Human Optic Nerve Head Astrocytes Research Articles

The endothelin receptor antagonist macitentan ameliorates endothelin-mediated vasoconstriction and promotes the survival of retinal ganglion cells in rats.

Glaucoma is a chronic and progressive eye disease, commonly associated with elevated intraocular pressure (IOP) and characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells (RGCs). The pathological changes in glaucoma are triggered by multiple mechanisms and both mechanical effects and vascular factors are thought to contribute to the etiology of glaucoma. Various studies have shown that endothelin-1 (ET-1), a vasoactive peptide, acting through its G protein coupled receptors, ETA and ETB, plays a pathophysiologic role in glaucoma. However, the mechanisms by which ET-1 contribute to neurodegeneration remain to be completely understood. Our laboratory and others demonstrated that macitentan (MAC), a pan endothelin receptor antagonist, has neuroprotective effects in rodent models of IOP elevation. The current study aimed to determine if oral administration of a dual endothelin antagonist, macitentan, could promote neuroprotection in an acute model of intravitreal administration of ET-1. We demonstrate that vasoconstriction following the intravitreal administration of ET-1 was attenuated by dietary administration of the ETA/ETB dual receptor antagonist, macitentan (5 mg/kg body weight) in retired breeder Brown Norway rats. ET-1 intravitreal injection produced a 40% loss of RGCs, which was significantly lower in macitentan-treated rats. We also evaluated the expression levels of glial fibrillary acidic protein (GFAP) at 24 h and 7 days post intravitreal administration of ET-1 in Brown Norway rats as well as following ET-1 treatment in cultured human optic nerve head astrocytes. We observed that at the 24 h time point the expression levels of GFAP was upregulated (indicative of glial activation) following intravitreal ET-1 administration in both retina and optic nerve head regions. However, following macitentan administration for 7 days after intravitreal ET-1 administration, we observed an upregulation of GFAP expression, compared to untreated rats injected intravitreally with ET-1 alone. Macitentan treatment in ET-1 administered rats showed protection of RGC somas but was not able to preserve axonal integrity and functionality. The endothelin receptor antagonist, macitentan, has neuroprotective effects in the retinas of Brown Norway rats acting through different mechanisms, including enhancement of RGC survival and reduction of ET-1 mediated vasoconstriction.

Sphingomyelinases in retinas and optic nerve heads: Effects of ocular hypertension and ischemia

Sphingomyelinases (SMase), enzymes that catalyze the hydrolysis of sphingomyelin to ceramide, are important sensors for inflammatory cytokines and apoptotic signaling. Studies have provided evidence that increased SMase activity can contribute to retinal injury. In most tissues, two major SMases are responsible for stress-induced increases in ceramide: acid sphingomyelinase (ASMase) and Mg2+-dependent neutral sphingomyelinase (NSMase). The purposes of the current study were to determine the localization of SMases and their substrates in the retina and optic nerve head and to investigate the effects of ocular hypertension and ischemia on ASMase and NSMase activities.Tissue and cellular localization of ASMase and NSMase were determined by immunofluorescence imaging. Tissue localization of sphingomyelin in retinas was further determined by Matrix-Assisted Laser Desorption/Ionization mass spectrometry imaging. Tissue levels of sphingomyelins and ceramide were determined by liquid chromatography with tandem mass spectrometry. Sphingomyelinase activities under basal conditions and following acute ischemic and ocular hypotensive stress were measured using the Amplex Red Sphingomyelinase Assay Kit. Our data show that ASMase is in the optic nerve head and the retinal ganglion cell layer. NSMase is in the optic nerve head, photoreceptor and retinal ganglion cell layers. Both ASMase and NSMase were identified in human induced pluripotent stem cell-derived retinal ganglion cells and optic nerve head astrocytes. The retina and optic nerve head each exhibited unique distribution of sphingomyelins with the abundance of very long chain species being higher in the optic nerve head than in the retina. Basal activities for ASMase in retinas and optic nerve heads were 54.98 ± 2.5 and 95.6 ± 19.5 mU/mg protein, respectively. Ocular ischemia significantly increased ASMase activity to 86.2 ± 15.3 mU/mg protein in retinas (P = 0.03) but not in optic nerve heads (81.1 ± 15.3 mU/mg protein). Ocular hypertension significantly increased ASMase activity to 121.6 ± 7.3 mU/mg protein in retinas (P < 0.001) and 267.0 ± 66.3 mU/mg protein in optic nerve heads (P = 0.03). Basal activities for NSMase in retinas and optic nerve heads were 12.3 ± 2.1 and 37.9 ± 8.7 mU/mg protein, respectively. No significant change in NSMase activity was measured following ocular ischemia or hypertension.Our results provide evidence that both ASMase and NSMase are expressed in retinas and optic nerve heads; however, basal ASMase activity is significantly higher than NSMase activity in retinas and optic nerve heads. In addition, only ASMase activity was significantly increased in ocular ischemia or hypertension. These data support a role for ASMase-mediated sphingolipid metabolism in the development of retinal ischemic and hypertensive injuries.

Evidence of an AnnexinA4 mediated plasma membrane repair response to biomechanical strain associated with glaucoma pathogenesis.

Glaucoma is a common neurodegenerative blinding disease that is closely associated with chronic biomechanical strain at the optic nerve head (ONH). Yet, the cellular injury and mechanosensing mechanisms underlying the resulting damage have remained critically unclear. We previously identified Annexin A4 (ANXA4) from a proteomic analyses of human ONH astrocytes undergoing pathological biomechanical strain that mimics glaucomatous conditions. Annexins are a family of calcium-dependent phospholipid binding proteins with key functions in plasma membrane repair (PMR); an active mechanism to limit and mend cellular injury that involves membrane and cytoskeletal reorganizations. However, a role for direct membrane damage and PMR has not been well studied in the context of biomechanical strain, such as that associated with glaucoma. Here we report that this moderate strain surprisingly damages cell membranes to increase permeability in a calcium-dependent manner, and induces rapid aggregation of ANXA4 at injury sites. ANXA4 loss-of-function increases permeability, while exogenous ANXA4 reduces it. Furthermore, ANXA4 aggregation is associated with F-actin dynamics in vitro, and remarkably this interaction and aggregation signature is also observed in the glaucomatous ONH in patient samples. Together these studies link moderate biomechanical strain with direct membrane damage and actin dynamics, and identify an active PMR role for ANXA4 in new model of cell injury associated with glaucoma pathogenesis.

Evidence for ceramide induced cytotoxicity in retinal ganglion cells

Ceramides are bioactive compounds that play important roles in regulating cellular responses to extracellular stimuli and stress. Previous studies have shown that ceramides contribute to retinal degeneration associated with ischemic and ocular hypertensive stress. Acid sphingomyelinase (ASMase) is one of the major enzymes responsible for the stress-induced generation of ceramides. The goals of this study are to investigate the effects of ceramides on retinal ganglion cells (RGCs) and of ASMase inhibition in ocular hypertensive mice. Induced pluripotent stem cell (iPSC)-derived RGCs and primary cultures of human optic nerve head astrocytes were used to characterize the response to C2-ceramide. Microbead-induced ocular hypertension in the ASMase heterozygote mouse model was used to confirm the physiological relevance of in vitro studies. In mice, RGC function and morphology were assessed with pattern ERG (pERG) and immunofluorescence. The addition of C2-ceramide to iPSC-derived RGCs produced a significant concentration- and time-dependent reduction in cell numbers when compared to control cultures. While the addition of C2-ceramide to astrocytes did not affect viability, it resulted in a 2.6-fold increase in TNF-α secretion. The addition of TNF-α or conditioned media from C2-ceramide-treated astrocytes to RGC cultures significantly reduced cell numbers by 56.1 ± 8.4% and 24.7 ± 4.8%, respectively. This cytotoxic response to astrocyte-conditioned media was blocked by TNF-α antibody. In ASMase heterozygote mice, functional and morphological analyses of ocular hypertensive eyes reveal significantly less RGC degeneration when compared with hypertensive eyes from wild-type mice. These results provide evidence that ceramides can induce RGC cell death by acting directly, as well as indirectly via the secretion of TNF-α from optic nerve head astrocytes. In vivo studies in mice provide evidence that ceramides derived through the activity of ASMase contribute to ocular hypertensive injury. Together these results support the importance of ceramides in the pathogenesis of ocular hypertensive injury to the retina.

Decorin-An Antagonist of TGF-β in Astrocytes of the Optic Nerve.

During the pathogenesis of glaucoma, optic nerve (ON) axons become continuously damaged at the optic nerve head (ONH). This often is associated with reactive astrocytes and increased transforming growth factor (TGF-β) 2 levels. In this study we tested the hypothesis if the presence or absence of decorin (DCN), a small leucine-rich proteoglycan and a natural inhibitor of several members of the TGF family, would affect the expression of the TGF-βs and connective tissue growth factor (CTGF/CCN2) in human ONH astrocytes and murine ON astrocytes. We found that DCN is present in the mouse ON and is expressed by human ONH and murine ON astrocytes. DCN expression and synthesis was significantly reduced after 24 h treatment with 3 nM CTGF/CCN2, while treatment with 4 pM TGF-β2 only reduced expression of DCN significantly. Conversely, DCN treatment significantly reduced the expression of TGF-β1, TGF-β2 and CTGF/CCN2 vis-a-vis untreated controls. Furthermore, DCN treatment significantly reduced expression of fibronectin (FN) and collagen IV (COL IV). Notably, combined treatment with DCN and triciribine, a small molecule inhibitor of protein kinase B (AKT), attenuated effects of DCN on CTGF/CCN2, TGF-β1, and TGF-β2 mRNA expression. We conclude (1) that DCN is an important regulator of TGF-β and CTGF/CCN2 expression in astrocytes of the ON and ONH, (2) that DCN thereby regulates the expression of extracellular matrix (ECM) components and (3) that DCN executes its negative regulatory effects on TGF-β and CTGF/CCN2 via the pAKT/AKT signaling pathway in ON astrocytes.

Histone Deacetylases Regulation by δ-Opioids in Human Optic Nerve Head Astrocytes.

PurposeWe determined whether δ-opioid receptor agonist (SNC-121) regulates acetylation homeostasis via controlling histone deacetylases (HDACs) activity and expression in optic nerve head (ONH) astrocytes.MethodsONH astrocytes were treated with SNC-121 (1 µM) for 24 hours. The HDAC activity was measured using HDAC-specific fluorophore-conjugated synthetic substrates, Boc-Lys(Ac)-AMC and (Boc-Lys(Tfa)-AMC). Protein and mRNA expression of each HDAC was determined by Western blotting and quantitative real-time PCR. IOP in rats was elevated by injecting 2.0 M hypertonic saline into the limbal veins.ResultsDelta opioid receptor agonist, SNC-121 (1 µM), treatment increased acetylation of histone H3, H2B, and H4 by 128 ± 3%, 45 ± 1%, and 68 ± 2%, respectively. The addition of Garcinol, a histone-acetyltransferase inhibitor, fully blocked SNC-121–induced histone H3 acetylation. SNC-121 reduced the activities of class I and IIb HDACs activities significantly (17 ± 3%) and this decrease in HDACs activities was fully blocked by a selective δ-opioid receptors antagonist, naltrindole. SNC-121 also decrease the mRNA expression of HDAC-3 and HDAC-6 by 19% and 18%, respectively. Furthermore, protein expression of HDAC 1, 2, 3, and 6 was significantly (P < 0.05) decreased by SNC-121 treatment. SNC-121 treatment also reduced lipopolysaccharide-induced TNF-α production from ONH astrocytes and glial fibrillary acidic protein immunostaining in the optic nerve of ocular hypertensive animals.ConclusionsWe provided evidence that δ-opioid receptor agonist activation increased histone acetylation, decrease HDACs class I and class IIb activities, mRNA, and protein expression, lipopolysaccharide-induced TNF-α production in ONH astrocytes. Our data also demonstrate that SNC-121 treatment decrease glial fibrillary acidic protein immunostaining in the optic nerves of animals with ocular hypertension.

Isolation and characterization of human optic nerve head astrocytes and lamina cribrosa cells

The lamina cribrosa is the initial site of glaucomatous injury. Pathological changes to the lamina cribrosa include posterior displacement of the lamina cribrosa, loss of trophic support, and remodeling of the extracellular matrix. Optic nerve head (ONH) astrocytes and lamina cribrosa cells synthesize extracellular matrix proteins to support and maintain the lamina cribrosa under physiological conditions. During glaucoma, these cells respond to mechanical strain and other stimuli, which leads to pathological remodeling of the ONH. Although ONH astrocytes and lamina cribrosa cells have been previously cultured, there is no well-accepted, straightforward technique to isolate both cell types from a single dissected human ONH. To better understand the pathophysiology of glaucoma, we obtained and cultured lamina cribrosa explants from human donor eyes. Initially, cells that grew out from the explant were ONH astrocytes and lamina cribrosa cells. Using a specialized medium, we isolated pure populations of lamina cribrosa cells and ONH astrocytes. ONH astrocytes expressed glial fibrillary acidic protein (GFAP). Lamina cribrosa cells expressed alpha-smooth muscle actin (α-SMA), but were negative for GFAP. This method of ONH cell isolation and cell-culture will provide a technique to better understand the molecular and cell-specific changes in glaucomatous damage to the ONH.

Statins Suppress TGF-β2-Mediated MMP-2 and MMP-9 Expression and Activation Through RhoA/ROCK Inhibition in Astrocytes of the Human Optic Nerve Head.

PurposeMatrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) maintenance and remodeling. The present study aimed to determine whether transforming growth factor (TGF)-β2 regulates MMP-2 and MMP-9 levels and activities in astrocytes derived from the optic nerve head (ONH) and the role of statins in such modulation.MethodsPrimary astrocytes cultured from the lamina cribrosa of human donor ONHs were incubated with three types of statins (5 µg/mL) for 1 hour followed by recombinant TGF-β2 (5 ng/mL) for various periods to test their effects. Levels and activities of MMP-2 and MMP-9 in astrocytes in vitro were determined by western blotting and zymography, respectively. Levels of phosphorylated myosin phosphatase target subunit 1 (MYPT1) in astrocyte lysates were determined by western blotting, and those of phosphorylated myosin light chain (MLC) were determined by western blotting and immunocytochemistry.ResultsMMP-2 and MMP-9 levels were upregulated by TGF-β2 in human ONH astrocytes. Prior incubation with simvastatin, lovastatin, and atorvastatin inhibited TGF-β2-mediated MMP-2 and MMP-9 expression and activities. Prior incubation with statins downregulated the TGF-β2-induced phosphorylation of MYPT1 and MLC, which are downstream substrates of RhoA and ROCKs.ConclusionsStatins inhibited the TGF-β2-mediated regulation of MMP-2 and MMP-9 by inhibiting the RhoA/ROCK signaling pathway. Considering the role of MMP in ECM remodeling, the present findings support the notion that statins positively impact ECM remodeling within the ONH.

Statins reduce TGF-beta2-modulation of the extracellular matrix in cultured astrocytes of the human optic nerve head

Statins are cholesterol lowering drugs and have shown beneficial effects on glaucoma. With regard to the mechanism of statin action on glaucoma, we investigated the effects of statins on transforming growth factor-beta 2 (TGF-β2)-induced expression of extracellular matrix (ECM) proteins in human astrocytes of the optic nerve head (ONH) lamina cribrosa (LC). By using primary human ONH astrocytes, we found that both simvastatin and lovastatin inhibited TGF-β2-mediated expression of ECM proteins such as connective tissue growth factor, collagen I, fibronectin, and plasminogen activator inhibitor-1. Suppression of ECM related proteins is due to inhibition of Smad2/3 activation as statins inhibit TGF-β2-induced Smad2 phosphorylation and Smad2/3 nuclear accumulation. In ONH astrocytes, TGF-β2 does not induce MAPK activation. In this study we found an anti-fibrotic effect of statins in human astrocytes of the ONH and identified TGF-β2 as a mediator of statin action, which may support a beneficial role for statins in blocking glaucomatous axonal damage induced by ECM remodeling.

Biomechanical insult switches PEA-15 activity to uncouple its anti-apoptotic function and promote erk mediated tissue remodeling

Biomechanical insult contributes to many chronic pathological processes, yet the resulting influences on signal transduction mechanisms are poorly understood. The retina presents an excellent mechanotransduction model, as mechanical strain on sensitive astrocytes of the optic nerve head (ONH) is intimately linked to chronic tissue remodeling and excavation by matrix metalloproteinases (MMPs), and apoptotic cell death. However, the mechanism by which these effects are induced by biomechanical strain is unclear. We previously identified the small adapter protein, PEA-15 (phosphoprotein enriched in astrocytes), through proteomic analyses of human ONH astrocytes subjected to pathologically relevant biomechanical insult. Under resting conditions PEA-15 is regulated through phosphorylation of two key serine residues to inhibit extrinsic apoptosis and ERK1/2 signaling. However, we surprisingly observed that biomechanical insult dramatically switches PEA-15 phosphorylation and function to uncouple its anti-apoptotic activity, and promote ERK1/2-dependent MMP-2 and MMP-9 secretion. These results reveal a novel cell autonomous mechanism by which biomechanical strain rapidly modifies this signaling pathway to generate altered tissue injury responses.

Increased mitochondrial fission and volume density by blocking glutamate excitotoxicity protect glaucomatous optic nerve head astrocytes.

Abnormal structure and function of astrocytes have been observed within the lamina cribrosa region of the optic nerve head (ONH) in glaucomatous neurodegeneration. Glutamate excitotoxicity-mediated mitochondrial alteration has been implicated in experimental glaucoma. However, the relationships among glutamate excitotoxicity, mitochondrial alteration and ONH astrocytes in the pathogenesis of glaucoma remain unknown. We found that functional N-methyl-d-aspartate (NMDA) receptors (NRs) are present in human ONH astrocytes and that glaucomatous human ONH astrocytes have increased expression levels of NRs and the glutamate aspartate transporter. Glaucomatous human ONH astrocytes exhibit mitochondrial fission that is linked to increased expression of dynamin-related protein 1 and its phosphorylation at Serine 616. In BAC ALDH1L1 eGFP or Thy1-CFP transgenic mice, NMDA treatment induced axon loss as well as hypertrophic morphology and mitochondrial fission in astrocytes of the glial lamina. In human ONH astrocytes, NMDA treatment in vitro triggered mitochondrial fission by decreasing mitochondrial length and number, thereby reducing mitochondrial volume density. However, blocking excitotoxicity by memantine (MEM) prevented these alterations by increasing mitochondrial length, number and volume density. In glaucomatous DBA/2J (D2) mice, blocking excitotoxicity by MEM inhibited the morphological alteration as well as increased mitochondrial number and volume density in astrocytes of the glial lamina. However, blocking excitotoxicity decreased autophagosome/autolysosome volume density in both astrocytes and axons in the glial lamina of glaucomatous D2 mice. These findings provide evidence that blocking excitotoxicity prevents ONH astrocyte dysfunction in glaucomatous neurodegeneration by increasing mitochondrial fission, increasing mitochondrial volume density and length, and decreasing autophagosome/autolysosome formation.

Osteopontin Is Induced by TGF-β2 and Regulates Metabolic Cell Activity in Cultured Human Optic Nerve Head Astrocytes

The aqueous humor (AH) component transforming growth factor (TGF)-β2 is strongly correlated to primary open-angle glaucoma (POAG), and was shown to up-regulate glaucoma-associated extracellular matrix (ECM) components, members of the ECM degradation system and heat shock proteins (HSP) in primary ocular cells. Here we present osteopontin (OPN) as a new TGF-β2 responsive factor in cultured human optic nerve head (ONH) astrocytes. Activation was initially demonstrated by Oligo GEArray microarray and confirmed by semiquantitative (sq) RT-PCR, realtime RT-PCR and western blot. Expressions of most prevalent OPN receptors CD44 and integrin receptor subunits αV, α4, α 5, α6, α9, β1, β3 and β5 by ONH astrocytes were shown by sqRT-PCR and immunofluorescence labeling. TGF-β2 treatment did not affect their expression levels. OPN did not regulate gene expression of described TGF-β2 targets shown by sqRT-PCR. In MTS-assays, OPN had a time- and dose-dependent stimulating effect on the metabolic activity of ONH astrocytes, whereas TGF-β2 significantly reduced metabolism. OPN signaling via CD44 mediated a repressive outcome on metabolic activity, whereas signaling via integrin receptors resulted in a pro-metabolic effect. In summary, our findings characterize OPN as a TGF-β2 responsive factor that is not involved in TGF-β2 mediated ECM and HSP modulation, but affects the metabolic activity of astrocytes. A potential involvement in a protective response to TGF-β2 triggered damage is indicated, but requires further investigation.

Delta-Opioid Receptors Attenuate TNF-α–Induced MMP-2 Secretion From Human ONH Astrocytes

We examined the signaling mechanisms involved in δ-opioid-receptor agonist, SNC-121-mediated attenuation of TNF-α-induced matrix metalloproteinase-2 (MMP-2) secretion from human optic nerve head (ONH) astrocytes. Human ONH astrocytes were treated with SNC-121 (1 μmol/L) for 15 minutes followed by TNF-α (25 ng/mL) treatment for 6 or 24 hours. Cells were pretreated with inhibitors of p38 mitogen-activated protein (MAP) kinase (SB-203580) or NF-κB (Helenalin) prior to TNF-α treatment. Changes in phosphorylation and expression of p38 MAP kinase, IκBα, NF-κB, and MMP-2 were measured by Western blotting. Translocation of NF-κB was determined by immunocytochemistry. TNF-α treatment increased MMP-2 secretion from ONH astrocytes to 236% ± 17% and 142% ± 8% at 6 and 24 hours, respectively; while SNC-121 treatment reduced MMP-2 secretion to 149% ± 11% and 108% ± 7% at 6 and 24 hours, respectively. The SNC-121-mediated inhibitory response was blocked by the δ-opioid-receptor antagonist naltrindole. TNF-α treatment resulted in a sustained phosphorylation of p38 MAP kinase up to 24 hours (226% ± 15% over control levels), which was reduced to 150% ± 20% by SNC-121 treatment. TNF-α treatment increased the expression of NF-κB to 179% ± 21% and 139% ± 6% at 6 and 24 hours, respectively, which was significantly blocked by SNC-121 treatment. Furthermore, TNF-α-induced MMP-2 secretion was blocked by 100% and 78% in the presence of SB-203580 and Helenalin, respectively. Evidence is provided that SNC-121 attenuated TNF-α-induced MMP-2 secretion from ONH astrocytes. Data also supported the idea that p38 MAP kinase and NF-κB played central roles in TNF-α-induced MMP-2 secretion, and both were negatively regulated by SNC-121.

Multikinase-Inhibitoren als therapeutischer Ansatz bei neovaskulärer AMD: In-vitro-Evaluation der Sicherheit von Axitinib, Pazopanib und Sorafenib zur intraokularen Anwendung

Multikinase inhibitors (MKI) interfere effectively at different levels of the neovascularisation cascade. Early clinical and experimental data suggest that MKIs represent a promising novel approach for the treatment of neovascular age-related macular degeneration (AMD). However, so far little is known about the biocompatibility of MKIs regarding human ocular cells. This in vitro study investigates and compares the biocompatibility of three MKIs, axitinib, pazopanib, and sorafenib regarding ocular cells of the anterior and posterior segments, as well as organ-cultured donor corneas. Primary human optic nerve head astrocytes (ONHA), trabecular meshwork cells (TMC), and retinal pigment epithelium (RPE), human corneal endothelial and lens epithelial cells (CEC and LEC) were treated with different concentrations of axitinib, pazopanib, or sorafenib (0.1 to 100 µg/mL). To simulate oxidative stress, the cells were additionally co-incubated with 400 µM hydrogen peroxide. Induction of cell death and cellular viability were examined by live-dead assay and tetrazolium dye reduction assay (MTT). In addition, the influence of the three substances on human corneal endothelium was evaluated in seropositive donor corneas in organ culture by phase contrast microscopy. Up to a concentration of 7.5 mg/mL of the substances tested in any cell type examined, no toxic effects were found. Even after 10 days of incubation of organ-cultured donor corneas with 7.5 µg/mL, axitinib, pazopanib, or sorafenib, no evidence for endothelial toxicity was found. All three MKIs tested, axitinib, pazopanib, and sorafenib showed a good biocompatibility on the investigated ocular cells. Even under conditions of oxidative stress, there were no toxic effects up to a concentration of 7.5 µg/mL. Only at higher concentrations, there was a dose-dependent decrease in cellular viability and pronounced induction of cell death. These effects on cellular viability and induction of cell death appeared to be stronger with pazopanib, followed by sorafenib, than with axitinib.

Idebenone Prevents Human Optic Nerve Head Astrocytes From Oxidative Stress, Apoptosis, and Senescence by Stabilizing BAX/Bcl-2 Ratio

Oxidative stress plays an important role in the pathogenesis of several neurodegenerative diseases including glaucoma. Astrocytes are supposed to play a role in glaucoma pathogenesis. This study investigates the antiapoptotic and cytoprotective effects of idebenone on optic nerve head astrocytes (ONHA) under oxidative stress. ONHA were treated with 1 to 150 µM idebenone. Cell viability (MTT assay and live-dead assay), induction of intracellular reactive oxygen species, senescence-associated β-galactosidase activity were investigated. In addition, apoptosis (detection of histone-associated DNA fragmentation), and expression of BAX and Bcl-2, and their mRNA were determined after 48 hours and after hydrogen peroxide (H2O2) treatment. Idebenone concentrations from 1 to 50 µM showed no effects on ONHA viability. Pretreatment with 10 µM idebenone led to an increase in viability of ONHA after H2O2 treatment. In addition, idebenone pretreatment significantly attenuated the increase of histone-associated DNA fragmentation, induction of senescence-associated β-galactosidase, and intracellular reactive oxygen species after treatment with H2O2. When ONHA cells were treated with idebenone and H2O2, real-time polymerase chain reaction and Western blot analysis yielded an increased expression of Bcl-2 and a decrease of BAX compared with those cells that were treated with H2O2 only. Idebenone reduced senescence, oxidative stress, and apoptotic cell death in cultured ONHA in vitro. Our results suggest that idebenone may help to protect ONHA in vivo, and therefore might be helpful in preventing the progression of glaucomatous degeneration.

Antioxidants reduce TGF-beta2-induced gene expressions in human optic nerve head astrocytes

The goal of the present study was to investigate whether the antioxidants vitamin E, vitamin C and vitamin B1 can reduce the transforming growth factor-beta2 (TGF-β2)-induced gene expressions in cultured human optic nerve head (ONH) astrocytes. Cultured human ONH astrocytes were pretreated with different concentrations of vitamin E, vitamin C and vitamin B1 and then exposed to 1.0 ng/ml TGF-β2 for 24 hr. Expression of the heat shock proteins Hsp27 and αB-crystallin, the extracellular matrix (ECM) component fibronectin and the ECM-modulating protein connective tissue growth factor (CTGF) was detected by immunohistochemistry or real-time PCR analysis. TGF-β2 increased the expression of Hsp27, αB-crystallin, fibronectin and CTGF in human ONH astrocytes. Pretreatment with different concentrations of vitamin E, vitamin C and vitamin B1 reduced the TGF-β2-stimulated gene expressions. In cultured human ONH astrocytes, the TGF-β2-stimulated gene expressions could be reduced by pretreatment with vitamin E, vitamin C and vitamin B1. Therefore, the use of antioxidants in glaucomatous optic neuropathy might be a promising approach to prevent TGF-β2-induced cellular changes in ONH astrocytes.

Purification and characterization of adult optic nerve head astrocytes

To establish a method of purifying and characterizing adult astrocytes from optic nerve head (ONH). Experimental study. The lamina cribrosa tissue from ONH of human eye was isolated under anatomic microscopy, and then 4 to 6 little explants were incubated in each culture plate containing culture medium DMEM/F12. After 8 to 10 weeks, the cells were removed by digesting cells with 0.25% trypsogen. Selective astrocyte culture medium is subsequently used. After two passages, astrocytes were identified by the observation of cell morphology and immunofluorescent staining of GFAP and NCAM. After 2 to 3 weeks of explants planting, cells showed an obvious migration procession by crawling in succession from the verge of the explants and rapidly splitting. Most cells displayed a flat star shape or polygon after digested with trypsogen. Several cells are long fusiformis. Almost all cells presented a flat star shape and simultaneously expressed GFAP and NCAM when the cells cultured with selective astrocyte culture medium. Cultured human ONH astrocytes can be obtained by precisely separating lamina cribrosa and placing the explants on the margin of culture medium, a method that promotes cell adherence. Using selective astrocyte culture medium is very effective and convenient in purifying primary astrocytes.

TGF-β2- and H2O2-Induced Biological Changes in Optic Nerve Head Astrocytes Are Reduced by the Antioxidant Alpha-Lipoic Acid

Background/Aims: The goal of the present study was to determine whether transforming growth factor-β₂ (TGF-β₂)- and oxidative stress-induced cellular changes in cultured human optic nerve head (ONH) astrocytes could be reduced by pretreatment with the antioxidant α-lipoic acid (LA). Methods: Cultured ONH astrocytes were treated with 1.0 ng/ml TGF-β₂ for 24 h or 200 µM hydrogen peroxide (H₂O₂) for 1 h. Lipid peroxidation was measured by a decrease in cis-parinaric acid fluorescence. Additionally, cells were pretreated with different concentrations of LA before TGF-β₂ or H₂O₂ exposure. Expressions of the heat shock protein (Hsp) αB-crystallin and Hsp27, the extracellular matrix (ECM) component fibronectin and the ECM-modulating protein connective tissue growth factor (CTGF) were examined with immunohistochemistry and real-time PCR analysis. Results: Both TGF-β₂ and H₂O₂ increased lipid peroxidation. Treatment of astrocytes with TGF-β₂ and H₂O₂ upregulated the expression of αB-crystallin, Hsp27, fibronectin and CTGF. Pretreatment with different concentrations of LA reduced the TGF-β₂- and H₂O₂-stimulated gene expressions. Conclusion: We showed that TGF-β₂- and H₂O₂-stimulated gene expressions could be prevented by pretreatment with the antioxidant LA in cultured human ONH astrocytes. Therefore, it is tempting to speculate that the use of antioxidants could have protective effects in glaucomatous optic neuropathy.

Proteomics Analyses of Human Optic Nerve Head Astrocytes Following Biomechanical Strain

We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor β1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin.

Opioid Receptor Activation: Suppression of Ischemia/Reperfusion-Induced Production of TNF-α in the Retina

The detrimental role of TNF-α in ischemia-induced tissue damage is known. The authors study examined whether opioid receptor activation alters TNF-α levels in the postischemic retina. Retinal ischemia was induced by raising the intraocular pressure above systolic blood pressure (155-160 mm Hg) for 45 minutes. Rats were pretreated with the opioid receptor agonist morphine (1 mg/kg; intraperitoneally) before injury. Selected animals were pretreated with the opioid antagonist naloxone (3 mg/kg; intraperitoneally). Human optic nerve head (ONH) astrocytes and rat microglial cells were treated with morphine (0.1-1 μM) for 24 hours and then treated with 10 μg/mL or 30 ng/mL lipopolysaccharide (LPS), respectively. TNF-α was measured by ELISA. Opioid receptor subtypes in astrocytes and microglia were determined by Western blot analysis. There was a time-dependent increase in TNF-α production; the maximum production occurred at 4 hours after ischemia and localized to the inner retinal regions. Ischemia-induced TNF-α production was significantly inhibited by morphine. In astrocytes and microglia, LPS triggered a robust increase in the release of TNF-α, which was significantly inhibited (P < 0.05) by morphine. Naloxone reversed the morphine-induced suppression of TNF-α production in vivo and in vitro. Both ONH astrocytes and microglial cells expressed δ-, κ-, and μ-opioid receptor subtypes. These data provide evidence that the production of TNF-α after ischemia/reperfusion injury is an early event and that opioid receptor activation reduces the production of TNF-α. Immunohistochemistry data and in vitro studies provide evidence that ONH astrocytes and microglial cells are the primary sources for the TNF-α production under ischemic/inflammatory conditions. Activation of one or more opioid receptors can reduce ischemic/reperfusion injury by the suppression of TNF-α production.