雌激素对缺氧视网膜Müller细胞PEDF和VEGF表达的影响

目的：探讨雌激素对缺氧视网膜Müller细胞色素上皮衍生因子（PEDF）和血管内皮生长因子（VEGF）表达的影响。方法：采用反转录聚合酶链反应（RT-PCR）和Western-blot印迹分析方法分别测定不同浓度雌二醇（E2）作用于缺氧Müller细胞后,细胞内PEDF mRNA,VEGF mRNA及相应的蛋白表达水平。结果：缺氧24h后PEDFmRNA及蛋白表达明显降低,10-5mmol/L和10-6mmol/LE2作用于Müller细胞后可明显缓解由于缺氧引起的细胞内PEDF mRNA及蛋白表达的降低,并与E2的浓度有关。缺氧24h后VEGF mRNA及蛋白表达明显升高,10-5mmol/L和10-6mmol/LE2作用于Müller细胞后可以明显降低细胞内VEGF mRNA及蛋白表达水平,并与E2的浓度有关。结论：雌激素可以调控缺氧条件下视网膜Müller细胞内PEDF和VEGF的表达,对视网膜病理性新生血管的形成具有保护作用。     

Role for extracellular signal-responsive kinase-1 and -2 in retinal angiogenesis

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent mitogen for micro- and macrovascular endothelial cells (ECs). Evidence points to a possible role for two mitogen-activated protein (MAP) kinases, the extracellular-signal responsive kinases (ERK)-1 and -2, in VEGF signaling in ECs. This study was undertaken to begin to define the precise role of MAP kinases in VEGF signal transduction related to angiogenesis. METHODS: Bovine retinal microvascular endothelial cells (BRMECs) and a well-established rat model of retinopathy of prematurity (ROP) were used to investigate the role of ERK-1/2 in EC proliferation and tube formation and in retinal angiogenesis in vivo. RESULTS: Administration of VEGF to BRMEC cultures increased ERK-1/2 phosphorylation, cell proliferation, and tube formation in a dose-dependent manner. Phosphorylation of retinal ERK-1/2 also was increased in the ROP model. An inhibitor of ERK, AG126, and an inhibitor of ERK kinase (MEK), PD98059, exhibited a dose-dependent reduction of ERK phosphorylation and EC proliferation, but not tube formation, in VEGF-stimulated BRMECs. In the ROP model, intravitreous injection of 10 micro M AG126 or PD98059 reduced the retinal neovascular area by 71% and 48%, respectfully. No effect was seen on intraretinal blood vessel growth. CONCLUSIONS: These experiments point to a critical role for ERK and MEK in proliferation of ECs, but not in tube formation. Furthermore, inhibition of either of these two signal intermediates can significantly retard retinal neovascularization. This suggests that the MAPK pathway may provide rational targets for therapeutic intervention in ocular and other diseases with an angiogenic component.     

高糖对视网膜Müller细胞VEGF,EPO和EPOR mRNA表达的影响

目的:观察高糖条件下VEGF mRNA,EPO mRNA和EPORmRNA在体外培养的Mller细胞中的表达情况。方法:胰蛋白酶将新生小鼠视网膜组织吹打消化后,制成单细胞悬液,体外培养Mller细胞,RT-PCR测定高糖条件下视网膜Mller细胞VEGF,EPO和EPOR基因的表达。结果:成功获得视网膜Mller细胞,传代后90%以上的细胞呈兔抗鼠谷氨酰胺合酶(GS)染色阳性。Mller细胞VEGF mRNA,EPO mRNA和EPOR mRNA在高糖条件下表达升高,且呈浓度依赖性(P<0.05),但糖浓度50mmol/L组较40mmol/L组差异无统计学意义,无明显时间依赖性。结论:Mller细胞在高糖条件下VEGF,EPO,EPOR的表达增加。     

Effects of high-concentration insulinon expressionof vascular endothelial groｗth factor in cultured Ｍüller cells in vitro

to studythe effectof high-concentration insulinonthe exPressionof vascular endothelial growth factor (VEGF) in cultured rabbit retinal Müller cells in vitro . METHODS: Müller cells were cultured with insulinof different concentrations (4×10³ , 8×10³ , 12×10³ U/L). Immunocytochemistry,in situ hybridization and ELISA were conductedto assaythe exPressionof VEGF in cultured Müller cells in vitro at different insulin concentrations qualitatively and quantitatively. RESULTS: VEGF exPression was enhancedobviously by high concentrationof insulin. CONCLUSION: Insulin Plays an imPortant role in neovascularizationof diabetic retinoPathy(DR) by stimulatingthetranscriPtionofthe VEGF gene in Müller cells so asto enhancethe exPressionof VEGF Protein.     

Müller Cells Do Not Influence Leukocyte Adhesion to Retinal Endothelial Cells

Purpose: Diabetic retinopathy is associated with inflammation. The authors investigated the influence of Müller cells on leukocyte adhesion to retinal endothelial cells. Methods: ICAM-1 levels were assessed by Western blotting and immunocytochemistry. Leukocyte adhesion was quantified using a fluorescence assay. Results: High glucose and oxidative/nitrosative stress conditions increased ICAM-1 levels in endothelial cells and leukocyte adhesion. Under the influence of Müller cells (co-cultures/conditioned medium), the effects were comparable to those found when endothelial cells were exposed, alone, to similar conditions. Conclusions: These results show that Müller cells do not influence leukocyte adhesion under the in vitro conditions used in this study.     

Müller cells in the healthy and diseased retina

Müller glial cells span the entire thickness of the tissue, and ensheath all retinal neurons, in vertebrate retinae of all species. This morphological relationship is reflected by a multitude of functional interactions between neurons and Müller cells, including a 'metabolic symbiosis' and the processing of visual information. Müller cells are also responsible for the maintenance of the homeostasis of the retinal extracellular milieu (ions, water, neurotransmitter molecules, and pH). In vascularized retinae, Müller cells may also be involved in the control of angiogenesis, and the regulation of retinal blood flow. Virtually every disease of the retina is associated with a reactive Müller cell gliosis which, on the one hand, supports the survival of retinal neurons but, on the other hand, may accelerate the progress of neuronal degeneration: Müller cells protect neurons via a release of neurotrophic factors, the uptake and degradation of the excitotoxin, glutamate, and the secretion of the antioxidant, glutathione. However, gliotic Müller cells display a dysregulation of various neuron-supportive functions. This contributes to a disturbance of retinal glutamate metabolism and ion homeostasis, and causes the development of retinal edema and neuronal cell death. Moreover, there are diseases evoking a primary Müller cell insufficiency, such as hepatic retinopathy and certain forms of glaucoma. Any impairment of supportive functions of Müller cells, primary or secondary, must cause and/or aggravate a dysfunction and loss of neurons, by increasing the susceptibility of neurons to stressful stimuli in the diseased retina. On the contrary, Müller cells may be used in the future for novel therapeutic strategies to protect neurons against apoptosis (somatic gene therapy), or to differentiate retinal neurons from Müller/stem cells. Meanwhile, a proper understanding of the gliotic responses of Müller cells in the diseased retina, and of their protective vs. detrimental effects, is essential for the development of efficient therapeutic strategies that use and stimulate the neuron-supportive/protective-and prevent the destructive-mechanisms of gliosis.     

Pigment epithelium derived factor as an anti-inflammatory factor against decrease of glutamine synthetase expression in retinal Müller cells under high glucose conditions

Background  

The pathogenesis of diabetic retinopathy (DR) is similar to that of a chronic inflammatory disease. A predominant function of Müller cells is to regulate glutamate levels, but in DR the function is compromised. The present study was performed to investigate the role of pigment epithelial derived factor (PEDF) on the expression of glutamine synthetase (GS) in rat retinal Müller cells under high glucose conditions, and to study the possible mechanism for PEDF against decrease of GS expression in retinal Müller cells under high glucose conditions.     

Müller细胞在糖尿病视网膜病变中的研究进展

Müller细胞是脊椎动物视网膜最主要的神经胶质细胞,从内界膜到外界膜纵贯视网膜全层,参与构成血-视网膜屏障,积极参与视网膜发育并通过许多细胞内机制促进和维持视网膜稳态。Müller细胞在糖尿病视网膜病变的发生发展过程中扮演重要角色,其病理生理改变仍有待深入研究。本文就Müller细胞在糖尿病视网膜病变中的病理生理改变以及近年研究进展作一综述。     

Dystrobrevin localization in photoreceptor axon terminals and at blood-ocular barrier sites

PURPOSE: Dystrobrevin is a newly discovered dystrophin-associated protein with multiple sites for phosphorylation on tyrosine residues. In the present study, the cellular distribution and subcellular localization of dystrobrevin were examined in the adult rat retina, cornea, lens, iris, ciliary body, and cultured Müller cells. METHODS: Immunoblot analysis, confocal laser scanning microscopy, and immunoelectron microscopy were used to examine dystrobrevin expression. RESULTS: Immunoblot analysis showed that an approximately 87-kDa band was expressed predominantly in the lens, retina, iris and ciliary body, whereas an approximately 60-kDa band was expressed in cultured Müller cells, cornea, retina, iris, and ciliary body. Confocal microscopy demonstrated dystrobrevin in the inner limiting membrane, outer plexiform layer, and retinal pigment epithelium and around blood vessels in the retina. At the ultrastructural level, dystrobrevin was localized under cell membranes of rod spherules and cone pedicles of photoreceptor cell terminals but often was found in the cytoplasm of endothelial cells and Müller cells. Furthermore, dystrobrevin was colocalized with beta-dystroglycan in corneal endothelium; lens, iris, and ciliary epithelia; and cultured Müller cells. CONCLUSIONS: The present study demonstrates that dystrobrevin is expressed in neurons, glia, and endothelial cells in the rat retina. In addition, dystrobrevin is localized at the blood-ocular barrier sites in extraocular tissue. These data suggest that dystrobrevin plays an important role in visual function.     

The significance of neuronal and glial cell changes in the rat retina during oxygen-induced retinopathy

Retinopathy of prematurity is a devastating vascular disease of premature infants. A number of studies indicate that retinal function is affected in this disease. Using the rat model of oxygen-induced retinopathy, it is possible to explore more fully the complex relationship between neuronal, glial and vascular pathology in this condition. This review examines the structural and functional changes that occur in the rat retina following oxygen-induced retinopathy. We highlight that vascular pathology in rats is characterized by aberrant growth of blood vessels into the vitreous at the expense of blood vessel growth into the body of the retina. Moreover, amino acid neurochemistry, a tool for examining neuronal changes in a spatially complete manner reveals widespread changes in amacrine and bipolar cells. In addition, neurochemical anomalies within inner retinal neurons are highly correlated with the absence of retinal vessels. The key cell types that link blood flow with neuronal function are macroglia. Macroglia cells, which in the retina include astrocytes and Müller cells, are affected by oxygen-induced retinopathy. Astrocyte loss occurs in the peripheral retina, while Müller cells show signs of reactive gliosis that is highly localized to regions that are devoid of intraretinal blood vessels. Finally, we propose that treatments, such as blockade of the renin–angiotensin system, that not only targets pathological angiogenesis, but that also promotes re-vascularization of the retina are likely to prove important in the treatment of those with retinopathy of prematurity.     

Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase: a role in high glucose-induced apoptosis in retinal Müller cells

PURPOSE: A recent study demonstrated that retinal Müller cells undergo hyperglycemia-induced apoptosis in vitro. Translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the cytosol to the nucleus is a critical step in the induction of apoptosis in neuronal cells. R-(-)-deprenyl prevents nuclear translocation of GAPDH and subsequent apoptosis in neuronal cells. In this study, the role of nuclear translocation of GAPDH in hyperglycemia-induced apoptosis in retinal Müller cells and the ability of R-(-)-deprenyl to inhibit the translocation of GAPDH and apoptosis were investigated. METHODS: Transformed rat Müller cells (rMC-1) and isolated human Müller cells were cultured in normal glucose, high glucose, and high glucose plus R-(-)-deprenyl for up to 5 days. Subcellular distribution of GAPDH was determined in vitro and in vivo by immunocytochemistry. Apoptosis in tissue cultures was determined by annexin-V staining and caspase-3 activity. RESULTS: Hyperglycemia significantly increased the amount of GAPDH protein in the nucleus above normal within the first 48 hours in rMC-1 and human Müller cells. The addition of R-(-)-deprenyl to these cells incubated in high glucose reduced the amount of GAPDH protein in the nucleus and decreased hyperglycemia-induced apoptosis in both cell types. In vivo studies confirmed the accumulation of GAPDH in nuclei of Müller cells in diabetes. CONCLUSIONS: The nuclear translocation of GAPDH in rMC-1 and human Müller cells is closely associated with the induction of apoptosis. R-(-)-deprenyl inhibits nuclear accumulation of GAPDH and subsequent apoptosis in these cells. Therefore, R-(-)-deprenyl offers a strategy to explore the role of GAPDH translocation into the nucleus in the development of diabetic retinopathy.     

Ciliary neurotrophic factor induces glial fibrillary acidic protein in retinal Müller cells through the JAK/STAT signal transduction pathway

PURPOSE: Intravitreal injection of ciliary neurotrophic factor (CNTF) is known to induce glial intermediate filament protein (GFAP) expression in retinal Müller cells. Because CNTF binding can activate multiple signaling kinases, we have examined the involvement of JAK/STAT pathway in GFAP induction in Müller cells. METHODS: CNTF was injected intravitreally into mouse eyes. Immunocytochemistry and immunoblotting were used to study GFAP and STAT3-p (phosphorylated STAT3) levels either in mouse eyes, retinal explant cultures or in a Müller cell line, rMC-1. RESULTS: In protein extracts of CNTF-injected eyes, retinal explants and the Müller cells, there was a substantial increase in STAT3-p level. Immunocytochemistry showed that STAT3-p was now present in many cell bodies in the INL and the GCL. To prove that CNTF acted via the JAK-STAT pathway, rMC-1 cells were transfected with a dominant-negative STAT3 mutant prior to treatment with CNTF. In the immunoblots of transfected cells, there was decrease in GFAP level. CONCLUSIONS: The results establish that CNTF can induce GFAP expression in retinal Müller cells through the JAK/STAT signaling pathway.     

Quantitative properties of Müller cells in rabbit retina as revealed by histochemical demonstration of NADH-diaphorase activity

Müller cells in rabbit retina were selectively stained by demonstrating NADH-diaphorase activity. After the sections were measured by light microscopy, the following properties of Müller cells were found: (a) independent of the retinal localization, one Müller cell corresponds with a retinal volume of 15,000 μm³, i.e., with 11 photoreceptor cells, 2 neurons of the inner nuclear layer, and 0.3–1 ganglion cells; (b) the Müller cell population represents about 6.8% of the total retinal volume; (c) the sorbitol accumulation of Müller cells in diabetic retinopathy is said to be unable to cause osmotic damage.     

Diabetes-induced dysfunction of the glutamate transporter in retinal Müller cells

PURPOSE: A decrease in the ability of Müller cells to remove glutamate from the extracellular space may play a critical role in the disruption of glutamate homeostasis that occurs in the diabetic retina. Because this amino acid is toxic to retinal neurons and is likely to exacerbate oxidative stress, elucidation of the mechanisms by which glutamate levels are elevated in diabetes may help in the understanding of the pathogenesis of diabetic retinopathy. This study tested the hypothesis that the function of the glutamate transporter in Müller cells of the diabetic retina is compromised by a mechanism involving oxidation. METHODS: Müller cells were freshly isolated from normal rats and those made diabetic by streptozotocin injection. The activity of the Müller cell glutamate transporter, which is electrogenic, was monitored by the perforated-patch configuration of the patch-clamp technique. RESULTS: Four weeks after the onset of hyperglycemia, a significant dysfunction of the Müller cell glutamate transporter was detected. After 13 weeks of streptozotocin-induced diabetes, the activity of this transporter was decreased by 67%. Consistent with oxidation's causing this dysfunction, exposure to a disulfide-reducing agent rapidly restored the activity of the glutamate transporter in Müller cells of diabetic retinas. CONCLUSIONS: Early in the course of diabetic retinopathy, the function of the glutamate transporter in Müller cells is decreased by a mechanism that is likely to involve oxidation.     

Pigment epithelial cell changes precede vascular transformations in the dystrophic rat retina.

In the Royal College of Surgeons rat with inherited retinal dystrophy, vascularization of the retinal pigment epithelium (RPE) is preceded by migration and proliferation of Müller cell processes in the subretinal space where they contact the RPE. Later, RPE cells envelope subretinal vessels which have lost their perivascular Müller cell sheath. To characterize RPE cell changes and interactions in relation to glial and vascular transformations in retinal dystrophy, we used immunocytochemical techniques and antibodies against cytokeratin (CK) and glial fibrillary acidic protein (GFAP). Prior to the proliferation of Müller cell processes in the dystrophic retina, CK filaments in RPE cells formed a circumferential meshwork with intense cytoplasmic and perinuclear labeling as in control RPE cells. Following entry of Müller cell processes into the membranous debris zone and formation of RPE-Müller cell contact, RPE cells became pleomorphic and extended prominent apical processes in the debris zone. Some CK-reactive RPE cells detached from Bruch's membrane and migrated into the debris zone. Electron microscopic study showed extensive areas of close RPE-Müller cell contact at this time. Obvious junctional specializations of the plasma membranes were not seen but prominent tubulo-vesicular profiles occupied the cytoplasm of altered RPE and Müller cell processes. Following RPE vascularization, hypertrophic CK-positive cells surrounded blood vessels and accompanied them into the inner retina. Electron microscopic analysis showed that RPE-associated vessels were fenestrated and devoid of their perivascular glial sheath. Apparent proliferation of RPE cells and redistribution of CK filaments were observed. Our study shows that RPE cell alterations accompany Müller cell and vascular changes which result in altered RPE-Müller cell and RPE-endothelial cell relationships in the dystrophic rat retina. The altered relationships among RPE, Müller and endothelial cells may result in increased cellular interaction and promote proliferation and transformation of all three cells types in diseased retinas.     

The pathogenesis of vitreoretinal diseases from the standpoint of molecular biology

It is important to study the pathogenesis in vitreoretinal diseases to develop new medical therapy. We investigated the molecular mechanisms underlying the genesis of idiopathic macular hole, exudative age-related macular degeneration (AMD), and diabetic retinopathy. We observed high levels of chymase and tryptase activity in the vitreous humor of patients with idiopathic macular hole. This activity was significantly higher than in other vitreoretinal diseases. Immunohistochemical study using monkey eyes showed the possibility that Müller cells in foveal lesions have properties similar to retinal stem cells. Intravitreal injection of chymase induced apoptosis of foveal retinal cells and fibrous change of vitreoretinal interface in the macular area. Biochemical study using cultured human Müller cells revealed that chymase caused the inhibition of growth and the induction of apoptosis in dedifferentiated Müller cells treated with basic fibroblast growth factor (bFGF). These findings show that increased production of chymase and tryptase in mast cells could be related to the pathogenesis of idiopathic macular hole. Oxidative stress and arterosclerosis may be the major causes of exudative AMD. Paraoxonase (PON) is a polymorphic protein known to prevent oxidation of low-density lipoprotein (LDL). We analyzed PON genotypes and found that two types of polymorphism were significantly different between patients with AMD and control subjects. We also investigated serum oxidized low-density lipoprotein(oxLDL) levels, PON activity, and extracellular superoxide dismutase(EC-SOD) levels. All these factors were significantly higher in patients with AMD than in controls. Titers of IgA and IgG antibodies against chlamydia pneumoniae in the serum of AMD patients were also significantly higher than in controls. These results indicate that genetic factors related to PON polymorphisms, vascular damage caused by increment of serum oxLDL and malfunction of EC-SOD, and chronic inflammation provoked by clamydia pneumoniae infection may be involved in the pathogenesis of AMD. Excess accumulation of advanced glycation end products(AGEs) has a causative role in the development of diabetic complications. We determined the concentrations of three AGEs (pentosidine, carboxy-methyllysine, and crossline) and two cytokines (VEGF, IL-6) using ELISA. The levels of the three AGEs and two cytokines in the vitreous of patients with proliferative diabetic retinopathy(PDR) were significantly higher than in controls. The concentrations of VEGF and IL-6 were strongly correlated with the level of these AGEs. Cultured human Müller cells expressed both VEGF and IL-6 mRNA and these expressions were augmented after the treatment of AGEs, while also acting as photosensitizers and accelerating the degradation of hyaluronic acid in vitro. AGEs may consequently play an important role in the pathogenesis of diabetic retinopathy by inducing the production of VEGF and IL-6 in retinal Müller cells and the acceleration of vitreous liquefaction.