADP), -5’-monophosphate (AMP), adenosine and inosine. Intravitreal levels of proinflammatory and pro-thrombotic ADP were lower in simvastatin-treated diabetic patients (simvastatin vs. non-statin, mean ± SEM 42.9 ± 15.1 vs 7.7 ± 2.7 nM; p= 0.026, Table 1), while the data on other purinergic factors (ATP 34.5 ± 17.1 vs 6.1 ± 2.1; AMP 1974.3 ± 253.7 vs 1634.9 ± 253.4; adenosine 1066.3 ± 235.7 vs 1744.5 ± 419.5 and inosine 1163.0 ± 456.1 vs 2351.8 ± 1206.2, respectively, Table 1) led to a non-significant trend towards lower pro-inflammatory ATP and higher anti-inflammatory adenosine levels due to simvastatin treatment. These results suggest that simvastatin may favour dephosphorylation of extracellular ADP and shift the balance to an anti-inflammatory direction in the eye. Our findings are parallel with the previous report that statin use was associated with low plasma ADP levels in patients with atherosclerotic disease [8]. These data provide a novel insight into the potential vasculoprotective mechanisms of simvastatin involving extracellular purine nucleotides in patients with sight-threatening diabetic retinopathy. * Raimo Tuuminen raimo.tuuminen@helsinki.fi

Progranulin promotes the retinal precursor cell proliferation and the photoreceptor differentiation in the mouse retina.

Progranulin (PGRN) is a secreted growth factor associated with embryo development, tissue repair, and inflammation. In a previous study, we showed that adipose-derived stem cell-conditioned medium (ASC-CM) is rich in PGRN. In the present study, we investigated whether PGRN is associated with retinal regeneration in the mammalian retina. We evaluated the effect of ASC-CM using the N-methyl-N-nitrosourea-induced retinal damage model in mice. ASC-CM promoted the differentiation of photoreceptor cells following retinal damage. PGRN increased the number of BrdU+ cells in the outer nuclear layer following retinal damage some of which were Rx (retinal precursor cell marker) positive. PGRN also increased the number of rhodopsin+ photoreceptor cells in primary retinal cell cultures. SU11274, a hepatocyte growth factor (HGF) receptor inhibitor, attenuated the increase. These findings suggest that PGRN may affect the differentiation of retinal precursor cells to photoreceptor cells through the HGF receptor signaling pathway.

The Translational Repressor 4E-BP1 Contributes to Diabetes-Induced Visual Dysfunction.

PurposeThe translational repressor 4E-BP1 interacts with the mRNA cap-binding protein eIF4E and thereby promotes cap-independent translation of mRNAs encoding proteins that contribute to diabetic retinopathy. Interaction of 4E-BP1 with eIF4E is enhanced in the retina of diabetic rodents, at least in part, as a result of elevated 4E-BP1 protein expression. In the present study, we examined the role of 4E-BP1 in diabetes-induced visual dysfunction, as well as the mechanism whereby hyperglycemia promotes 4E-BP1 expression.MethodsNondiabetic and diabetic wild-type and 4E-BP1/2 knockout mice were evaluated for visual function using a virtual optomotor test (Optomotry). Retinas were harvested from nondiabetic and type 1 diabetic mice and analyzed for protein abundance and posttranslational modifications. Similar analyses were performed on cells in culture exposed to hyperglycemic conditions or an O-GlcNAcase inhibitor (Thiamet G [TMG]).ResultsDiabetes-induced visual dysfunction was delayed in mice deficient of 4E-BP1/2 as compared to controls. 4E-BP1 protein expression was enhanced by hyperglycemia in the retina of diabetic rodents and by hyperglycemic conditions in retinal cells in culture. A similar elevation in 4E-BP1 expression was observed with TMG. The rate of 4E-BP1 degradation was significantly prolonged by either hyperglycemic conditions or TMG. A PEST motif in the C-terminus of 4E-BP1 regulated polyubiquitination, turnover, and binding of an E3 ubiquitin ligase complex containing CUL3.ConclusionsThe findings support a model whereby elevated 4E-BP1 expression observed in the retina of diabetic rodents is the result of O-GlcNAcylation of 4E-BP1 within its PEST motif.

PEPito-driven PEDF Expression Ameliorates Diabetic Retinopathy Hallmarks.

Diabetic retinopathy (DR) is one of the major complications of diabetes mellitus. It is characterized by retinal microvascular changes caused by chronic exposure to hyperglycemia, leading to low tissue oxygenation and ultimately to neovascularization. Laser photocoagulation and vitrectomy are the most efficient treatments for DR, but display severe side effects such as the destruction of the healthy retina. Another clinical approach uses antiangiogenic agents to prevent and delay progression of neovascularization, but these require recurrent local administrations that increase the possibility of retinal detachment, vitreous hemorrhage, and cataract formation. Studies in human diabetic retinas have revealed an imbalance between proangiogenic factors such as the vascular endothelial growth factor (VEGF) and antiangiogenic factors, such as pigment epithelial-derived factor (PEDF). This imbalance favors pathological angiogenesis contributing to DR, and can constitute a therapeutic target. Gene therapy was recently shown to be an adequate intervention for long-term treatment of several retinal pathologies. We have previously shown the newly engineered episomal vector pEPito to be able of sustained gene expression in the mouse retina. We here show that pEPito was able to overexpress PEDF for up to three months, both in in vitro cultures of human retinal pigment epithelial cells and in the retina of diabetic mice after a single subretinal injection. In vivo, in parallel with the increase in PEDF we observed a decrease in VEGF levels in injected compared with noninjected eyes and a significant effect on two hallmarks of DR: reduction of glucose transport (by glucose transporter GLUT1), and reduction of inflammation by decreased reactivity of microglia. Jointly, these results point to a significant therapeutic potential of gene therapy with pEPito-PEDF for the treatment of DR.

Mitochondrial Uncoupling Protein 2 (UCP2) Regulates Retinal Ganglion Cell Number and Survival.

In the brain, mitochondrial uncoupling protein 2 (UCP2) has emerged as a stress signal associated with neuronal survival. In the retina, UCP2 is expressed primarily by retinal ganglion cells. Here, we investigated the functional relevance of UCP2 in the mouse retina. Increased expression of UCP2 significantly reduced apoptosis during the critical developmental period resulting in elevated numbers of retinal ganglion cells in the adult. Elevated UCP2 levels also protected against excitotoxic cell death induced by intraocular injection of either NMDA or kainic acid. In monolayer cultures of retinal cells, elevated UCP2 levels increased cell survival and rendered the cells independent of the survival-promoting effects of the neurotrophic factors BDNF and CNTF. Taken together, these data implicate UCP2 as an important regulator of retinal neuron survival both during development and in adult animals.

Linoleic Acid is a Diabetes-relevant Stimulator of Retinal Inflammation in Human Retinal Muller Cells and Microvascular Endothelial Cells.

To determine the effect of oleic acid and linoleic acid on the production and secretion of specific diabetic retinopathy- (DR-) related cytokines: vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and interleukin-8 (IL-8) by human retinal glial cells, retinal endothelial cells, and retinal pigment epithelial cells. These expression profiles will be compared to those obtained by treatment of the same cell types with elevated D-glucose, a diabetes-relevant stimulus often used in retinal cell culture experiments. Primary cultures of human retinal Müller cells, astrocytes, and microvascular endothelial cells (RMEC) and a human retinal pigment epithelial cell line (ARPE-19) were treated with oleic acid, linoleic acid, elevated D-glucose, or L-glucose as an osmotic control. VEGF, IL-6, and IL-8 concentrations in conditioned media were determined by colorimetric ELISA and normalized to total cellular protein. In the conditioned medium of human Müller cells, linoleic and oleic acid increased VEGF production by 6.4-fold and 9.9-fold, respectively. Linoleic acid also significantly increased IL-6 by 2.9-fold and IL-8 by 5.7-fold. L-glucose and D-glucose both increased VEGF by 3.1-fold in Müller cell conditioned medium. Linoleic acid increased IL-8 concentrations by 56% in human RMEC conditioned medium. Human retinal astrocytes and ARPE-19 were unaffected by all stimuli. Linoleic and oleic acid induce inflammatory mediators believed to be involved in the pathogenesis of diabetic retinopathy (DR). In culture, the free fatty acid insults, particularly linoleic acid, significantly increased cytokine production by Müller cells. In summary, these data identified Müller cells as the primary producer of these inflammatory mediators when treated with unsaturated fatty acids. This study also demonstrates that elevated glucose is an inadequate stimulus for assessing the production of inflammatory mediators. Therefore this study provides a novel in vitro model system of the dyslipidemia-induced inflammation occurring in DR.