Galectin-1 and -3 in high amounts inhibit angiogenic properties of human retinal microvascular endothelial cells in vitro.

PURPOSE: Galectin-1 and -3 are ß-galactoside binding lectins with varying effects on angiogenesis and apoptosis. Since in retinal pigment epithelial cells high amounts of human recombinant galectin (hr-GAL)1 and 3 inhibit cell adhesion, migration and proliferation, we investigated if hr-GAL1 and 3 have homologous effects on human retinal microvascular endothelial cells (HRMEC) in vitro. METHODS: To investigate the effect of galectin-1 and -3 on HRMEC, proliferation, apoptosis and viability were analyzed after incubation with 30, 60 and 120 µg/ml hr-GAL1 or 3 by BrdU-ELISA, histone-DNA complex ELISA, live/dead staining and the WST-1 assay, respectively. Further on, a cell adhesion as well as tube formation assay were performed on galectin-treated HRMEC. Migration was investigated by the scratch migration assay and time-lapse microscopy. In addition, immunohistochemical staining on HRMEC for ß-catenin, galectin-1 and -3 were performed and ß-catenin expression was investigated by western blot analysis. RESULTS: Incubation with hr-GAL1 or 3 lead to a decrease in proliferation, migration, adhesion and tube formation of HRMEC compared to the untreated controls. No toxic effects of hr-GAL1 and 3 on HRMEC were detected. Intriguingly, after treatment of HRMEC with hr-GAL1 or 3, an activation of the proangiogenic Wnt/ß-catenin signaling pathway was observed. However, incubation of HRMEC with hr-GAL1 or 3 drew intracellular galectin-1 and -3 out of the cells, respectively. CONCLUSION: Exogenously added hr-GAL1 or 3 inhibit angiogenic properties of HRMEC in vitro, an effect that might be mediated via a loss of intracellular endogenous galectins.

Alkylphosphocholines: a new approach to inhibit cell proliferation in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy represents the major complication in retinal detachment surgery and occurs in about 5-15% of cases resulting in a significant loss of vision despite multiple surgical procedures. Although successful anatomical reattachment is usually achieved, the reduction in central vision often remains permanent due to the intraretinal changes induced by retinal detachment and the subsequent proliferative response within the retina. Retinal Muller glial cells play a pivotal role in this process together with retinal pigment epithelial cells which are dispersed in the vitreous and stimulated by growth factors and serum in the vitreous after the breakdown of the blood-retinal barrier.

The effect of alkylphosphocholines on intraretinal proliferation initiated by experimental retinal detachment.

PURPOSE: To determine the effect of alkylphosphocholines (APCs) on intraretinal proliferation induced by experimental retinal detachment in the rabbit. METHODS: Retinal detachments were created in adult pigmented rabbits. APCs, either liposome bound (liposome, L-APC) or unbound (free, F-APC), were injected intravitreally on either day 1 or day 2 after detachment. BrdU was injected on day 3, 4 hours before death. After fixation, retinas were triple labeled with anti-BrdU, anti-vimentin, and the isolectin B4. The number of anti-BrdU-labeled cells was counted per millimeter of retina from sections imaged by laser scanning confocal microscopy. Toxicity was examined using toluidine blue-stained sections imaged by light microscopy and by electron microscopy for ultrastructural evaluation. RESULTS: Retinal detachment initiated proliferation of all non-neuronal cells. After intravitreal injection on day 1 or 2 after experimental induction of retinal detachment, APCs significantly reduced the number of dividing cells at day 3. Liposome-bound drug given on day 2 was more effective on Müller cell proliferation than was unbound drug. Injection of F-APC on day 1 was more effective than when given on day 2. No apparent effect was seen on Müller cell hypertrophy as indicated by vimentin expression. In addition, no evidence of toxicity was observed in the retina at day 3 for any of the conditions. CONCLUSIONS: APCs significantly reduce the number of Müller cells that are stimulated to divide as a result of retinal detachment. The preliminary results indicate no evidence of significant toxicity; however, further studies are needed. APCs have the potential to be used as part of a therapeutic approach if they can be combined with other agents that can suppress the fibrosis that is also a critical event in the pathogenesis of proliferative vitreoretinal diseases such as proliferative vitreoretinopathy (PVR).

Inhibition of human retinal pigment epithelial cell attachment, spreading, and migration by alkylphosphocholines.

PURPOSE: To investigate the effect of alkylphosphocholines (APCs) on human retinal pigment epithelium (RPE) attachment, spreading, migration, and microfilament assembly in vitro. METHODS: Cultured RPE cells of five human donors were treated with one of four APCs (C18:1-PC, C20:1-PC, C21:1-PC, or C22:1-PC) in the presence of fetal calf serum. Cell viability was tested by the trypan blue exclusion assay. Attachment was assessed after a 2-hour incubation of RPE cells on coated 96-well-plates and subsequent MTT testing. Cellular spreading is characterized by cytoplasmic halo formation and was quantified by counting four separate fields of RPE cells allowed to spread on coated 24-well plates for 4 hours. Migration was measured by a modification of the Boyden chamber method in microchemotaxis chambers with polycarbonated filters. Microfilament assembly was assessed by immunofluorescence analysis after incubation with rhodamine-phalloidin. RESULTS: All four APCs inhibited RPE cell attachment by more than 70% of their IC50 (C18:1-PC: 30 microM; C20:1-PC: 10 microM; C21:1-PC: 10 microM; and C22:1-PC: 10 microM). Also, APCs inhibited RPE cell spreading by more than 80% and migration by more than 90% at similar concentrations. Trypan blue staining revealed a toxicity within control limits within the concentration interval tested. Microfilament organization was significantly disturbed after incubation of RPE cells with one of the four APCs close to its IC50. CONCLUSIONS: APCs inhibit RPE cell attachment and spreading in vitro at nontoxic concentrations. As a possible mechanism of action, APCs disturb microfilament assembly, since they are known to interfere with protein kinase C (PKC) function. This could represent a novel method of preventing even early stages of proliferative vitreoretinal diseases like proliferative vitreoretinopathy (PVR).

Toxicity study of erucylphosphocholine in a rat model.

PURPOSE: To investigate the effect of intraocular erucylphosphocholine (ErPC) on the retina, the retinal pigment epithelium (RPE), and the choroid in an in vivo rat model. METHODS: Adult male Brown Norway rats were injected intravitreally with ErPC dissolved in balanced salt solution (BSS) at a final concentration of 10 or 100 microM with BSS serving as control. Adverse effects on the anterior and posterior segment were assessed by slit-lamp biomicroscopy and ophthalmoscopy. Retinal toxicity was assessed by electroretinography (ERG), retinal ganglion cell (RGC) quantification, and histology 7 days after intravitreal administration of ErPC. RESULTS: There was neither a statistically significant difference in the clinical examination nor in the ERG waves of treated versus control rats 7 days after intravitreal administration of ErPC. Correspondingly, the number of RGC after BSS injection did not differ significantly from ErPC-injected animals. Histologic sections of the posterior segment of 10 and 100 microM ErPC-injected rats did not show any signs of retinal toxicity. Electron microscopy did not display a difference between the 10 microM and the control group. Only the 100 microM-injected animals showed a discrete irregularity of the Müller cell and the retinal ganglion cell cytoplasm at the ultrastructural level. CONCLUSIONS: ErPC can safely be injected into the vitreous of adult rats at a concentration of 10 microM without any retinal toxicity. Even a 10-fold increase in ErPC concentration leads only to a discrete cytoplasmic irregularity of the innermost retinal layers.

Alkylphosphocholines: a new therapeutic option in glaucoma filtration surgery.

PURPOSE: To investigate the effect of alkylphosphocholines (APCs) on human Tenon fibroblast (HTF) proliferation, migration, and cell-mediated collagen gel contraction. METHODS: HTFs were isolated from tissue samples of three patients obtained during surgery and cultured in DMEM and 10% fetal calf serum (FCS). HTFs (passage 3-6) were treated with one APC in different concentrations spanning the 50% inhibitory concentration (IC(50)), as determined previously. Inhibition of cell proliferation was assessed by the tetrazolium dye reduction assay. Migration was determined in chemoattractant chambers with fibronectin-coated polycarbonated membranes. For inhibition of contraction, three-dimensional collagen gels were seeded with HTFs, and the gel size was measured. Cell viability was determined by the trypan blue exclusion assay. For analysis of the mechanism of action, protein kinase C (PKC) activity was measured. RESULTS: The IC(50) varied between 7.0 and 10.5 microM for all APCs tested. At this concentration, all four APCs inhibited HTF migration and cell-mediated collagen gel contraction in the presence of serum. The inhibitory effects on HTF proliferation, migration, and contraction were observed at nontoxic concentrations. PKC activity was reduced to 50% of control level at the IC(50) of all APCs applied. CONCLUSIONS: APCs are effective inhibitors of HTF proliferation, migration, and cell-mediated contraction of collagen gels at nontoxic concentrations. Their mechanism of action seems to involve the inhibition of the PKC pathway.

Alkylphosphocholines inhibit proliferation of human retinal pigment epithelial cells.

PURPOSE: To investigate the effect and mechanism of action of alkylphosphocholines (APCs) on proliferation of human retinal pigment epithelium (RPE) cells and RPE-mediated collagen matrix contraction in vitro. METHODS: Cultured RPE cells of five human donors were treated with four APCs in the presence of fetal calf serum. Proliferation was assessed by the tetrazolium dye-reduction (MTT) assay and by counting the number of cells dividing in culture. The effect of APCs on RPE-mediated matrix contraction was determined in three-dimensional collagen gels. Cell viability was tested by the trypan blue exclusion assay. As a possible mechanism of APC action, protein kinase C (PKC) activity was quantified by scintillation counting of (32)P-labeled phosphate transferred to a PKC-specific substrate. RESULTS: All APCs inhibited RPE proliferation and RPE-mediated collagen matrix contraction in a dose-dependent manner in vitro. The antiproliferative and anticontractile effect of APCs increased with elongation of the fatty acid chain beyond C20. IC(50)s of all APCs varied between 8.5 micro M (erucyl-phosphocholine, C22:1-PC), 9.0 micro M (Z)-12-heneicosenyl-phosphocholine, C21:1-PC), 11.0 micro M (Z)-10-eicosenyl-phosphocholine, C20:1-PC), and 26.5 micro M (oleyl-phosphocholine, C18:1-PC). Trypan blue staining revealed a toxicity below 5% for all APCs within the concentration interval tested. PKC activity was significantly reduced by all four APCs, with C22:1-PC being the most effective. CONCLUSIONS: APCs inhibit proliferation of RPE cells and RPE-mediated matrix contraction in vitro at nontoxic concentrations. This effect seems to be exerted through inhibition of PKC activity. Therefore, APCs are promising candidates for treatment of RPE-mediated proliferative processes such as proliferative vitreoretinopathy.

Alkylphosphocholines for intraocular lens coating.

PURPOSE: To assess the effect of alkylphosphocholine (APC)-coated intraocular lenses (IOLs) on human lens epithelial cell (LEC) proliferation in vitro and the corneal biocompatibility of APC in an organ culture model of human donor eyes. SETTING: Research Laboratory for Experimental Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental study. METHODS: Six foldable IOLs differing in optic material were incubated with APC, washed in phosphate-buffered saline, and dried overnight. Intraocular lenses of the same lot served as uncoated controls. Each rehydrated IOL was placed in 1 well of a 24-well plate containing proliferating human LECs. Cell survival was tested by the tetrazolium-dye reduction assay 5 days later. Biocompatibility of the human corneal endothelium was assessed by a 24-hour incubation of human donor corneas with different concentrations of APC before the live/dead assay was performed. RESULTS: Human LEC proliferation was attenuated by APC-coated IOLs (P=.001). Coated hydrophilic acrylic IOLs were more effective inhibitors of human LEC proliferation than coated hydrophobic acrylic or silicone IOLs (P=.001). Alkylphosphocholines were well tolerated by the corneal endothelium in an organ culture model of human donor corneas up to a concentration of 1 mM (n = 12). CONCLUSIONS: Results show that APCs are suitable agents for IOL coating without linker molecules. Coated IOLs can inhibit human LEC proliferation and were well tolerated by human donor corneas in organ culture.

Quality of life in the follow-up of uveal melanoma patients after CyberKnife treatment.

To assess quality of life in uveal melanoma patients within the first and second year after CyberKnife radiosurgery. Overall, 91 uveal melanoma patients were evaluated for quality of life through the Short-form (SF-12) Health Survey at baseline and at every follow-up visit over 2 years after CyberKnife radiosurgery. Statistical analysis was carried out using SF Health Outcomes Scoring Software and included subgroup analysis of patients developing secondary glaucoma and of patients maintaining a best corrected visual acuity (BCVA) of the treated eye of 0.5 log(MAR) or better. Analysis of variance, Greenhouse-Geisser correction, Student's t-test, and Fisher's exact test were used to determine statistical significance. Physical Functioning (PF) and Role Physical (RP) showed a significant decrease after CyberKnife radiosurgery, whereas Mental Health (MH) improved (P=0.007, P<0.0001 and P=0.023). MH and Social Functioning (SF) increased significantly (P=0.0003 and 0.026) in the no glaucoma group, MH being higher compared with glaucoma patients (P=0.02). PF and RP were significantly higher in patients with higher BCVA at the second follow-up (P=0.02). RP decreased in patients with BCVA<0.5 log(MAR) (P=0.013). Vitality (VT) increased significantly in patients whose BCVA could be preserved (P=0.031). Neither tumor localization nor size influenced the development of secondary glaucoma or change in BCVA. Although PF and RP decreased over time, MH improved continuously. Prevention of secondary glaucoma has a significant influence on both SF and MH, whereas preservation of BCVA affects VT. Emotional stability throughout follow-up contributes positively toward overall quality of life. CyberKnife radiosurgery may contribute to attenuation of emotional distress in uveal melanoma patients.

Immunocytochemical and ultrastructural evidence of glial cells and hyalocytes in internal limiting membrane specimens of idiopathic macular holes.

PURPOSE: To provide new information on epiretinal cell proliferation and the cells' origin in idiopathic macular holes and to overcome the effects of embedding and sectioning preparation procedures on cell-distribution patterns. METHODS: Interference and phase-contrast microscopy, immunocytochemistry, and scanning and transmission electron microscopy were performed on surgically excised whole-mounted internal limiting membrane (ILM) specimens removed from 60 eyes with idiopathic macular holes. Cell distribution and cell morphology were correlated with immunocytochemical staining characteristics. Twelve cell type-specific antibodies were used to detect glial cells, hyalocytes, retinal pigment epithelial cells, retinal ganglion cells, and immune cells. Cell viability was analyzed. RESULTS: Epiretinal cell proliferation was found in all ILM specimens, irrespective of the stage of the macular hole. Cell density showed a broad variety. Immunocytochemistry frequently revealed simultaneous expression of GFAP/CD45, GFAP/CD64, GFAP/CD68, GFAP/CRALBP, and GFAP/CD90. Some cells presented with intracellular contractile filaments (anti-αSMA); others were not immunoreactive to any antibody examined. The percentage of viable cells showed a broad variety with a mean of 73% (SD 29%). Electron microscopy demonstrated glial cells, hyalocytes, and myofibroblast-like cells. CONCLUSIONS: The presence of epiretinal cells at the ILM in all macular hole stages strongly suggests a substantial involvement of cell migration and proliferation in the course of macular hole development. Glial cells and hyalocytes play the predominant role in epiretinal cell proliferation. Given the co-expression of glial cell and hyalocyte markers, transdifferentiation of epiretinal cells needs further elucidation, especially with respect to αSMA-positive cells leading to traction at the vitreoretinal interface.

Sequential epiretinal membrane removal with internal limiting membrane peeling in brilliant blue G-assisted macular surgery.

PURPOSE: To assess the selectivity of brilliant blue G (BBG) staining by analysing the morphological components of unstained and stained tissue obtained during epiretinal membrane (ERM) removal with internal limiting membrane (ILM) peeling in BBG-assisted macular surgery. METHODS: Twenty-six surgical specimens were removed from 13 eyes with epiretinal gliosis during vitrectomy using BBG for ERM and ILM peeling. We included eyes with idiopathic macular pucker, idiopathic macular hole and vitreomacular traction syndrome. The dye was injected into the fluid-filled globe. Unstained and stained epiretinal tissue was harvested consecutively and placed into separate containers. All specimens were processed for conventional transmission electron microscopy. RESULTS: The first surgical specimen of all eyes showed no intraoperative staining with BBG and corresponded to masses of cells and collagen. The second surgical specimen demonstrated good staining characteristics and corresponded to the ILM in all patients included. In seven eyes, the ILM specimens were seen with minor cell proliferations such as single cells or a monolayer of cells. Myofibroblasts, fibroblasts and astrocytes were present. In five cases, native vitreous collagen fibrils were found at the ILM. In six of the eyes, ILM specimens were blank. CONCLUSION: Our clinicopathological correlation underlines the selective staining properties of BBG. The residual ILM is selectively stained by BBG even when a small amount of cells and collagen adheres to its vitreal side. To reduce the retinal exposure to the dye, the surgeon might choose to remove the ERM without using the dye, followed by a BBG injection to identify residual ILM.

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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Alkylphosphocholines as a potential pharmacologic prophylaxis for posterior capsule opacification.

PURPOSE: To evaluate the effect of alkylphosphocholines (APCs) on human lens epithelial cell (LEC) proliferation, attachment, and migration in a well-established in vitro model. SETTING: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. METHODS: The immortalized human LEC line HLE-B3 was incubated for 24 hours with APC in different concentrations in the presence of Eagle's modified essential medium supplemented with fetal calf serum under standard cell-culture conditions. The trypan blue exclusion test and live-dead assay were performed to exclude toxic concentrations. To determine cell proliferation, cells were incubated with APCs at the maximum slope of the growth curve for 24 hours before the tetrazolium dye-reduction assay (MTT test) was performed. After cells were seeded on coated 24-well plates, incubated with APCs, and rinsed with phosphate-buffered saline, cell attachment was assessed by the MTT test. Migration was determined by a modified Boyden chamber method after incubation of LECs with APCs. RESULTS: Alkylphosphocholines were effective inhibitors of human LEC proliferation, attachment, and migration at nontoxic concentrations in vitro. The 50% inhibitory concentration was close to 0.1 mM. An APC concentration of 1.0 mM accounted for the following: inhibition of cell proliferation of more than 80%, reduction in cell attachment to 66.5%, and inhibition of cell migration of more than 90%. All effects were dose dependent. No toxic effects were detected compared with controls. CONCLUSIONS: Alkylphosphocholines might have the potential for topical application as a single-dose agent to prevent posterior capsule opacification formation. However, further studies are needed before a clinical application can be considered.

Intracameral moxifloxacin: in vitro safety on human ocular cells.

PURPOSE: The fourth-generation fluoroquinolone, moxifloxacin, covers most gram-positive and gram-negative isolates causing endophthalmitis. It is safe and effective for systemic and topical use, but only limited data are available on prophylactic intracameral administration to prevent endophthalmitis. This study uses a cell culture model to investigate the safety of moxifloxacin for intracameral application. METHODS: Endothelial toxicity of moxifloxacin was evaluated in cultured human corneas. Possible toxic effects of moxifloxacin (10-750 microg/mL) in corneal endothelial cells (CEC), primary human trabecular meshwork cells (TMC), and primary human retinal pigment epithelial (RPE) cells were evaluated after 24 hours and under conditions of oxidative and inflammatory stress by treatment with tumor necrosis factor alpha, lipopolysaccharides, or interleukin-6. Toxicity was evaluated by tetrazolium dye reduction assay, and cell viability was quantified by a microscopic live-dead assay. RESULTS: No corneal endothelial toxicity could be detected after 30 days of treatment with 500 microg/mL moxifloxacin. Concentrations up to 150 microg/mL had no influence on CEC, TMC, or RPE cell proliferation or on cell viability when administered for 24 hours. After preincubation with tumor necrosis factor alpha, lipopolysaccharides, or interleukin-6 for 24 hours and subsequent treatment with moxifloxacin at concentrations from 10 to 150 microg/mL for 24 hours, no significant decrease in proliferation or viability was observed. Hydrogen peroxide exposure did not increase cellular toxicity. CONCLUSIONS: This study showed no significant toxicity for moxifloxacin on CEC, TMC, RPE cells, or human corneal endothelium for concentrations up to 150 microg/mL. The minimum inhibitory concentration of moxifloxacin to inhibit 90% of pathogens commonly encountered in endophthalmitis is known to be in the range of 0.25-2.5 microg/mL. Therefore, prophylactic intracameral use of moxifloxacin at concentrations up to 150 microg/mL may be safely used to prevent endophthalmitis after intraocular surgery.

Cytoprotective effects of a blue light-filtering intraocular lens on human retinal pigment epithelium by reducing phototoxic effects on vascular endothelial growth factor-alpha, Bax, and Bcl-2 expression.

PURPOSE: To compare the possible protective effects of the ultraviolet (UV)-filtering and blue light-filtering SN60AT intraocular lens (IOL) and the untinted UV-filtering SA60AT IOL with regard to light-induced stress on human retinal pigment epithelium (RPE). SETTING: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. METHODS: Primary human RPE cells were exposed to white light, and a tinted or untinted IOL was placed in the light beam. After 15 to 60 minutes of irradiation, cell viability was determined by a colorimetric test (tetrazolium dye-reduction assay) and a microscopic live/dead assay. The expression of vascular endothelial growth factor-alpha (VEGF-alpha), Bax, and Bcl-2 and their mRNA was determined by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: Without an IOL, white-light exposure decreased cell viability compared with the decrease with the nonirradiated control in a time-dependent manner. Light-induced cell death was significantly reduced by both the tinted IOL and untinted IOL. The combined UV and blue-light filtering attenuated light-induced cell damage significantly more than UV filtering alone. Results of RT-PCR and Western blotting showed a significant time-dependent decrease in Bcl-2 and increase in Bax and VEGF-alpha that were significantly less with the tinted IOL than with the untinted IOL. CONCLUSIONS: Both IOLs reduced light-induced RPE damage. The UV- and blue light-filtering IOL reduced damage more than the conventional IOL. This supports the hypothesis that blue light-filtering IOLs may prevent retinal damage in clinical use.

The role of alkylphosphocholines in retinal Müller glial cell proliferation.

PURPOSE: Alkylphosphocholines (APCs) are investigated for their effect on Müller glial cell proliferation and F-actin stress fiber distribution in vitro. MATERIALS AND METHODS: Müller cells were incubated with APCs (C18:1-PC and C22:1-PC) +/- fetal calf serum. Proliferation was assessed by BrdU labeling and with the tetrazolium dye-reduction assay. Toxicity was measured using the trypan blue exclusion assay. The distribution of F-actin stress fibers was determined using FITC-phalloidin staining. RESULTS: APCs are effective inhibitors of human and rat Müller glial cell proliferation and hypoxia-induced up-regulation of F-actin stress fibers in vitro in non-toxic concentrations. CONCLUSIONS: APCs might prevent intraretinal changes as a result of serum stimulation and hypoxia following retinal detachment.

Hypoxia/reoxygenation and TGF-beta increase alphaB-crystallin expression in human optic nerve head astrocytes.

Reactive astrocytes in glaucomatous optic nerve changes are characterized by an increased expression of alphaB-crystallin and transforming growth factor-beta (TGF-beta). 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-beta on the expression of alphaB-crystallin in cultured human astrocytes of the optic nerve head (ONH). Cultured human astrocytes were incubated under hypoxic conditions (1% O2 for 4-12 h) with subsequent reoxygenation (12-24 h). Additionally, cells were treated with 1.0 ng/ml TGF-beta1 and TGF-beta2 for 12-48 h. Expression of alphaB-crystallin was examined by Northern- and Western-blotting. Levels of TGF-beta1 and TGF-beta2 were analyzed by RT-PCR analysis and ELISA. The effect of TGF-beta blocking on the hypoxia/reoxygenation modulated expression of alphaB-crystallin was investigated by simultaneous incubation with neutralizing antibodies against TGF-beta during the reoxygenation phase. Hypoxia/reoxygenation increased the expression of alphaB-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-beta1 and TGF-beta2 for 12-48 h markedly enhanced alphaB-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-beta isoforms prevented the hypoxia/reoxygenation-mediated elevation of alphaB-crystallin. The process of hypoxia/reoxygenation is capable of inducing the expression of alphaB-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.