PEDF derived from glial Müller cells: a possible regulator of retinal angiogenesis

A precise balance between stimulators and inhibitors of angiogenesis, such as vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), respectively, is essential for angiogenic homeostasis in ocular tissues. Retinal hypoxia is accompanied by some pathological conditions that may promote intraocular neovascularization. Here we demonstrate that retinal glial (Müller) cells express and release pigment epithelium-derived factor (PEDF). Decreasing oxygen concentrations cause strong attenuation of PEDF release resulting in enhanced VEGF/PEDF ratios. Exposure of Müller cells to VEGF suppressed PEDF release in a dose-dependent manner. This may represent a novel mechanism of ocular angiogenic homeostasis sufficient in the control of PEDF levels during normoxia or mild hypoxia but supplemented by other (hitherto unknown) mechanisms in cases of strong hypoxia. In spite of the enhanced VEGF/PEDF ratios resulting from hypoxia, conditioned media of Müller cells failed to stimulate additional proliferation of retinal endothelial cells. These findings suggest that in the ischemic retina, Müller cells generate a permissive condition for angiogenesis by secreting more VEGF and less PEDF, but the onset of retinal endothelial cell proliferation requires another triggering signal that remains to be identified.     

Conversion of mammalian Müller glial cells into a neuronal lineage by in vitro aggregate-culture

Mammalian Müller glial cells are major glial cells in the retina. Here we report that these glial cells can be redirected towards a neuronal lineage by an aggregate-culture in vitro. Rat and macaque Müller glial cells did not express neuronal markers except after transfer to adhesive conditions. Furthermore, this expression could only take place in the presence of platelet-derived growth factor and valproic acid. We compared a normal monolayer-culture and an aggregate-culture, and rat Müller glial cells could only differentiate into neurons under non-adhesive conditions. However, Müller glial cells did not express the photoreceptor markers in vitro. After transplantation into the subretinal space, a retina-specific niche, rat Müller glial cells expressed the photoreceptor-specific marker, opsin (RET-P1). We demonstrate the potential of mammalian Müller glial cells as a source of photoreceptors, which may possibly contribute to the treatment of degenerative retinal diseases such as retinitis pigmentosa.     

Atoh7 promotes retinal Müller cell differentiation into retinal ganglion cells

Glaucoma is one of the leading eye diseases due to the death of retinal ganglion cells. Increasing evidence suggests that retinal Müller cells exhibit the characteristics of retinal progenitor cells and can differentiate to neurons in injured retinas under certain conditions. However, the number of ganglion cells differentiated from retinal Müller cells falls far short of therapeutic needs. This study aimed to promote the differentiation of retinal Müller cells into ganglion cells by introducing Atoh7 into the stem cells dedifferentiated from retinal Müller cells. Rat retinal Müller cells were isolated and dedifferentiated into stem cells, which were transfected with PEGFP-N1 or PEGFP-N1-Atoh7 vector, and then further induced to differentiate into ganglion cells. The proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of control transfected or untransfected cells. In summary, Atoh7 promotes the differentiation of retinal Müller cells into retinal ganglion cells. This may open a new avenue for gene therapy of glaucoma by promoting optic nerve regeneration.     

BDNF regulates GLAST and glutamine synthetase in mouse retinal Müller cells

This study investigated whether brain-derived neurotrophic factor (BDNF) regulates the L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) in mouse retinal Müller cells (RMCs) under normal and hypoxic conditions. Mouse RMCs were treated with recombinant human BDNF (50, 75, 100, 125, or 150 ng/ml) for 24 h or underwent hypoxia induced by CoCl(2) (125 μM; 6, 12, 24, 48, or 72 h). An additional group underwent combined treatment with BDNF (100 ng/ml; 24, 48, 72, or 96 h) and CoCl(2) (125 μM/ml; 72 h). GLAST and GS mRNA and protein expression, L-[3,4-3H]-glutamic acid uptake, and apoptosis were assessed. BDNF dose-dependently up-regulated GLAST and GS mRNA and protein and increased glutamate uptake. Similarly, in early-stage CoCl(2)-induced hypoxia, GLAST and GS were up-regulated and glutamate uptake increased, but these decreased over time. BDNF also up-regulated GLAST and GS and increased glutamate uptake when RMCs under CoCl(2) induced hypoxic condition. However, BDNF treatment 24 h before CoCl(2) had no effect on GLAST or GS expression. CoCl(2) alone or combined with BDNF did not induce apoptosis. Hypoxia rapidly increased GLAST and GS expressions. This effect was transient, perhaps due to compensatory mechanisms that reduce GLAST and GS by 72 h. BDNF can up-regulate GLAST and GS and increase glutamate uptake during hypoxia, and these functions may underlie its neuroprotective effects.     

The glutathione content of retinal Müller (glial) cells: effect of pathological conditions

Maintenance of isolated retinal Müller (glial) cells in glutamate-free solutions over 7 h causes a significant loss of their initial glutathione content; this loss is largely prevented by the blockade of glutamine synthesis using methionine sulfoximine (5 mM). Anoxia does not reduce the glutathione content of Müller cells when glucose (11 mM), glutamate and cystine (0.1 mM each) are present. In contrast, simulation of total ischemia (i.e., anoxia plus removal of glucose) decreases the glutathione levels dramatically, even in the presence of glutamate and cystine. Less severe effects are caused by high extracellular K+ (40 mM). Reactive oxygen species are generated in the retina under various conditions, such as anoxia, ischemia, and reperfusion. One of the crucial substances protecting the retina against reactive oxygen species is glutathione, a tripeptide constituted of glutamate, cysteine and glycine. It was recently shown that glutathione can be synthesized in retinal Müller glial cells and that glutamate is the rate-limiting substance. In this study, glutathione levels were determined in acutely isolated guinea-pig Müller cells using the glutathione-sensitive fluorescent dye monochlorobimane. The purpose was to find out how the glial glutathione content is affected by anoxia/ischemia and accompanying pathophysiological events such as depolarization of the cell membrane. Our results further strengthen the view that glutamate is rate-limiting for the glutathione synthesis in glial cells. During glutamate deficiency, as caused by e.g., impaired glutamate uptake, this amino acid is preferentially delivered to the glutamate-glutamine pathway, at the expense of glutathione. This mechanism may contribute to the finding that total ischemia (but not anoxia) causes a depletion of glial glutathione. In situ depletion may be accelerated by the ischemia-induced increase of extracellular K+, decreasing the driving force for glutamate uptake. The ischemia-induced lack of glutathione is particularly fatal considering the increased production of reactive oxygen species under this condition. Therefore the therapeutic application of exogenous free radical scavengers is greatly recommended.     

An alternative isomerohydrolase in the retinal Müller cells of a cone-dominant species

Cone photoreceptors have faster light responses than rods and a higher demand for 11-cis retinal (11cRAL), the chromophore of visual pigments. RPE65 is the isomerohydrolase in the retinal pigment epithelium (RPE) that converts all-trans retinyl ester to 11-cis retinol, a key step in the visual cycle for regenerating 11cRAL. Accumulating evidence suggests that cone-dominant species express an alternative isomerase, likely in retinal Müller cells, to meet the high demand for the chromophore by cones. In the present study, we describe the identification and characterization of a novel isomerohydrolase, RPE65c, from the cone-dominant zebrafish retina. RPE65c shares 78% amino acid sequence identity with RPE-specific zebrafish RPE65a (orthologue of human RPE65) and retains all of the known key residues for the enzymatic activity of RPE65. Similar to the other RPE-specific RPE65, RPE65c was present in both the membrane and cytosolic fractions, used all-trans retinyl ester as its substrate and required iron for its enzymatic activity. However, immunohistochemistry detected RPE65c in the inner retina, including Müller cells, but not in the RPE. Furthermore, double-immunostaining of dissociated retinal cells using antibodies for RPE65c and glutamine synthetase (a Müller cell marker), showed that RPE65c co-localized with the Müller cell marker. These results suggest that RPE65c is the alternative isomerohydrolase in the intra-retinal visual cycle, providing 11cRAL to cone photoreceptors in cone-dominant species. Identification of an alternative visual cycle will contribute to the understanding of the functional differences of rod and cone photoreceptors.     

Genetic Pathways Regulating Glutamate Levels in Retinal Müller Cells

Müller cells serve many functions including the regulation of extracellular glutamate levels. The product of two genes, Slc1a3 [aka solute carrier family 1 (glial high affinity glutamate transporter), member 3] and Glul (aka glutamine synthetase) are the primary role players that transport glutamate into the Müller cell and convert it into glutamine. In this study, we sought to identify the genetic regulation of both genes. Given their tightly coupled biological functions, we predicted that they would be similarly regulated. Using an array of 75 recombinant inbred strains of mice, we determined that Slc1a3 and Glul are differentially regulated by distinct chromosomal regions. Interestingly, despite their independent regulation, gene ontology analysis of tightly correlated genes reveals that the enriched and statistically significant molecular function categories of both directed acyclic graphs have substantial overlap, indicating that the shared functions of correlates of Slc1a3 and Glul include production and usage of ATP.     

Murine Müller cells are progenitor cells for neuronal cells and fibrous tissue cells

Mammalian Müller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Müller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Müller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Müller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Müller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.     

Cultured retinal neuronal cells and Müller cells both show net production of lactate

Glucose has long been considered the substrate for energy metabolism in the retina. Recently, an alternative hypothesis (metabolic coupling) suggested that mitochondria in retinal neurons utilize preferentially the lactate produced specifically by Müller cells, the principal glial cell in the retina. These two views of retinal metabolism were examined using confluent cultures of photoreceptor cells, Müller cells, ganglion cells, and retinal pigment epithelial cells incubated in modified Dulbecco's minimal essential medium containing glucose or glucose and lactate. The photoreceptor and ganglion cells represented neural elements, and the Müller and pigment epithelial cells represented non-neural cells. The purpose of the present experiments was two-fold: (1) to determine whether lactate is a metabolic product or substrate in retinal cells, and (2) to examine the evidence that supports the two views of retinal energy metabolism. Measurements were made of lactic acid production, cellular ATP levels, and cellular morphology over 4 h. Results showed that all cell types incubated with 5 mM glucose produced lactate aerobically and anaerobically at linear rates, the anaerobic rate being 2-3-fold higher (Pasteur effect). Cells incubated with both 5 mM glucose and 10 mM lactate produced lactate aerobically and anaerobically at rates similar to those found when cells were incubated with glucose alone. Anaerobic ATP content in the cells was maintained at greater than 50% of the control, aerobic value, and cellular morphology was well preserved under all conditions. The results show that the cultured retinal cells produce lactate, even in the presence of a high starting ambient concentration of lactate. Thus, the net direction of the lactic dehydrogenase reaction is toward lactate formation rather than lactate utilization. It is concluded that retinal cells use glucose, and not glial derived lactate, as their major substrate.     

Regulation of potassium levels by Müller cells in the vertebrate retina

The membrane properties of Müller cells, the principal glial cells of the vertebrate retina, have been characterized in a series of physiological experiments on freshly dissociated cells. In species lacking a retinal circulation (tiger salamander, rabbit, guinea pig), the end-foot of the Müller cell has a much higher K+ conductance than do other cell regions. In species with retinal circulation (mouse, cat, owl monkey) the K+ conductance of the end-foot is greater than the conductance of the proximal process of the cell. In these species, however, the K+ conductance of the soma and distal process is equal to, or greater than, the end-foot conductance. Müller cells also possess four voltage-dependent ion channels, including an inward rectifying K+ channel. These membrane specializations may aid in the regulation of extracellular K+ levels by Müller cells in the retina. High end-foot conductance shunts excess K+ out through the end-foot, where it diffuses into the vitreous humor. In vascularized retinae, excess K+ may also be transferred to the ablumenal wall of capillaries, where it could be transported into the blood.     

Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma

Glaucoma is one of the leading eye diseases resulting in blindness due to the death of retinal ganglion cells. This study aimed to develop novel protocol to promote the differentiation of retinal Müller cells into ganglion cells in vivo in a rat model of glaucoma. The stem cells dedifferentiated from rat retinal Müller cells were randomized to receive transfection with empty lentivirus PGC-FU-GFP or lentivirus PGC-FU-Atoh7-GFP, or no transfection. The stem cells were induced further to differentiate. Ocular hypertension was induced using laser photocoagulation. The eyes were injected with Atoh7 expression vector lentivirus PGC-FU-Atoh7-GFP. Eyeball frozen sections, immunohistochemistry, RT-PCR, Western bolt, and apoptosis assay were performed. We found that the proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of the other two groups. The mean intraocular pressure of glaucomatous eyes was elevated significantly compared with those of contralateral eyes. Some retinal Müller cells in the inner nuclear layer entered the mitotic cell cycle in rat chronic ocular hypertension glaucoma model. Atoh7 contributes to the differentiation of retinal Müller cells into retinal ganglion cells in rat model of glaucoma. In conclusion, Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma, thus opening up a new avenue for gene therapy and optic nerve regeneration in glaucoma.     

Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling

Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y₁ and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase, P2Y₁, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic calcium responses and cellular volume.     

Effect of high glucose concentration on VEGF and PEDF expression in cultured retinal Müller cells

To explore the effect of high glucose concentration on the expression of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in the cultured rat retinal Müller cells. Rat Müller cells were cultured and RT-PCR and Western-blot analysis were used to measure the levels of VEGF and PEDF in cultured Müller cells at different high glucose concentrations. Under 10, 20, 30 mmol/L high glucose conditions, the levels of VEGF mRNA and protein increased and the levels of PEDF mRNA and protein decreased. These results suggest that the VEGF and PEDF expression in Müller cells are unbalance under high glucose concentration, which contribute to retinal neovascularization in diabetic retinopathy.     

Genome-wide analysis of Müller glial differentiation reveals a requirement for Notch signaling in postmitotic cells to maintain the glial fate

Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10-14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle.     

Dehydroascorbic acid uptake and intracellular ascorbic acid accumulation in cultured Müller glial cells (TR-MUL)

Vitamin C is mainly transported across the inner blood–retinal barrier (inner BRB) as dehydroascorbic acid (DHA) via a facilitative glucose transporter (GLUT) 1, and accumulates as ascorbic acid (AA) in the retina. Müller cells, huge glial cells, exhibit passive structural and metabolic functions for retinal neurons and the inner BRB. We characterized DHA transport and its corresponding transporter in a rat Müller cell line (TR-MUL5 cells). [¹⁴C]DHA uptake by TR-MUL5 cells took place in a time-dependent and Na⁺-independent manner. [¹⁴C]DHA uptake was inhibited by substrates and inhibitors of GLUTs, suggesting that Müller cells take up DHA via GLUTs. HPLC analysis revealed that most of the DHA taken up by TR-MUL5 cells was converted to AA and accumulated as AA in TR-MUL5 cells. [¹⁴C]DHA uptake by TR-MUL5 cells took place in a concentration-dependent manner with a Michaelis–Menten constant of 198 μM and was inhibited by cytochalasin B in a concentration-dependent manner with a 50% inhibition concentration of 0.283 μM. Although GLUT1, 3, and 4 mRNA are expressed in TR-MUL5 cells, quantitative real-time PCR revealed that GLUT1 mRNA expression was 5.85- and 116-fold greater than that of GLUT3 and 4, respectively. Western blot analysis supports the expression of GLUT1 protein with 45 kDa in TR-MUL5 cells. In conclusion, DHA is taken up by facilitative glucose transporters, most likely GLUT1, and converted to AA in TR-MUL5 cells.     

MAPK signaling during Müller glial cell development in retina explant cultures

The Müller cell is the only glial cell type generated from the retinal neuroepithelium. This cell type controls normal retina homeostasis and has been suggested to play a neuroprotective role. Recent evidence suggests that mammalian Müller cells can de-differentiate and return to a progenitor or stem cell stage following injury or disease. In vivo exploration of the molecular mechanisms of Müller cell differentiation and proliferation will add essential information to manipulate Müller cell functions. Signal transduction pathways that regulate Müller cell responses and activity are a critical part of their cellular machinery. In this study, we focus on mitogen-activated protein kinase (MAPK) signaling pathway during Müller glial cell differentiation and proliferation. We found that both MAPK and STAT3 signaling pathways are present during Müller glial cell development. Ciliary neurotrophic factor (CNTF)-stimulated Müller glial cell proliferation is associated with early developmental stages. Specific inhibition of MAPK phosphorylation significantly reduced the number of Müller glial cells with or without CNTF stimulation. These results suggested that the MAPK signal transduction pathway is important in the formation of Müller glial cells during retina development.