Purpose:This study was performed to determine whether angiopoietin (Ang)-1 and Ang-2 influence the survival of retinal neurons via Müller cell-mediated mechanisms. Methods:Expression of Ang-1, Ang-2, and Tie-2 in retinal cells and their hypoxia-dependent regulation were investigated using (quantitative) reverse transcription (RT)-PCR (qPCR), immunofluorescence staining, and Western blotting. Survival of Ang-stimulated R28 cells was assessed in homotypic cultures and co-cultures with rat Müller cells (RMCs). Ang-1, Ang-2, PEDF in RMCs and PEDF receptor (PEDF-R) in R28 cells were knocked down by siRNA. Tie-2-PI3 kinase (PI3K)/Akt activation was demonstrated using signaling blockers and by detecting Tie-2-Akt phosphorylation. PEDF expression and secretion were determined by qPCR and ELISA. Results:Ang-1, Ang-2, and Tie-2 are expressed in primary retinal cells, in particular, RMCs and human Müller cells of gliosis tissue. Under hypoxia, Ang-1 and Ang-2 expression declined in RMCs and R28 cell viability was reduced. Although both angiopoietins did not significantly influence R28 cell survival in homotypic cultures, co-cultures with Ang-deficient RMCs revealed that Müller cell-derived Ang-1 promoted, whereas Ang-2 impaired, R28 cell survival. Ang-1 induced Tie-2 and PI3K/Akt activation in RMCs and prevented hypoxia-induced PEDF downregulation. Ang-2 suppressed Tie-2-Akt phosphorylation and PEDF expression. Impaired R28 cell survival under hypoxia or caused by Ang-1 knockdown in co-cultured RMCs was mitigated by Ang-2-deficient RMCs or rescued by supplemental Ang-1 in a PEDF-/PEDF-R-dependent fashion. Conclusions:Ang-regulated signaling and PEDF expression constitute a significant pathway in Müller cells to control retinal neuronal survival. This has implications for effector functions of the therapeutic antibody, Faricimab.

PURPOSE . The purpose of this study was to examine possible involvement of vascular endothelial growth factor (VEGF) receptor (VEGFR)-1/Flt-1 in pigment epithelium- derived factor (PEDF)-promoted survival of retinal neurons. METHODS . Survival of growth factor-deprived retinal ganglion cells (RGCs) and R28 cells and activation of ERK-1/-2 MAP kinases were assessed in the presence of PEDF, placental growth factor (PlGF), and VEGF using cell cultures, viability assays and quantitation of ERK-1/-2 phosphorylation. VEGFR-1/Flt-1 expression was determined using quantitative PCR (qPCR) and Western blotting. VEGFR-1/Flt-1 was knocked down in R28 cells by small interfering RNA (siRNA). Binding of a PEDF-IgG Fc fusion protein (PEDF-Fc) to retinal neurons, immobilized VEGFR-1/Flt-1 and VEGFR-1/Flt-1-derived peptides was studied using binding assays and peptide scanning. RESULTS . PEDF in combination with PlGF stimulated increased cell survival and ERK1/-2 MAP kinase activation compared to effects of either factor alone. VEGFR-1/Flt-1 expression in RGCs and R28 cells was significantly upregulated by hypoxia, VEGF, and PEDF. VEGFR-1/Flt-1 ligands (VEGF and PlGF) or soluble VEGFR-1 (sflt-1) competed with PEDF-Fc for binding to R28 cells. Depleting R28 cells of VEGFR-1/Flt-1 resulted in reduced PEDF-Fc binding when comparing VEGFR-1/Flt-1 siRNAand control siRNAtreated cells. PEDF-Fc interacted with immobilized sflt-1, which was specifically blocked by VEGF and PlGF. PEDF-Fc binding sites were mapped to VEGFR-1/Flt-1 extracellular domains D3 and D4. Peptides corresponding to D3 and D4 specifically inhibited PEDFFc binding to R28 cells. These peptides and sflt-1 significantly inhibited PEDF-promoted survival of R28 cells. CONCLUSIONS . These results suggest that PEDF can target VEGFR-1/Flt-1 and this interaction plays a significant role in PEDF-mediated neuroprotection in the retina.

Progressive retinal ganglion cell (RGC) loss underlies a number of retinal neurodegenerative disorders, which may lead to permanent vision loss. However, secreted neuroprotective factors, such as PEDF, VEGF and IL-6, which are produced by Müller cells, have been shown to promote RGC survival. Assuming that the communication of RGCs with Müller cells involves a release of glioactive substances we sought to determine whether retinal neurons are able to modulate expression levels of Müller cell-derived PEDF, VEGF and IL-6. We demonstrate elevated mRNA levels of these factors in Müller cells in co-cultures with RGCs or R28 cells when compared to homotypic Müller cell cultures. Furthermore, R28 cells were more protected from apoptosis when co-cultured with Müller cells. IL-6 and VEGF were upregulated in Müller cells under hypoxia. Both cytokines, as well as PEDF, induced an altered neuronal expression of members of the Bcl-2 family, which are central molecules in the regulation of apoptosis. These results suggest that in retinal ischemia, via own secreted mediators, RGCs can resist a potential demise by stimulating Müller cells to increase production of neuroprotective factors, which counteract RGC apoptosis.

The demise of retinal ganglion cells (RGCs) is characteristic of diseases of the retina such as glaucoma and diabetic or ischemic retinopathies. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted protein that mediates neuroprotection and inhibition of angiogenesis in the retina. We have studied expression and regulation of two of several receptors for PEDF, patatin-like phospholipase 2 gene product/PEDF-R and laminin receptor (LR), in serum-starved RGC under normoxia and hypoxia and investigated their involvement in the survival of retinal neuronal cells. We show that PEDF-R and LR are co-expressed in RGC and R28 retinal precursor cells. Expression of both receptors was enhanced in the presence of complex secretions from retinal glial (Müller) cells and upregulated by VEGF and under hypoxic conditions. PEDF-R- and LR-knocked-down cells demonstrated a markedly attenuated expression of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL) and neuroprotective mediators (PEDF, VEGF, BDNF) suggesting that both PEDF-R and LR mediate pro-survival effects of PEDF on RGC. While this study does not provide evidence for a differential survival-promoting influence of either PEDF-R or LR, it nevertheless highlights the importance of both PEDF receptors for the viability of retinal neurons.

Age-relatedmacular degeneration (AMD) is a leading cause of blindness in people over 50 years of age in many developed countries. Drusen are yellowish extracellular deposits beneath retinal pigment epithelium (RPE) found in aging eyes and considered as a biomarker of AMD. However, the biogenesis of drusen has not been elucidated. We reported previously that multicellular spheroids of human RPE cells constructed a well-differentiated monolayer of RPE with a Bruch's membrane. We determined that RPE spheroids exhibited drusen formation between the RPE and Bruch's membrane with expression of many drusen-associated proteins, such as amyloid beta and complement components, the expression of which was altered by a challenge with oxidative stress. Artificial lipofuscin-loaded RPE spheroids yielded drusen more frequently. In the current study, we showed that drusen originates from the RPE. This culture system is an attractive tool for use as an in vitro drusen model, which might help elucidate the biogenesis of drusen and the pathogenesis of related diseases, such as AMD.

Cell surface molecules of retinal pigment epithelial (RPE) cells participate in the pathogenesis of retinal diseases. In an attempt to identify cell surface proteins that play a role in RPE cell-cell interactions, we have considered studying the expression, regulation, and signaling of ADGRE5/CD97, an adhesion G protein-coupled receptor family member, based on its known adhesive function in other cell types such as leukocytes. We showed that RPE cells express three isoforms of CD97 and identified inflammation-related cytokines as important mediators regulating CD97 expression. Whereas TNF-alpha and IFN-gamma upregulated CD97, TGF-beta decreased CD97 expression. Due to interaction with CD55, ARPE-19 cells firmly adhered to monocytes and T lymphocytes when overexpressing CD97, suggesting a role for CD97 in controlling leukocyte infiltration across the RPE-based blood-retinal barrier. CD97-mediated signaling acted synergistically with PDGF-BB and IFN-gamma to regulate cell growth and survival, ensuring a cellular balance under inflammatory conditions. These findings suggest that CD97 on RPE cells serves to control leukocyte activation and trafficking in uveoretinal inflammation while at the same time regulating second messenger-mediated gene expression, cell growth, and survival.

Introduction Muller glial cells carry out different tasks to warrant normal retinal functions. The aim of this study was to investigate if Muller cells also support retinal ganglion cells (RGC). Materials and Methods RGC were cultured for 24 hours in the presence or absence of Muller glial cells under normoxic (20% O-2, 5% CO2) or hypoxic (0.2% O-2, 5% CO2, 94.8% N-2) culture conditions. The number of vital RGC and the length of the newly developed neurites were evaluated. Results Under normoxic conditions, RGC vitality was significantly higher (p < 0.01) when cultured with Muller cells (62.85 +/- 2.06%) than without (47.29 +/- 2.83%). Under hypoxia, RGC vitality was significantly higher (p < 0.01) in co-cultures (41.07 +/- 2.28%) than in homotypic RGC cultures (28.49 +/- 2.16%). The maximum length of the newly developed neurites was found in the normoxic co-culture (90.7 +/- 7.4 mu m), but showed only a minor difference (p = 0.04) when compared to the normoxic homotypic RGC culture. Conclusion Muller glial cells support RGC under normoxic and hypoxic culture conditions. Length of newly developed neurites and number of surviving RGC are both parameters to evaluate cell vitality.

Zusammenfassung Einleitung Die Müllerʼschen Gliazellen übernehmen in der Netzhaut vielfältige Aufgaben und gewährleisten damit deren regelrechte Funktion. Wie stark die neuroprotektive Wirkung von Müller-Zellen auf retinale Ganglienzellen (RGC) ist, soll untersucht werden. Material und Methoden RGC wurden für 24 Stunden alleine oder in Kokultur mit Müllerʼschen Gliazellen unter normoxischen (20% O2, 5% CO2) und hypoxischen (0,2% O2, 5% CO2, 94,8% N2) Bedingungen kultiviert. Die Anzahl lebender RGC und die Länge der neu ausgebildeten Neuriten dienten zur Beurteilung der Zellvitalität. Ergebnisse Unter normoxischen Kulturbedingungen war die Anzahl vitaler RGC in Kokultur (62,85 ± 2,06%) signifikant höher (p < 0,01) als in der homotypischen RGC-Kultur (47,29 ± 2,83%). Unter Hypoxie war die Anzahl vitaler RGC in der Kokultur (41,07 ± 2,28%) ebenfalls signifikant höher (p < 0,01) als in der homotypischen RGC-Kultur (28,49 ± 2,16%). Die Länge der neu gebildeten Neuriten war in der normoxischen Kokultur am größten (90,7 ± 7,4 µm), zeigte aber nur im Vergleich zwischen normoxischer Kokultur und homotypischer RGC-Kultur einen signifikanten Unterschied (p = 0,04). Schlussfolgerung Müllerʼsche Gliazellen unterstützen das Überleben von RGC unter normoxischen und hypoxischen Bedingungen. Ein weiterer Parameter zur Bewertung der Zellvitalität neben der Anzahl lebender RGC ist die Länge der neu ausgebildeten Neuriten.

Background/ Aims: This study was performed to reveal signaling pathways exploited by pigment epithelium-derived factor (PEDF) derived from retinal (glial) Müller cells to protect retinal ganglion cells (RGCs) from cell death. Methods: The survival of RGCs was determined in the presence of conditioned culture media (MCM) from or in co-cultures with Müller cells. The significance of PEDF-induced STAT3 activation was evaluated in viability assays and using Western blotting analyses and siRNA-transfected cells. Results: Secreted mediators of Müller cells increased survival of RGCs under normoxia or hypoxia to a similar degree as of PEDF- or IL-6-exposed cells. PEDF and MCM induced an increased STAT3 activation in RGCs and R28 cells, and neutralization of PEDF in MCM attenuated STAT3 activation. Inhibition of STAT3 reduced PEDF-promoted survival of RGCs. Similar to IL-6, PEDF induced STAT3 activation, acting in a dose-dependent manner via the PEDF receptor (PEDF-R) encoded by the PNPLA2 gene. Ablation of PEDF-R attenuated MCM-induced STAT3 activation and compromised the viability of PEDF-exposed R28 cells. Conclusions: Müller cells are an important source of PEDF, which promotes RGC survival through STAT3 activation and, at least in part, via PEDF-R. Enhancing the secretory function of Müller cells may be useful to promote RGC survival in retinal neurodegenerative diseases.

Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

Background/Aims: By the release of antiangiogenic factors, Müller glial cells provide an angiostatic environment in the normal and ischemic retina. We determined whether Müller cells produce thrombospondin-1 (TSP-1), a known inhibitor of angiogenesis. Methods: Secretion of TSP-1 by cultured Müller cells was determined with ELISA. Slices of rat retinas and surgically excised retinal membranes of human subjects were immunostained against TSP-1 and the glial marker vimentin. The effects of TSP-1 on the growth of bovine retinal endothelial cells (BRECs) and activation of ERK1/2 were determined with DNA synthesis and migration assays, and Western blotting, respectively. Results: Cultured Müller cells secrete TSP-1 under normoxic and hypoxic (0.2% O2) conditions. Secretion of TSP-1 was increased in hypoxia compared to normoxia. In rat retinal slices, glial, retinal ganglion, and possibly horizontal cells were stained for TSP-1. Retinal glial cells in preretinal membranes from human subjects with nonhypoxic epiretinal gliosis (macular pucker) and proliferative diabetic retinopathy, respectively, were immunopositive for TSP-1. Exogenous TSP-1 reduced the VEGF-induced proliferation and migration of BRECs and decreased the phosphorylation level of ERK1/2 in BRECs. Conclusion: The data suggest that Müller cells are one major source of TSP-1 in the normal and ischemic retina. Glia-derived TSP1 may inhibit angiogenic responses in the ischemic retina.

The death of retinal ganglion cells (RGC) leads to visual impairment and blindness in ocular neurodegenerative diseases, primarily in glaucoma and diabetic retinopathy; hence, mechanisms that contribute to protecting RGC from ischemia/hypoxia are of great interest. We here address the role of retinal glial (Müller) cells and of pigment-epithelium-derived factor (PEDF), one of the main neuroprotectants released from the glial cells. We show that the hypoxia-induced loss in the viability of cultured purified RGC is due to apoptosis, but that the number of viable RGC increases when co-cultured with Müller glial cells suggesting that glial soluble mediators attenuate the death of RGC. When PEDF was ablated from Müller cells a significantly lower number of RGC survived in RGC-Müller cell co-cultures indicating that PEDF is a major survival factor allowing RGC to escape cell death. We further found that RGC express a PEDF receptor known as patatin-like phospholipase domain-containing protein 2 (PNPLA2) and that PEDF exposure, as well as the presence of Müller cells, leads to an activation of nuclear factor (NF)-κB in RGC. Furthermore, adding an NF-κB inhibitor (SN50) to PEDF-treated RGC cultures reduced the survival of RGC. These findings strongly suggest that NF-κB activation in RGC is critically involved in the pro-survival action of Müller-cell derived PEDF and plays an important role in maintaining neuronal survival.

Neovascularization is a sight‐threatening complication of ischemic proliferative retinopathies. Transforming growth factor (TGF)‐β, a cytokine with multiple functions in the retina, participates in the control of pathological angiogenesis and neovascularization. Retinal glial (Müller) cells produce TGF‐β2 under physiological and post‐ischemic conditions. To characterize glial cell‐derived mediators of angiogenesis regulation in glial‐endothelial interactions in the retina, we co‐cultured primary Müller cells and bovine microvascular retinal endothelial cells (BRECs). Müller cell‐derived TGF‐β2 was bound by the BRECs, which were found to express serine/threonine kinase TGF‐β receptors, and stimulated TGF‐β‐dependent anti‐proliferative signaling pathways. The proliferation of BRECs was attenuated by exogenous TGF‐β2 as well as by the presence of Müller cell culture media. The following intracellular signaling mechanisms were found to be involved in the anti‐angiogenic action of Müller cell‐derived TGF‐β2: (i) binding of TGF‐β2 to BRECs is mediated by the type‐II TGF‐β receptor, leading to (ii) activation and phosphorylation of receptor‐activated Smads; (iii) Müller cell‐derived TGF‐β2 activates Smad2 and Smad3 to (iv) attenuate the phosphorylation state of the MAP kinases, extracellular signal‐regulated kinase (ERK)‐1/‐2. Neutralizing TGF‐β or TGF‐β type‐II receptor or blocking the activation of Smads partially abrogated the effect of Müller cell‐conditioned media on BRECs. Together, our data suggest that Müller cells release TGF‐β2, inhibiting the proliferation of retinal endothelial cells via activation of Smad2/Smad3 and attenuation of ERK signaling. Given the context‐dependent action of TGF‐β2 on angiogenesis, our results may have implications for understanding the pathogenesis of retinal angiopathies, such as diabetic retinopathy, and the anti‐angiogenic role of TGF‐β therein. GLIA 2014;62:1476–1485

Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine with pro-angiogenic and neurotrophic effects. The angioregulatory role of this molecule may become especially significant in retinal neovascularization, which is a hallmark of a number of ischemic eye diseases. This study was undertaken to reveal expression characteristics of bFGF, produced by retinal glial (Müller) cells, and to determine conditions under which glial bFGF may stimulate the proliferation of retinal microvascular endothelial cells. Immunofluorescence labeling detected bFGF in Müller cells of the rat retina and in acutely isolated Müller cells with bFGF levels, which increased after ischemia-reperfusion in postischemic retinas. In patients with proliferative diabetic retinopathy or myopia, the immunoreactivity of bFGF co-localized to glial fibrillary acidic protein (GFAP)-positive cells in surgically excised retinal tissues. RT-PCR and ELISA analyses indicated that cultured Müller cells produce bFGF, which is elevated under hypoxia or oxidative stress, as well as under stimulation with various growth factors and cytokines, including pro-inflammatory factors. When retinal endothelial cells were cultured in the presence of media from hypoxia (0.2%)-conditioned Müller cells, a distinct picture of endothelial cell proliferation emerged. Media from 24-h cultured Müller cells inhibited proliferation, whereas 72-h conditioned media elicited a stimulatory effect. BFGF-neutralizing antibodies suppressed the enhanced endothelial cell proliferation to a similar extent as anti-VEGF antibodies. Furthermore, phosphorylation of extracellular signal-regulated kinases (ERK-1/-2) in retinal endothelial cells was increased when the cells were cultured in 72-h conditioned media, while neutralizing bFGF attenuated the activation of this signaling pathway. These data provide evidence that retinal (glial) Müller cells are major sources of bFGF in the ischemic retina. Müller cells under physiological conditions or transient hypoxia seem to provide an anti-angiogenic environment, but long-lasting hypoxia causes the release of bFGF, which might significantly co-stimulate neovascularization in the retina.

PURPOSE:Aging changes in the RPE involve lipid accumulation and membranous basal deposits onto the underlying Bruch's membrane, which may be related to AMD. Conventional in vitro cell culture is limited in its ability to observe the epithelial functions on the basal side. The purpose of this study was to develop a three-dimensional culture system to observe basolateral functions of the RPE.METHODS:Isolated human RPE cells were cultured in a viscous medium on a rounded-bottom culture dish, resulting in spheroid formation. The appearance and size of the spheroids were assessed by light microscopy. Spheroids were fixed in 4% paraformaldehyde for immunohistochemistry or sampled for Western blotting. For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), spheroids were postfixed in 1% osmium tetroxide.RESULTS:The spheroids had a differentiated RPE monolayer with a thin elastic layer, a main layer of Bruch's membrane, on their surface and showed outward deposition of lipoproteins with apoB-100. TEM revealed widely spaced collagen, which was identified as condensation of collagen fibrils by SEM. SEM showed deposition of membranous debris and lipid particles, which have been observed in human Bruch's membrane. Western blotting showed expression of RPE differentiation markers and components of Bruch's membrane and RPE lipoproteins.CONCLUSIONS:This model provides direct views of epithelialization processes involving elastogenesis and functions at the basolateral side such as lipoprotein deposition and may elucidate not only unknown epithelial behaviors but also the pathogenesis of RPE-related diseases.

Retinal glial (Müller) cells are involved in a wide range of developmental mechanisms, including axon guidance and angiogenesis. This study was undertaken to explore whether Netrin‐4, an axonal guidance molecule, is expressed by Müller cells and promotes angiogenesis‐related activities. Netrin‐4 was found through all retinal layers, and its expression was demonstrated in Müller cells, retinal pigment epithelium cells and bovine retinal endothelial cells (BRECs). Co‐localization of Netrin‐4 with Müller cell‐specific molecules [cellular retinaldehyde‐binding protein (cRALBP), vimentin] was observed in the ganglion cell layer, nerve fiber layer, and at the outer limiting membrane. Under hypoxic conditions, the release of Netrin‐4 from Müller cells was increased, with mRNA levels upregulated in a hypoxia‐inducible factor‐1‐dependent manner and dependent on the concomitantly induced release of vascular endothelial growth factor. These findings were consistent with an intensified immunofluorescence of Netrin‐4 labeling in the postischemic retinas after ischemia‐reperfusion. Netrin‐4 stimulated BRECs to increase phosphorylation of the mitogen‐activated protein (MAP) kinases, extracellular signal‐regulated kinase (ERK)‐1/‐2, and p38, in a dose‐dependent manner. Synthetic inhibitors of the MAP kinases were able to suppress Netrin‐4‐induced migration and proliferation of BRECs suggesting that both MAP kinases are differentially involved in Netrin‐4‐induced angiogenesis. Two receptors for Netrins, i.e., deleted in colorectal cancer (DCC) and uncoordinated‐5‐homolog 1 (Unc5H1), were detected in BRECs. DCC is at least partially required for Netrin‐4‐induced activation of ERK‐1/‐2. These data suggest that Müller glial cells contribute to, and may modulate, retinal Netrin‐4 levels. This may be a novel pathway of Müller cell‐mediated control of retinal angiogenesis, particularly under hypoxic/ischemic conditions when the cells upregulate Netrin‐4 expression. © 2012 Wiley Periodicals, Inc.

Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Müller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.