Human Optic Nerve Head Astrocytes Research Articles

Minocycline is cytoprotective in human trabecular meshwork cells and optic nerve head astrocytes by increasing expression of XIAP, survivin, and Bcl-2.

Primary open-angle glaucoma (POAG) is one of the leading causes of blindness. Activation of optic nerve head astrocytes (ONHA) and loss of trabecular meshwork cells (TMC) are pathognomonic for this neurodegenerative disease. Oxidative stress and elevated levels of transforming growth factor beta (TGFbeta) play an important role in the pathogenesis of POAG. This study investigates the possible antiapoptotic and cytoprotective effects of minocycline on TMC and ONHA under oxidative stress and increased TGFbeta levels. TMC and ONHA were treated with minocycline 1-150 muM. Possible toxic effects and IC(50) were evaluated after 48 hours. Cell proliferation and viability were examined in order to assess the protective effects of minocycline on TMC and ONHA. Expression of Bcl-2, XIAP, and survivin, as well as their mRNA expression, were assessed by real time polymerase chain reaction (RT-PCR) and Western Blot analysis 48 hours after treatment with minocycline alone and additional incubation with TGFbeta-2 or oxidative stress. Minocycline 1-75 muM showed no toxic effects on TMC and ONHA. Under conditions of oxidative stress, both TMC and ONHA showed an increase in viability and an ability to proliferate when treated with minocycline 20-40 muM. RT-PCR and Western blotting yielded an overexpression of Bcl-2, XIAP, and survivin when TMC or ONHA were treated with minocycline 20-40 muM under conditions of oxidative stress and when additionally incubated with TGFbeta-2. Minocycline up to 75 muM does not have toxic effects on TMC and ONHA. Treatment with minocycline 20-40 muM led to increased viability and proliferation under oxidative stress and TGFbeta-2, as well as overexpression of Bcl-2, XIAP, and survivin. This protective pathway may help to prevent apoptotic cell death of TMC and ONHA and therefore be a promising approach to avoidance of progression of glaucomatous degeneration.

Sorafenib protects human optic nerve head astrocytes from light-induced overexpression of vascular endothelial growth factor, platelet-derived growth factor, and placenta growth factor

Objectives: Growth factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and placenta growth factor (PlGF) are key players in the development of diabetic retinopathy, age-related macular degeneration, and other retinal neovascular diseases. Glial cells provide a significant source of retinal growth factor production under physiologic and pathologic conditions. Cumulative light exposure has been linked to increased retinal growth factor expression. Previous reports indicate that sorafenib, an oral multikinase inhibitor, might have a beneficial effect on retinal neovascularization. This study was designed to investigate the effects of sorafenib on light-induced overexpression of growth factors in human retinal glial cells.Methods: Primary human optic nerve head astrocytes (ONHAs) were exposed to white light and incubated with sorafenib. Viability, expression, and secretion of VEGF-A, PDGF-BB, and PlGF and their mRNA were determined by reverse transcription-polymerase chain reaction, immunohistochemistry, and enzyme-linked immunosorbent assay.Results: Light exposure decreased cell viability and increased VEGF-A, PDGF-BB, and PlGF expression and secretion. These light-induced effects were significantly reduced when cells were treated with sorafenib at a concentration of 1 μg/ml.Conclusion: Sorafenib significantly reduced light-induced overexpression of VEGF-A, PDGF-BB, and PlGF in primary human ONHAs. Sorafenib has promising properties as a potential supportive treatment for retinal neovascularization.

Network analysis of human glaucomatous optic nerve head astrocytes

BackgroundAstrocyte activation is a characteristic response to injury in the central nervous system, and can be either neurotoxic or neuroprotective, while the regulation of both roles remains elusive.MethodsTo decipher the regulatory elements controlling astrocyte-mediated neurotoxicity in glaucoma, we conducted a systems-level functional analysis of gene expression, proteomic and genetic data associated with reactive optic nerve head astrocytes (ONHAs).ResultsOur reconstruction of the molecular interactions affected by glaucoma revealed multi-domain biological networks controlling activation of ONHAs at the level of intercellular stimuli, intracellular signaling and core effectors. The analysis revealed that synergistic action of the transcription factors AP-1, vitamin D receptor and Nuclear Factor-kappaB in cross-activation of multiple pathways, including inflammatory cytokines, complement, clusterin, ephrins, and multiple metabolic pathways. We found that the products of over two thirds of genes linked to glaucoma by genetic analysis can be functionally interconnected into one epistatic network via experimentally-validated interactions. Finally, we built and analyzed an integrative disease pathology network from a combined set of genes revealed in genetic studies, genes differentially expressed in glaucoma and closely connected genes/proteins in the interactome.ConclusionOur results suggest several key biological network modules that are involved in regulating neurotoxicity of reactive astrocytes in glaucoma, and comprise potential targets for cell-based therapy.

INTRAVITREAL VORICONAZOLE

Fungal endophthalmitis is a rare but sight-threatening disease. Despite an expanding range of fungal pathogens, there are only few therapeutic agents for its treatment available. Voriconazole is a second-generation synthetic triazole with a broad action against yeasts and molds. The current study investigates the safety of Voriconazole for intravitreal application in a cell culture model. Primary human retinal pigment epithelium cells (RPE) and primary human optic nerve head astrocytes were treated with concentrations of Voriconazole ranging from 25 microg/mL to 10 mg/mL. Possible toxic effects and IC50 were evaluated after 24 hours and under conditions of oxidative stress. By treating the RPE cell lines with tumor-necrosisfactor alpha (TNF-alpha), lipopolysaccharides (LPS), and interleukin-6 (IL-6) the effects of Voriconazole on cellular viability under conditions of inflammation were investigated. Toxicity was evaluated by colorimetric measuring the inhibition of RPE cell proliferation (MTT). Additionally cell viability was quantified by a microscopic live-dead-assay. Concentrations <250 microg/mL Voriconazole had no influence neither on RPE nor on optic nerve head astrocytes cell proliferation and cell viability when administered for 24 hours and under oxidative stress. When preincubated with tumor-necrosis-factor alpha, lipopolysaccharides and interleukin-6 for 24 hours and subsequently treated with Voriconazole at concentrations up to 250 microg/mL for 24 hours no significant decrease in proliferation and viability was observed. This study showed that no significant toxicity existed for Voriconazole in vitro on primary RPE and optic nerve head astrocytes when administered in therapeutic concentrations up to 250 microg/mL.

Reactivation of Optic Nerve Head Astrocytes by TGF-β2 and H2O2Is Accompanied by Increased Hsp32 and Hsp47 Expression

Histologic studies have previously demonstrated increased expression of small heat shock proteins (Hsps) in reactive optic nerve head (ONH) astrocytes of patients with glaucoma. Transforming growth factor (TGF)-beta2 and hydrogen peroxide (H(2)O(2)) are known to induce ONH astrocyte reactivation. The goal of the present study was to determine whether new potentially involved Hsps, such as Hsp32, -47, -60, and -70, are expressed in the reactivation process of ONH astrocytes mediated by TGF-beta2 and H(2)O(2). Cultured human ONH astrocytes were treated with 1.0 ng/mL TGF-beta2 for up to 48 hours. In addition, the cells were exposed to 100, 200, or 400 microM H(2)O(2) for 1 hour. Expression of Hsp32, -47, -60, and -70 was examined by immunohistochemistry, real-time PCR, and Western blot analyses. Treatment with TGF-beta2 increased Hsp32 after 4 and 6 hours, whereas Hsp47 was upregulated after treatment with TGF-beta2 for 12, 24, and 48 hours. Exposure of the cells to H(2)O(2) could increase both Hsp32 and -47. No significant effects on the expression of Hsp60 and -70 were observed after treatment of the cells with TGF-beta2 or H(2)O(2). TGF-beta2 increased Hsp32 after short-term treatment and Hsp47 after longer periods in cultured human ONH astrocytes. H(2)O(2) increased both Hsp32 and -47 levels. No effects on Hsp60 and -70 levels were induced by TGF-beta2 and H(2)O(2). These results may provide further insights into the cellular stress responses of reactive human ONH astrocytes. Further extensive studies are needed to examine the potential roles of Hsps in the ONH of glaucomatous eyes.

Oxidative Stress and TGF-β2 Increase Heat Shock Protein 27 Expression in Human Optic Nerve Head Astrocytes

Reactive astrocytes of glaucomatous optic nerve heads (ONHs) are characterized by an increased expression of transforming growth factor (TGF)-beta2 and heat shock proteins (Hsps) such as Hsp27. The goal of the present study was to determine the effect of oxidative stress on TGF-beta2 and Hsp27 expression in human ONH astrocytes. Cultured ONH astrocytes were exposed to 100, 200, and 400 muM hydrogen peroxide (H(2)O(2)) for 1 hour. Levels of TGF-beta2 were analyzed by ELISA. Additionally, cells were treated with 1.0 ng/mL TGF-beta2 for 12, 24, and 48 hours, respectively. Expression of Hsp27 was detected by immunohistochemistry, real-time PCR, and Western blot analyses. To investigate the role of signal transduction proteins, cells were pretreated with the specific inhibitors SB203580, PD98059, U0126, and SP600125. Furthermore, induction of p38MAP kinase and its phosphorylated form were determined by Western blot analyses. H(2)O(2) exposure increased the secretion of TGF-beta2. Both H(2)O(2) and TGF-beta2 increased the Hsp27 expression. Pretreatment of cells with SB203580, an inhibitor for p38MAP kinase, reduced the H(2)O(2)- and TGF-beta2-stimulated Hsp27 expression, whereas pretreatment with PD98059 and U0126, specific inhibitors of ERK1/2, and SP600125, a specific c-Jun N-terminal kinase inhibitor, had no effects. H(2)O(2) and TGF-beta2 upregulated the phosphorylated p38MAP kinase. Oxidative stress increased TGF-beta2 secretion in cultured human ONH astrocytes. Both H(2)O(2) and TGF-beta2 increased Hsp27 expression via p38MAP kinase activation. Therefore, it is tempting to speculate that reduction of oxidative stress and TGF-beta2 may help to minimize the incidence of characteristic changes in the ONH of patients with glaucoma.

Activation of the BMP Canonical Signaling Pathway in Human Optic Nerve Head Tissue and Isolated Optic Nerve Head Astrocytes and Lamina Cribrosa Cells

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-beta superfamily that controls multiple functions in a variety of cells. We have previously shown that human optic nerve head (ONH) astrocytes and lamina cribrosa (LC) cells express BMP and BMP receptor mRNA and proteins. The purpose of the present study was to determine whether human ONH tissues express the canonical BMP signaling pathway and whether ONH cells secrete BMP-4 and respond to exogenous BMP-4 through this pathway. Well-characterized human ONH astrocytes (N = 2) and LC cells (N = 3) were treated with exogenous BMP-4 (20 ng/mL) for various times. Western immunoblot analysis was used to detect secreted BMP-4 in serum-free conditioned media of ONH cells and in human ONH tissues (N = 4) and Smad proteins in total cell lysate of ONH cells and tissues. Intracellular colocalization of p-R-Smad1 with Co-Smad4 and localization of inhibitory Smads (e.g., I-Smad6 and I-Smad7) were studied through immunocytochemistry. In addition, coimmunoprecipitation was used to verify the interaction of p-R-Smad1 with Co-Smad4. ONH astrocytes and LC cells secrete BMP-4 and synthesize R-Smad1, R-Smad5, I-Smad6, I-Smad7, and Co-Smad4 proteins. Exposure to BMP-4 for either 10 or 60 minutes resulted in increased p-R-Smad1 and p-R-Smad1/5/8 protein levels that declined after 12 hours of treatment. Immunocytochemistry and coimmunoprecipitation studies revealed that p-R-Smad1/5/8 and Co-Smad4 interact and colocalize in the nucleus. BMP-4 treatment resulted in increased coprecipitation of p-R-Smad1/5/ 8 and Co-Smad4. I-Smad6 and I-Smad7 are localized in the nucleus and cytoplasm of ONH astrocytes and LC cells. Proteins for BMP-4, p-R-Smad1/5/8, R-Smad1, R-Smad5, R-Smad8, and Co-Smad4 are present in human ONH tissues. In addition, phosphorylated Smad1 and Smad5 colocalize with Smad4 in the nuclei of ONH tissues. These results indicate that BMP-4 and Smad signaling proteins are present in human ONH tissues, isolated ONH astrocytes, and LC cells. In addition, exogenous BMP-4 treatment of ONH astrocytes and LC cells results in downstream signaling through the canonical Smad pathway. Thus, cells within the human ONH may respond to locally released BMP through paracrine or autocrine mechanisms.

Minocycline is cytoprotective in human trabecular meshwork cells and optic nerve head astrocytes by increasing expression of XIAP, Survivin and Bcl‐2

Abstract Purpose: Primary open‐angle glaucoma (POAG) is one of the leading cause for blindness. Activation of optic nerve head astrocytes (ONHA) and loss of trabecular meshwork cells (TMC) are pathognomonic for this disease. Oxidative stress (OS) and elevated levels of TGF‐beta play an important role in the pathogenesis of this neurodegenerative disease. This study investigates possible anti‐apoptotic and cytoprotective effects of Minocycline (M) on TMC and ONHA under OS and TGF‐beta. Methods: TMC and ONHA were treated with 1μM to 150μM M. Possible toxic effects and IC50 were evaluated after 24h (MTT). To investigate possible protective effects of M on TMC and ONHA, cell proliferation and viability was examined. Expression of XIAP, Survivin as well as Bcl‐2 and their mRNA was assessed by RT‐PCR 24h after treatment with M alone, additional incubation with TGF‐beta‐2 and OS. Results: M concentrations from 1μM to 75μM showed no toxic effects on TMC and ONHA. Under conditions of OS both TMC and ONHA showed an increase in viability and ability to proliferate when treated with 20‐40μM M. RT‐PCR yielded an overexpression of XIAP, Survivin and Bcl‐2, when TMC or ONHA cells were treated with 20‐40μM M for 24 hours, under OS and when additionally incubated with TGF‐beta2. Conclusions: We could show that M does not have toxic effects on TMC and ONHA cells up to 75μM. The observed increase in viability and proliferation under OS and TGF‐beta2 and the overexpression of XIAP, Survivin and Bcl‐2 after treatment with 20‐40μM M might prevent apoptotic‐cell‐death in response to cellular stress and may be a protective pathway for TMC and ONHA to avoid progression of glaucomatous degeneration.

Endothelin-1-Mediated Signaling in the Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Astrocytes

Endothelin (ET)-1 levels are increased in aqueous and vitreous humor in patients with glaucoma and animal models of glaucoma. Whether the elevated ET-1 induces extracellular matrix (ECM) remodeling in the optic nerve head is still unknown. In the present study, the regulation of matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases (MMPs/TIMPs) and ECM remodeling in ET-1-activated human optic nerve head astrocytes (hONAs) were determined. Primary hONAs were exposed to ET-1 for 1 day and 4 days. Incubation media were subjected to zymography and Western blot to detect activity and expression of MMPs and TIMPs. Fibronectin (FN) was monitored by Western blot and immunofluorescent staining. ET-1 increased the activity of MMP-2 and the expression of TIMP-1 and -2 in hONAs. The expression of TIMP-1 and -2 induced by ET-1 was abolished by application of inhibitors of mitogen-activated protein kinase (MAPK) or PKC, leading to enhanced activity of MMP-2. Knockdown of MMP-2, by using small interfering (si)RNA, not only decreased the activity of MMP-2 but also decreased the expression of TIMP-1 and -2. ET-1 increased the soluble (s)FN expression as well as FN matrix formation. However, the accumulation of sFN did not enhance FN matrix formation. Unlike ET-1's effects on MMP-2, blockade of MAPK and PKC did not alter the expression and deposition pattern of FN in hONAs. ET-1 increased the expression and activity of MMP-2 and TIMP-1 and -2. The ERK-MAPK and PKC pathways are involved in the regulation of expression of MMP-2 and TIMP-1 and -2. ET-1's effects on MMPs/TIMPs may be important, not only in regulating the expression of MMPs and TIMPs, but also in influencing ECM remodeling.

Bevacizumab wirkt nicht toxisch auf Zellen des menschlichen Auge

Intravitreal anti-vascular endothelial growth factor (VEGF) treatment with bevacizumab (Avastin) has emerged as a promising therapy for the treatment of choroidal neovascularisation in age-related macular degeneration. Intravitreal administration of bevacizumab is "off-label," and only very limited data regarding short-term toxicity exist. Therefore, we investigated the safety of different doses of bevacizumab on the anterior- and posterior-segment cells of the human eye. Primary human retinal pigment epithelium (RPE) cells, human optic nerve head astrocytes (ONHA), human trabecular meshwork cells (TMC), and cornea buttons not suitable for transplantation were treated with bevacizumab (25 microg/ml, 250 microg/ml, and 2,500 microg/ml) for 48 h, corresponding to 0.1x, 1x, and 10x the dosage used intravitreally. Bevacizumab-related toxicity was evaluated by a colorimetric test (MTT) measuring inhibition of RPE, ONHA, and TMC cell proliferation. Additionally, cell viability was quantified by live/dead fluorescence assay. Corneal endothelium was quantified by phase-contrast microscopy. Bevacizumab showed adverse effects on primary RPE cell proliferation as well as on cell viability at a concentration of 2,500 microg/ml. The lower concentrations of 25 microg/ml and 250 microg/ml had no influence on RPE cell proliferation or cell viability. There was no toxicity for any investigated concentration on human ONHA, TMC, or corneal endothelium. In this study, a 10-fold concentration (compared with common clinical use) of the VEGF-blocking antibody bevacizumab had toxic effects on primary RPE. There was no toxicity for lower concentrations or toxicity to other cell types of the anterior and posterior segments. Therefore, the clinical use of bevacizumab at concentrations of 1-1.25 mg intravitreally seems to be safe.

Pressure induces loss of gap junction communication and redistribution of connexin 43 in astrocytes

Astrocytes, the major glia in the nonmyelinated optic nerve head (ONH), connect via gap junctions built of connexin-43 (Cx43) to form a functional syncytium allowing communication and control of ionic and metabolic homeostasis of retinal ganglion cells (RGCs) axon. We examined gap junction intercellular communication (GJIC) by scrape loading assays in human ONH astrocytes exposed to hydrostatic (HP) or ambient pressure (CP) in vitro. Immunostaining, immunoprecipitation, and immunoblots were used to detect Cx43 distribution and phosphorylation in astrocytes exposed to HP with/without EGF receptor (EGFR) tyrosine kinase inhibitors AG1478 and AG82 and MAPK inhibitors U0126, PD98059, and SB203580. The data indicates that upon exposure to HP, astrocytes decrease GJIC and exhibit altered cellular localization and phosphorylation of Cx43. Inhibition of EGFR blocked the effects of HP on GJIC and HP-induced Cx43 tyrosine phosphorylation. Inhibitors of MAPK- ERK1/2 and -p38 caused partial closure of GJIC under CP and HP, which was maintained for 6 h. Inhibition of Big Mitogen-Activated Kinase 1/ERK5 (BMK1/ERK5) caused partial closure under CP and HP followed by full recovery after 6 h. Inhibition of MAPK did not affect the HP-induced increase in Cx43 serine 279/282 phosphorylation. We conclude that activation of the EGFR pathway in response to HP leads to decrease of GJIC via tyrosine phosphorylation of Cx43 in ONH astrocytes. In glaucoma under conditions of elevated intraocular pressure (IOP), astrocytes may lose GJIC altering the homeostasis of RGC axons, adopting the reactive phenotype, contributing to glaucomatous neuropathy.

Expression of ephrinB1 and its receptor in glaucomatous optic neuropathy

Objective:To determine ephrinB1, ephrinB2 and EphB1 expression in the optic nerve head (ONH) and retina of monkeys with glaucoma and in human ONH astrocytes.Methods:Using immunohistochemistry, the localisation of ephrinB1, ephrinB2...

Hypoxia/reoxygenation and TGF-β increase αB-crystallin expression in human optic nerve head astrocytes

Reactive astrocytes in glaucomatous optic nerve changes are characterized by an increased expression of αB-crystallin and transforming growth factor-beta (TGF-β). In the pathogenesis of glaucomatous optic nerve damage, ischemia/reperfusion injury may play an important role. The goal of the present study was to determine the influence of hypoxia/reoxygenation and TGF-β on the expression of αB-crystallin in cultured human astrocytes of the optic nerve head (ONH). Cultured human astrocytes were incubated under hypoxic conditions (1% O 2 for 4–12 h) with subsequent reoxygenation (12–24 h). Additionally, cells were treated with 1.0 ng/ml TGF-β1 and TGF-β2 for 12–48 h. Expression of αB-crystallin was examined by Northern- and Western-blotting. Levels of TGF-β1 and TGF-β2 were analyzed by RT-PCR analysis and ELISA. The effect of TGF-β blocking on the hypoxia/reoxygenation modulated expression of αB-crystallin was investigated by simultaneous incubation with neutralizing antibodies against TGF-β during the reoxygenation phase. Hypoxia/reoxygenation increased the expression of αB-crystallin at the mRNA (2.8- to 3.1-fold) and protein level (1.8- to 2.1-fold). Treatment with 1.0 ng/ml TGF-β1 and TGF-β2 for 12–48 h markedly enhanced αB-crystallin mRNA expression approximately three- to fourfold. Using Western blot analysis, this increase ranged from 2 to 3 times. Both cytokines showed a twofold increase after 12 and 24 h of reoxygenation at the mRNA and a two- to threefold increase at the protein level. Simultaneous treatment with neutralizing antibodies against both TGF-β isoforms prevented the hypoxia/reoxygenation-mediated elevation of αB-crystallin. The process of hypoxia/reoxygenation is capable of inducing the expression of αB-crystallin and TGF-ß in cultured ONH astrocytes. Therefore, optimization of conditions leading to hypoxia/reoxygenation in the ONH of glaucomatous patients may help to lower the incidence of characteristic changes in the optic nerve.

A neuroactive steroid 5α‐androstane‐3α,17β‐diol regulates androgen receptor level in astrocytes

Optic nerve head (ONH) astrocytes from patients with glaucomatous optic neuropathy exhibit increased production of 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), a neuroactive metabolite of 5alpha-dihydrotestosterone (5alpha-DHT). To determine whether ONH astrocytes are androgen target cells, and whether 3alpha-diol is capable of regulating astrocyte functions, we studied the response of human ONH astrocytes to 3alpha-diol compared with 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one (R1881), a synthetic 5alpha-DHT agonist. In ONH astrocytes, both 3alpha-diol and R1881 increased protein levels of androgen receptor (AR) and glial fibrillary acidic protein (GFAP), however, only R1881 also increased the AR mRNA level and astrocyte proliferation. Both R1881 and 3alpha-diol rapidly activate the mitogen-activated protein kinase (MAPK) signaling pathway in ONH astrocytes, as confirmed by phosphorylation of extracellular signal-regulated kinase (ERK). 3Alpha-diol also activates the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. 3Alpha-diol regulates the increase of AR protein level and the phosphorylation through the PI3K/Akt pathway, whereas R1881 regulates them through the MAPK/ERK pathway. Our findings demonstrate that human ONH astrocytes are androgen target cells and respond to androgens by the rapid activation of cell signaling. The activation of the PI3K/Akt pathway by 3alpha-diol may regulate various properties of astrocytes, including cell motility and survival, and may play a role in the formation and maintenance of the reactive phenotype of ONH astrocytes in glaucoma.

Transforming Growth Factor-β2 Modulated Extracellular Matrix Component Expression in Cultured Human Optic Nerve Head Astrocytes

To study whether glaucomatous extracellular matrix (ECM) modifications in the lamina cribrosa might be induced by TGF-beta 2, the effect of TGF-beta 2 on the expression of collagen types I (Col1 alpha 1), III (Col3 alpha 1), and IV (Col4 alpha 2); fibronectin (FN); tissue transglutaminase (TGM2); connective tissue growth factor (CTGF); and thrombospondin (TSP-1) in cultured human optic nerve head (ONH) astrocytes was investigated. Astrocytes were isolated from eyes of five human donors, and cultured monolayers were treated with 1.0 ng/mL TGF-beta 2 for 24 and 48 hours. Expression of Col1 alpha 1, Col3 alpha 1, Col4 alpha 2, FN, TGM2, CTGF, and TSP-1 was examined by semiquantitative RT-PCR and Northern and Western blot analyses. The effect of CTGF silencing on the TGF-beta 2-modulated expression of these genes was investigated by transfection of CTGF small interfering (si)RNA before TGF-beta 2 treatment. TGF-beta 2 treatment upregulated the expression of Col1 alpha 1, Col4 alpha 2, FN, CTGF, TGM2, and TSP-1 mRNA and protein in cultured astrocytes. Inductions ranged between 1.5- and 4-fold. Expression of Col3 alpha 1 remained unaffected. Transfection of 10 nM CTGF siRNA inhibited the TGF-beta 2-induced upregulation of CTGF, Col4 alpha 2, Col1 alpha 1, TGM2, and FN, whereas TSP-1 expression was not reduced. TGF-beta 2 is capable of inducing the expression of ECM and basement membrane components in cultured ONH astrocytes via CTGF and upregulated TSP-1, a protein naturally involved in the activation of latent TGF-beta. Therefore, TGF-beta 2 could be a factor in the initiation of the modification of ECM in the glaucomatous ONH. In addition, TSP-1 induction may be a mechanism by which TGF-beta 2 amplifies its own activation.

Effect of elevated intraocular pressure on endothelin-1 in a rat model of glaucoma

The role of endothelin-1 (ET-1) a potent vasoactive peptide, in glaucoma pathogenesis is receiving increasing attention, particularly in astroglial activation in optic nerve damage. Our laboratory has also shown that ET-1 treatment causes proliferation of cultured human optic nerve head astrocytes to possibly initiate astrogliosis. ET-1 is distributed in retina, optic nerve, and ciliary epithelium, however the effects of elevated intraocular pressure (IOP) (as seen in glaucoma) on ET-1 and ET B receptors are not clearly understood. In the present study, the levels of immunoreactive ET-1 (ir-ET-1) in aqueous humour (AH) and optic nerve head (ONH) were determined in the Morrison elevated IOP model of glaucoma. Additionally in the ONH of these rats, immunohistochemical analyses of ET B receptors and glial fibrillary acidic protein (GFAP; a marker for astroglial cells and for astrogliosis) were performed. There was 2- to 2.5-fold increase in AH ir-ET-1 levels for rats subjected to elevated IOP, compared to their respective controls. In the Morrison rat model of glaucoma, elevated IOP increased optic nerve ir-ET-1 with concomitant increases in ir-ET B and ir-GFAP labelling (possibly indicative of astrogliosis and hypertrophy). As seen in brain astrocytes subjected to neurotrauma, the present findings are suggestive of ET-1’s role in astroglial activation, particularly in response to elevated IOP in glaucoma.

Hypoxia augments TNF-$alpha;-mediated endothelin-1 release and cell proliferation in human optic nerve head astrocytes

The effect of hypoxia (24 h) on TNF-α-mediated release of endothelin-1 (ET-1) from human optic nerve head astrocytes (hONAs) and TNF-α- and ET-1-induced hONA proliferation was determined. ET-1 synthesis and release was quantitated using ELISA while TNF-α (10 nM)- and ET-1 (100 nM)-mediated hONA proliferation was assessed by CellTiter 96 aqueous one-solution cell proliferation assay, respectively. hONAs appeared to be more rounded with fewer processes following 24 h hypoxia compared to thodr seen in normoxia. Hypoxia enhanced TNF-α-mediated ET-1 synthesis and release (by 5-fold) and also significantly increased TNF-α- and ET-1-mediated hONA proliferation. PD142893 (1 μM), an ETA/B receptor antagonist, blocked ET-1-mediated hONA proliferation both under normoxia and hypoxia, while doing so only under normoxia following TNF-α treatment. Also, U0126 (10 μM; an upstream ERK1/2 inhibitor) completely blocked agonist-induced hONA proliferation in normoxia and partially blocked the same in hypoxia. These results demonstrate for the first time that hONAs secrete ET-1 and that TNF-α and hypoxia can regulate its levels. Moreover, hypoxia augments the proliferative responses of hONAs to TNF-α and ET-1. These agonist-mediated effects following hypoxia could contribute to astroglial activation as seen in glaucomatous optic nerve heads.

Hypoxia augments TNF-α-mediated endothelin-1 release and cell proliferation in human optic nerve head astrocytes

The effect of hypoxia (24 h) on TNF-α-mediated release of endothelin-1 (ET-1) from human optic nerve head astrocytes (hONAs) and TNF-α- and ET-1-induced hONA proliferation was determined. ET-1 synthesis and release was quantitated using ELISA while TNF-α (10 nM)- and ET-1 (100 nM)-mediated hONA proliferation was assessed by CellTiter 96 aqueous one-solution cell proliferation assay, respectively. hONAs appeared to be more rounded with fewer processes following 24 h hypoxia compared to thodr seen in normoxia. Hypoxia enhanced TNF-α-mediated ET-1 synthesis and release (by 5-fold) and also significantly increased TNF-α- and ET-1-mediated hONA proliferation. PD142893 (1 μM), an ET A/B receptor antagonist, blocked ET-1-mediated hONA proliferation both under normoxia and hypoxia, while doing so only under normoxia following TNF-α treatment. Also, U0126 (10 μM; an upstream ERK1/2 inhibitor) completely blocked agonist-induced hONA proliferation in normoxia and partially blocked the same in hypoxia. These results demonstrate for the first time that hONAs secrete ET-1 and that TNF-α and hypoxia can regulate its levels. Moreover, hypoxia augments the proliferative responses of hONAs to TNF-α and ET-1. These agonist-mediated effects following hypoxia could contribute to astroglial activation as seen in glaucomatous optic nerve heads.

DNA microarray analysis of gene expression in human optic nerve head astrocytes in response to hydrostatic pressure.

There is clinical and experimental evidence that elevated intraocular pressure (IOP), a mechanical stress, is involved in the pathogenesis of glaucomatous optic neuropathy. The mechanism by which astrocytes in the optic nerve head (ONH) respond to changes in IOP is under study. Gene transcription by ONH astrocytes exposed either to 60 mmHg hydrostatic pressure (HP) or control ambient pressure (CP) for 6, 24, and 48 h was compared using Affymetrix GeneChip microarrays to identify HP-responsive genes. Data were normalized across arrays within each gene. A linear regression model applied to test effect of time and HP on changes in expression level identified 596 genes affected by HP over time. Using GeneSpring analysis we selected genes whose average expression level increased or decreased more than 1.5-fold at 6, 24, or 48 h. Expression of selected genes was confirmed by real-time RT-PCR; protein levels were detected by Western blot. Among the genes highly responsive to HP were those involved in signal transduction, such as Rho nucleotide exchange factors, Ras p21 protein activator, tyrosine kinases and serine threonine kinases, and genes involved in transcriptional regulation, such as c-Fos, Egr2, and Smad3. Other genes that increased expression included ATP-binding cassettes, solute carriers, and genes associated with lipid metabolism. Among the genes that decreased expression under HP were genes encoding for dual activity phosphatases, transcription factors, and enzymes involved in protein degradation. These HP-responsive genes may be important in the establishment and maintenance of the ONH astrocyte phenotype under conditions of elevated IOP in glaucoma.

Altered expression of 3α-hydroxysteroid dehydrogenases in human glaucomatous optic nerve head astrocytes

3α-Hydroxysteroid dehydrogenase (3α-HSD) isoforms (AKR1C1–AKR1C4) are aldo-keto reductases that metabolize steroids and other substances in many tissues including the CNS. Here we demonstrated that in glaucomatous human optic nerve heads, increased expression of 3α-HSD was localized to reactive astrocytes in the lamina cribrosa. Similar, optic nerve head astrocytes exhibited increased expression of 3α-HSD in response to elevated intraocular pressure in a monkey model of experimental glaucoma, but not in monkeys with unilateral optic nerve transection. In vitro, glaucomatous optic nerve head astrocytes expressed higher levels of AKR1C1, AKR1C2, and AKR1C3 mRNA, than normal astrocytes, with significant differential increase of AKR1C2 expression, and exhibited higher enzymatic activity forming 3α-androstanediol a well-recognized neurosteroid. Normal astrocytes exposed to elevated hydrostatic pressure selectively increased AKR1C2 expression. Our findings of increased expression of 3α-HSDs in glaucomatous optic nerve head astrocytes offer new insights into possible roles for neurosteroids in the pathophysiology of glaucoma.