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Purpose: To investigate the roles of Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ), the major effector molecules of the Hippo pathway, in TGF-β2-mediated conjunctival fibrosis., Methods: Primary human conjunctival fibroblasts were treated with TGF-β2. The expression of YAP/TAZ was examined by Western blot analyses and immunocytochemistry. The expression of fibrotic proteins and genes were evaluated by Western blot analyses and quantitative real-time PCR, respectively. The effects of YAP/TAZ on fibrotic changes were examined by knockdown experiments and the YAP/TAZ inhibitor, verteporfin., Results: TGF-β2 stabilized YAP/TAZ and subsequently activated Smad2/3, which led to the transcription of fibrotic genes in human primary conjunctival fibroblasts. These fibrotic genes were differently regulated by YAP/TAZ. Notably, α-smooth muscle actin, fibronectin, collagen I, and collagen IV were primarily regulated by YAP. In contrast, CCN family proteins (CTGF and CYR61) depended on both YAP and TAZ. Mechanistically, YAP/TAZ were located in close proximity to Smad2/3, and in particular, YAP was required for TGF-β2-mediated phosphorylation and the nuclear translocation of Smad2/3. Furthermore, a YAP/TAZ inhibitor markedly suppressed TGF-β2-mediated fibrotic changes in conjunctival fibroblasts., Conclusions: YAP/TAZ acted as a molecular hub of TGF-β2 signaling in a cellular model of conjunctival fibrosis. Moreover, verteporfin, a YAP/TAZ inhibitor exerted potent antifibrosis effects by suppressing TGF-β2-YAP/TAZ-Smad signaling. Our study highlights YAP/TAZ as essential regulators of conjunctival fibrosis and shows that inhibition of YAP/TAZ might potentially improve the outcomes of glaucoma filtration surgery.

Purpose: To explore interactions between pilocarpine and the ROCK inhibitor, ripasudil, on IOP and pupil diameter in human eyes, and morphological and functional changes in outflow tissues in vitro., Methods: IOP and pupil diameter were measured after pilocarpine and/or ripasudil, which were topically applied in healthy subjects. Human trabecular meshwork (HTM) cells were used in a gel contraction assay, for the evaluation of phosphorylation of myosin light chain and cofilin, and immunostaining for cytoskeletal proteins. Porcine ciliary muscle (CM) was used in a CM contraction assay. The permeability of human Schlemm's canal endothelial (SCE) cells was evaluated by measuring transendothelial electrical resistance and fluorescein permeability., Results: Both pilocarpine and ripasudil significantly reduced IOP in human eyes, but pilocarpine interfered with ripasudil-induced IOP reduction when concomitantly introduced. Ripasudil significantly inhibited gel contraction, TGFβ2-induced stress fiber formation, α-smooth muscle actin expression, and phosphorylation of both myosin light chain and cofilin in HTM cells. Pilocarpine reduced these effects, significantly inhibited the ripasudil-induced HTM cell responses to TGFβ2 stimulation, and increased the permeability in SCE cells. In CM, ripasudil inhibited pilocarpine-stimulated contraction, but ripasudil did not have significant effects on pilocarpine-induced miosis., Conclusions: Pilocarpine interfered with the direct effects of ROCK inhibitor on the conventional outflow pathway leading to IOP reduction and cytoskeletal changes in trabecular meshwork cells, but did not affect the relaxation effect of the ROCK inhibitor. It is therefore necessary to consider possible interference between these two drugs, which both affect the conventional outflow.

Purpose: To identify determinants of Bruch's membrane opening (BMO), and BMO-minimum rim width (BMO-MRW) and circumpapillary retinal nerve fiber layer thickness (RNFLT) centered on BMO center and characterize these parameters in a normal Japanese population., Methods: Spectral-domain optical coherence tomography images of optic nerve head and circumpapillary and macular retina were obtained in 258 eyes of 258 normal Japanese with mean (standard deviation) age of 51.7 (18.2) years. BMO area, BMO-MRW, RNFLT (measured with a 3.5-mm-diameter circle scan) were all acquired and analyzed relative to the eye-specific fovea to BMO (FoBMO) axis. One randomly selected eye of each subject was analyzed. Multiple regression analysis was used to identify determinants to the parameters., Results: BMO area, global BMO-MRW, RNFLT, and FoBMO angle averaged 2.06 (0.45) mm2, 305.5 (50.0) μm, 101.8 (9.6) μm, and -7.8° (3.8°), respectively. There was a modest correlation between global BMO-MRW and RNFLT (r = 0.337; P < 0.001), while the sectorwise correlations were highest in the superior-temporal sector (r = 0.500; P < 0.001) and lowest in the nasal sector (r = 0.117; P = 0.063). Global BMO-MRW and RNFLT declined with age at -1.04 μm/y (P < 0.001) and -0.12 μm/y (P = 0.001), and the former correlated negatively (P = 0.001) and the latter positively (P < 0.001) with BMO area after adjustment for other factors (R2 = 0.191 and 0.272, respectively). BMO area correlated positively with axial length (P = 0.023) and negatively with age (P < 0.001) (R2 = 0.157)., Conclusions: BMO-MRW and RNFLT declined with age with a difference between them in their relationship to BMO area. BMO area positively correlated with axial length and negatively with age.

Purpose: Suberoylanilide hydroxamic acid (SAHA) has been shown to support the maintenance of experimental filtration blebs in animal models. This study was performed to investigate the molecular mechanisms underlying the bleb-maintaining effects of SAHA in modulating wound healing activities of conjunctival fibroblasts., Methods: Human conjunctival fibroblasts (HConFs) were pretreated with SAHA before treatment with TGF-β2. Microarray-based screening was used to investigate the gene expression profiles. Gene ontology (GO) analysis was conducted to categorize the gene functions. The expression of TGF-β-induced signaling molecules, α-smooth muscle actin, and extracellular matrix (ECM) proteins were evaluated by Western blot analyses. Multiplex immunoassay was performed to evaluate supernatant cytokine concentrations. Tube formation assay was used to evaluate angiogenesis using human umbilical vein endothelial cells., Results: GO analysis showed that SAHA, in the presence of TGF-β2, induced changes in expression of genes involved in the TGF-β receptor signaling pathway, cell proliferation, extracellular matrix organization, inflammatory responses, and angiogenesis. Subsequent in vitro experiments showed that SAHA partly inhibited the phosphorylation of Smad2, Smad3, and Akt. SAHA pretreatment potently suppressed TGF-β2-driven cell proliferation, myofibroblast differentiation, contraction, ECM production, and angiogenic cytokine expression. The supernatant of HConFs treated with SAHA inhibited tube formation., Conclusions: SAHA has been shown to suppress angiogenesis and activation of conjunctival fibroblasts partly via inhibition of Smad and non-Smad TGF-β signaling. This in vitro study provides new evidence for the molecular basis of the potential bleb-maintaining effects of SAHA, a novel candidate drug in modulating scar formation after glaucoma filtration surgery.

Purpose: The epigenetic mechanisms associated with ocular neurodegenerative diseases remain unclear. The present study aimed to determine the role of lysine-specific demethylase 1 (LSD1), which represses transcription by removing the methyl group from methylated lysine 4 of histone H3, in retinal ganglion cell (RGC) survival, and to investigate the details of the neuroprotective mechanism of tranylcypromine, a major LSD1 inhibitor., Methods: The authors evaluated whether tranylcypromine contributes to neuronal survival following stress-induced damage using primary cultured rat RGCs and in vivo N-methyl-D-aspartate (NMDA)-induced excitotoxicity. Additionally, the molecules associated with tranylcypromine treatment were assessed by microarray and immunoblot analysis., Results: Tranylcypromine significantly suppressed neuronal cell death following glutamate neurotoxicity and oxidative stress. Microarray and immunoblot analyses revealed that p38 mitogen-activated protein kinase (MAPK)γ was a key molecule involved in the neuroprotective mechanisms induced by tranylcypromine because the significant suppression of p38 MAPKγ by glutamate was reversed by tranylcypromine. Moreover, although pharmacologic inhibition of the phosphorylation of the total p38 MAPKs interfered with neuroprotective effects of tranylcypromine, the specific inhibition of p38 MAPKα and p38 MAPKβ did not influence RGC survival. This suggests that the non-p38 MAPKα/β isoforms have important roles in neuronal survival by tranylcypromine. Additionally, the intravitreal administration of tranylcypromine significantly saved RGC numbers in an in vivo glaucoma model employing NMDA-induced excitotoxicity., Conclusions: These findings indicate that tranylcypromine-induced transcriptional and epigenetic regulation modulated RGC survival via the promotion of p38 MAPKγ activity. Therefore, pharmacologic treatments that suppress LSD1 activity may be a novel therapeutic strategy that can be used to treat neurodegenerative diseases.

Purpose: The purpose of this study was to elucidate the differentiated expression patterns and phagocytic activities of two subtypes of microglia., Methods: A rat optic nerve crush model was used to identify the expression patterns of two subtypes of microglia. Primary microglia were isolated from rat mixed glial cultures. Subsequently, in vitro experiments evaluating the phagocytosis of axonal debris were performed to analyze responsiveness to immunologic modulators (lipopolysaccharide [LPS], interleukin [IL]-4 and interferon [IFN]-γ), and we assessed the effects of LPS and IL-4 in the optic nerve crush model. The expression levels of IL-4-associated signaling molecules were analyzed in immunoblotting experiments., Results: In the optic nerve crush model, increased numbers of microglia were found, and a minor and transient population was identified as type 1 microglia. The type 2 microglia phagocytosed more axonal debris than the type 1 microglia. The activities of both subtypes of microglia were enhanced by treatment with LPS and IFN-γ. However, the phagocytic activity of the type 1 microglia, which showed activated STAT6 signal transduction, was significantly inhibited by stimulation with the anti-inflammatory cytokine IL-4. LPS reduced the fragmentation of axons in crushed nerve fibers, whereas the axonal debris remained in IL-4-treated rats subjected to optic nerve crush., Conclusions: The present study revealed the time-dependent distribution of the two subpopulations of microglia in an optic nerve crush model and IL-4-dependent inhibition of the phagocytosis of axonal debris by type 1 but not type 2 microglia.

Purpose: To visualize intravital immune cell dynamics in the subconjunctival tissue during the wound-healing process using multiphoton microscopy., Methods: Gene-targeted mice expressing enhanced green fluorescent protein under the control of the endogenous lysozyme M promoter (LysM-eGFP mice) were anesthetized with isoflurane, and injured by a 10-0 nylon conjunctival suture. Vessels were visualized by intravenous injection of 70 kDa rhodamine-conjugated dextran. Using a multiphoton microscope, the three-dimensional images of the subconjunctival tissue were acquired every minute for 20 minutes before and 0.5, 3, 6, and 72 hours after injury. Raw imaging data were processed for four-dimensional images and analyzed for the number and the velocity of the LysM-eGFP-positive cells using Imaris software., Results: The intravital LysM-eGFP-positive cells and the red-labeled vessels were successfully visualized using a multiphoton microscope. The conjunctival and scleral collagen fibers were detected as secondary harmonic generation signals, which were colored blue. Compared with mice without injury, the number of LysM-eGFP-positive cells in the subconjunctival tissue after conjunctival surgery increased in a time-dependent manner. The cell velocities significantly increased until 3 hours after surgery (5.9 ± 3.2 μm/min; P < 0.0001) and the elevated level was sustained until 72 hours after injury (5.9 ± 3.3 μm/min)., Conclusion: This is the first report to visualize and evaluate intravital cellular dynamics during inflammation in the subconjunctival tissue using multiphoton microscopy. This technique may be a useful tool to characterize the molecular mechanisms of the wound-healing process after various ocular injuries, such as glaucoma surgery.

Purpose: To investigate aqueous humor proinflammatory cytokine levels of patients with neovascular glaucoma (NVG), and to analyze the effects of background factors in the expression of these molecules., Methods: This cross-sectional study enrolled 137 participants who were grouped into (1) primary open-angle glaucoma (POAG; n = 36) patients; (2) NVG patients (NVG; n = 33); and (3) cataract surgery patients as a comparative group (CG; n = 68). Aqueous humor samples were collected from the anterior chamber at the start of surgery, deposited in CryoTubes, registered, and stored at -80 °C until processing. Multiplex microparticle-based immunodetection was performed by using xMAP and the Human Cytokine/Chemokine Panel I. Bevacizumab was injected into the vitreous cavity 1 to 2 days before surgery in 22 NVG patients (IVB group), whereas 11 NVG patients received no antivascular endothelial growth factor (VEGF) therapy 3 months preoperatively (N group). The Wilcoxon rank sum test or Fisher's exact test for two variables and the Tukey-Kramer honestly significant difference test for multiple variables were used to compare the cytokine levels., Results: The NVG patients had higher levels of interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, tumor necrosis factor-α (TNF-α), and platelet-derived growth factor (PDGF)-AA compared to both the CG and POAG groups. The levels of IL-6, IL-8, MCP-1, and PDGF-AB/BB were higher in the IVB group than the N group, whereas the VEGF level was significantly lower in the IVB group (P < 0.01)., Conclusions: Intravitreal bevacizumab injection decreased VEGF levels, but not those of the other cytokines.

Purpose: The purpose of this study was to investigate the function of merlin, which is a binding partner of the hyaluronan receptor CD44, during the epithelial-to-mesenchymal transition (EMT) of human retinal pigment epithelium (ARPE-19) cells., Methods: Human retinal pigment epithelium cells were stimulated with TNF-α and treated using epithelial-to-mesenchymal-transition inhibitors (a dynamin inhibitor or transforming growth factor-β-activated kinase 1 [TAK1] inhibitor). Levels of protein expression were assessed by immunoblot analysis, and localization of the relevant proteins was determined by immunofluorescence microscopy. Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation assays. All experiments were performed in serum-free medium., Results: Tumor necrosis factor-α treatments downregulated the expression of merlin and led to the dissociation of CD44 and merlin. The ezrin-radixin-moesin (ERM) proteins were phosphorylated, and hyaluronan endocytosis was accelerated in merlin small interfering RNA (siMerlin)-transfected cells. Treatment with the endocytosis inhibitor dynasore blocked hyaluronan endocytosis, whereas treatment with TNF-α induced mesenchymal phenotypes and downregulation of merlin. Additionally, siMerlin transfection promoted p38MAPK phosphorylation, which was inhibited not only by TAK1 inhibitor treatment but also by TAK1 small interfering RNA (siRNA, siTAK1) transfection. The increased level of BrdU incorporation in siMerlin cells was reduced by additional siTAK1 transfection. Furthermore, TNF-α-induced mesenchymal differentiation and high motility were also inhibited by TAK1 inhibitor treatment and by siTAK1 transfection., Conclusions: Our findings demonstrated that merlin exerts inhibitory effects on TNF-α-induced EMT by regulating hyaluronan endocytosis and the TAK1-p38MAPK signaling pathway. The proliferative and mesenchymal characteristics of RPE cells play important roles in the development of intraocular fibrotic disorders, such as proliferative vitreoretinopathy (PVR), and our findings provide new therapeutic strategies to prevent the development of PVR.

Purpose: To investigate levels of biomarkers of Alzheimer's disease in the aqueous humor in patients with open-angle glaucoma (OAG)., Methods: Aqueous humor samples were collected from 38 patients with cataracts, 20 patients with POAG, and 32 patients with exfoliation glaucoma. Aqueous levels of apolipoprotein (Apo) AI, ApoCIII, ApoE, transthyretin (TTR), complement factor H, complement C3, and α2-macroglobulin (α2M) were determined by multiplex bead immunoassay. Clinical data were obtained from medical charts., Results: Age and sex did not differ significantly among groups. POAG patients had significantly higher aqueous levels of Apo AI, ApoCIII, ApoE, TTR, and α2M compared with cataract patients (P < 0.001, P = 0.036, P < 0.001, P < 0.001, and P < 0.001, respectively). Corresponding values for exfoliation glaucoma patients were also significantly higher compared with those for cataract patients (P = 0.003, P = 0.009, P < 0.001, P < 0.001, and P < 0.001), and those levels correlated positively with each other in eyes with OAG (P < 0.001 for all combinations). Complement factor H and complement C3 levels did not differ among groups. Mean deviation values for the Humphrey visual field test correlated positively with levels of Apo AI, ApoE, TTR, and complement factor H in OAG patients (P = 0.026, 0.012, 0.008, and 0.027, respectively), but age and IOP values were not correlated with protein levels., Conclusions: OAG patients had elevated levels of multiple biomarkers of Alzheimer's disease in the aqueous humor.

Purpose: To elucidate the potential of three-dimensional anterior segment optical coherence tomography (3D AS-OCT) for identifying filtration openings where aqueous humor flows from the sclera into the subconjunctival space., Methods: We used 3D AS-OCT and custom software to identify filtration openings, which were defined by pits and/or troughs in fluid-filled cavities in both horizontal and vertical rasters and corresponding C-scan images of scleral flap margin in the blebs. We measured bleb parameters, and at least three different reviewers surveyed the complete 3D images of the internal structure of the filtration blebs and associated findings., Results: We identified filtration openings in 118 (95%) of 124 eyes. Among these 118 eyes, we found only a single filtration opening in 90 eyes (76%) and two or more filtration openings in the remaining 28 eyes (24%). Filtration openings were located in the middle third of the scleral flap margins in 57 eyes (63%) of the 90 eyes. The pattern of incision and sutures used for closure of the conjunctival flap affected the location of the openings, but additional laser suture lysis did not. In 6 (5%) of the 124 eyes, filtration openings could not be identified because of high reflectivity and/or elevated bleb wall (2 eyes), and no fluid-filled cavities because a sponge-like structure masked the filtration openings just above the scleral flap (4 eyes)., Conclusions: 3D AS-OCT allows, in most cases, detailed evaluation of internal morphology of filtration blebs and precise identification of filtration openings on the scleral flap margins after trabeculectomy.

Purpose: To elucidate the impact of phacoemulsification on aqueous monocyte chemoattractant protein-1 (MCP-1) levels, and identify its cell origin., Methods: For clinical study, aqueous humor samples were collected before and after surgery (17.0 ± 4.0 months postoperatively) from 21 cataract cases that underwent phacoemulsification and intraocular lens (IOL) implantation. Aqueous MCP-1 levels were determined using a multiplex immunoassay. For animal experiments, rabbits underwent phacoemulsification (± IOL). Aqueous humor samples were collected from nonoperated eyes and operated eyes, and immunoassays were performed. Eyes were analyzed by reverse transcription-polymerase chain reaction and immunohistochemical studies., Results: In the clinical study, mean (± SD) aqueous MCP-1 levels were higher postoperatively (1773.5 ± 321.2 pg/mL) than preoperatively (796.9 ± 211.3 pg/mL; P < 0.0001). In animal experiments, mean aqueous MCP-1 levels (pg/mL) were higher in postoperative eyes on day 30 (207.1 ± 62.9) than in nonoperated eyes (31.2 ± 12.5; P = 0.018). IOL implantation did not affect the changes in MCP-1 levels. After phacoemulsification, MCP-1 mRNA expression was increased in the cornea, iris, ciliary body, and capsular bag. Expression of MCP-1 mRNA in the capsular bag, but not the other tissues, increased from day 30 to 90. Immunohistochemical studies showed positive immunoreactivity for MCP-1 in cells of the posterior capsule after phacoemulsification., Conclusions: aqueous MCP-1 levels were elevated in both human and animal eyes after phacoemulsification. Proliferated Lens epithelial cells on the capsule might be the major cell origin for prolonged MCP-1 production after phacoemulsification. (http://www.umin.ac.jp/ number, UMIN000005788.).

Purpose: We investigated the involvement of the RhoA/Rho kinase (ROCK) signal transduction pathway in dexamethasone (DEX)-induced changes in aqueous outflow., Methods: Using trabecular meshwork (TM) and Schlemm's canal endothelial (SCE) cells, RhoA activation was evaluated with a pull-down assay and myosin light chain phosphorylation was evaluated by Western blot analysis. Outflow facility was measured in perfused porcine anterior segment organ cultures treated with DEX and/or Y-27632, a selective ROCK inhibitor. The barrier function of the cultured cells on a micropore filter was evaluated by measuring the transendothelial electrical resistance. Collagen, fibronectin, and integrin mRNA expression levels were evaluated by quantitative real-time RT-PCR., Results: Relative RhoA activities increased following stimulation with 100 nM DEX in TM and SCE cells. Perfusion with DEX decreased outflow facility by 31.9 ± 14.3% compared to controls at 24 hours, but not by 50 μM Y-27632 in addition to DEX. The transendothelial electrical resistance of the SCE cell monolayer was increased by 48.6 ± 6.4% and 5.3 ± 5.0% following DEX treatments without and with 10 μM Y-27632, respectively, compared to controls. In TM cells, the mRNA expressions of COL4A1 and fibronectin were increased significantly by DEX treatment, but combined treatment with Y-27632 and DEX significantly inhibited the increase in COL4A1 and fibronectin expression., Conclusions: Activation of the Rho/ROCK pathway in SCE cells contributes to the mechanism of DEX-induced changes in aqueous outflow.

Purpose: To investigate the effect of monocyte chemoattractant protein-1 (MCP-1)/CC chemokine ligand 2 on aqueous humor outflow facility., Methods: Aqueous humor outflow facility was measured in enucleated porcine eyes in a constant pressure perfusion system with or without MCP-1 (1600 ng/mL). Expression of CCR2, an MCP-1 receptor, in Schlemm's canal endothelial (SCE) cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) assay. The effect of MCP-1 (0-1600 ng/mL) on SCE cell viability was evaluated using a WST-8 assay. The effect of MCP-1 (0-800 ng/mL) on SCE-cell monolayer permeability was evaluated with or without a CCR2 antagonist (10 nM) by measuring transendothelial electrical resistance (TEER). The intracellular localization of the gap junction protein ZO-1 was analyzed by immunofluorescence staining of SCE cells., Results: The aqueous humor outflow facility increased significantly from basal levels at 80 minutes after perfusion with MCP-1 compared with control eyes (21.2% ± 6.6% [MCP-1] vs. 5.7 ± 2.5% [control]; P = 0.048). CCR2 was detected by RT-PCR. Cell viability was not affected by MCP-1 treatment. TEER of SCE-cell monolayer at 3 hours after treatment with 800 ng/mL MCP-1 decreased by 21.6 ± 1.7% compared with controls (P = 0.014), and the TEER-decreasing effects of MCP-1 were attenuated by a CCR2 antagonist. Immunocytochemical staining revealed a modest disruption of ZO-1 in MCP-1-treated SCE cells., Conclusions: The present results revealed that MCP-1 increased aqueous humor outflow facility and decreased TEER via CCR2. These findings suggest that MCP-1 modulates aqueous humor outflow through the conventional pathway.

Purpose: To investigate the effect of a specific inhibitor of Rho-associated protein kinase, Y-27632, on monkey Schlemm's canal endothelial (SCE) cells., Methods: SCE cells were isolated from cynomolgus monkey eyes. The effects of Y-27632 on aqueous outflow facility were evaluated using enucleated monkey eyes and a constant-pressure perfusion system. The effect of Y-27632 on the barrier function of the confluent SCE-cell monolayer was evaluated by measuring transendothelial electrical resistance (TEER) and fluorescein permeability. Y-27632-induced changes in the intracellular localization of ZO-1, claudin-5, β-catenin, pan-cadherin, and filamentous actin (F-actin) were examined by immunofluorescence. Gene-expression changes induced by Y-27632 were analyzed with microarray, and the functional categories of changed genes were identified by gene ontology analysis. The concentrations of intracellular calcium ions were estimated using Fluo-4/AM and a fluorescence microscope system., Results: Y-27632 significantly increased the outflow facility and the number of associated giant vacuoles, decreased TEER of the SCE-cell monolayer, and increased the transendothelial flux of fluorescein. Y-27632 disrupted ZO-1 and claudin-5 expression in a confluent SCE-cell monolayer. Among 12,544 genes, Y-27632 treatment increased the expression of 57 genes and decreased the expression of 15 genes. Gene ontology analysis revealed that changed genes were related to various cellular functions, including regulation of calcium ion transport into the cytosol. Y-27632 partially diminished the A23187-induced increase in intracellular calcium ions., Conclusions: Y-27632 increased the permeability of the SCE-cell monolayer in association with disruption of the tight junction, F-actin depolymerization, and changes in various cell functions, including calcium transfer.

PURPOSE. Heparan sulfate (HS) is abundantly expressed in the developing neural retina; however, its role in the intraretinal axon guidance of retinal ganglion cells (RGCs) remains unclear. In this study, the authors examined whether HS was essential for the axon guidance of RGCs toward the optic nerve head. METHODS. The authors conditionally ablated the gene encoding the exostosin-1 (Ext1) enzyme, using the dickkopf homolog 3 (Dkk3)-Cre transgene, which disrupted HS expression in the mouse retina during directed pathfinding by RGC axons toward the optic nerve head. In situ hybridization, immunohistochemistry, DiI tracing, binding assay, and retinal explant assays were performed to evaluate the phenotypes of the mutants and the roles of HS in intraretinal axon guidance. RESULTS. Despite no gross abnormality in RGC distribution, the mutant RGC axons exhibited severe intraretinal guidance errors, including optic nerve hypoplasia, ectopic axon penetration through the full thickness of the neural retina and into the subretinal space, and disturbance of the centrifugal projection of RGC axons toward the optic nerve head. These abnormal phenotypes shared similarities with the RGC axon misguidance caused by mutations of genes encoding Netrin-1 and Slit-1/2. Explant assays revealed that the mutant RGCs exhibited disturbed Netrin-1-dependent axon outgrowth and Slit-2-dependent repulsion. CONCLUSIONS. The present study demonstrated that RGC axon projection toward the optic nerve head requires the expression of HS in the neural retina, suggesting that HS in the retina functions as an essential modulator of Netrin-1 and Slit-mediated intraretinal RGC axon guidance.

Purpose: Disruption of the axonal transport of neurotrophic factors plays a critical role in the apoptosis of retinal ganglion cells (RGCs) in glaucomatous optic neuropathy. Live-cell imaging in vitro was used in this study, to visualize and evaluate the axonal transport of brain-derived neurotrophic factor (BDNF) in both living and axon-damaged RGCs., Methods: Rat RGCs were purified by a two-step immunopanning method. Cultivated RGCs were transfected with a plasmid encoding BDNF tagged with green fluorescent protein (GFP), and the dynamics of BDNF-GFP in the axons and dendrites were analyzed by time-lapse imaging. Changes in the axonal transport of BDNF-GFP were examined after treatment with 1 mM colchicine, and RGC death after treatment was evaluated with ethidium homodimer-1., Results: The expression of BDNF-GFP showed a vesicular pattern in the axons and dendrites of cultivated RGCs. Time-lapse imaging revealed that the mean velocity of BDNF-GFP in the axons (0.86 ± 0.37 μm/s; maximum = 2.03 μm/s) was significantly greater (P < 0.0001) than that in the dendrites (mean = 0.49 ± 0.19 μm/s). Colchicine significantly inhibited the axonal transport of BDNF-GFP at 2 and 3 hours after treatment (P = 0.003 and 0.0002, respectively) without affecting cell viability; however, RGC death was detected 24 hours after treatment., Conclusions: Live-cell imaging revealed the dynamics of the axonal transport of BDNF in living RGCs, which clearly differed from the movements in dendrites. Furthermore, it was possible to confirm the disruption of axonal transport in colchicine-treated RGCs before cell death.

Purpose: CD44 is a cell-surface adhesion molecule and receptor for hyaluronan (HA), one of the major extracellular matrix components. The purpose of the present study was to clarify a role of HA and CD44 in the development of choroidal neovascularization (CNV)., Methods: Laser photocoagulation was used to induce CNV in C57BL/6 mice or CD44-deficient mice. The mRNA expression of CD44 and HA synthase (HAS)-2 in the retinal pigment epithelium (RPE)-choroid complex was evaluated by DNA microarray and real-time RT-PCR analyses 3 days after laser treatment. HA synthesis and CD44 expression were examined by immunohistochemistry 1 week after photocoagulation. Mice with laser-induced CNV were systemically administered the HA synthesis inhibitor 4-methylumbelliferone (MU) or an anti-CD44-neutralizing antibody. The response of CNV was analyzed by volumetric measurements 1 week after photocoagulation. Macrophage infiltration into CNV lesions was evaluated by real-time RT-PCR for F4/80 3 days after laser-induced injury., Results: The induction of CNV led to a significant increase in expression of CD44 and HAS2 mRNA. HA and CD44 were immunopositive in the CNV lesions. Compared with vehicle treatment, the systemic application of MU significantly attenuated CNV volume in a dose-dependent fashion, together with macrophage infiltration into the lesions. Consistently, antibody-based blockade of CD44 resulted in a significant reduction of CNV volume, compared with the isotype control. In contrast, genetic ablation of CD44 significantly augmented CNV formation together with HA accumulation and macrophage infiltration, compared with wild-type mice., Conclusions: These results indicate a significant role of HA and its receptor CD44 in the development of CNV.

Purpose: To elucidate the role of the scavenger receptor, lectin-like oxidized low-density lipoprotein receptor type 1 (LOX-1), in the formation of choroidal neovascularization (CNV)., Methods: CNV was induced by laser photocoagulation of the ocular fundus in mice. The expression of LOX-1 mRNA and protein after laser injury was determined by real-time RT-PCR and Western blot analysis. Gelatin zymography was used to measure the activity of matrix metalloproteinase (MMP)-2 and pro-MMP-9, and ELISA was used to determine monocyte chemoattractant protein (MCP)-1 and vascular endothelial growth factor (VEGF) levels. At 14 days after laser injury, the extent of CNV was evaluated by fluorescein angiography and lectin staining using confocal microscopy., Results: In wild-type mice, the relative expression level of LOX-1 mRNA compared with the control increased significantly 6 hours after laser injury and peaked 12 hours after laser injury (P = 0.011 and P = 0.0006, respectively), and the expression of LOX-1 protein was also detected 1 and 3 days after laser injury. Increases in MMP-2, pro-MMP2, and pro-MMP-9 after laser injury were reduced in LOX-1-deficient mice compared with wild-type mice. At 3 days after laser injury, increases in MCP-1 and VEGF significantly decreased in LOX-1-deficient mice compared with wild-type mice (P = 0.014 and P = 0.001, respectively). Morphometric analyses revealed that the induction of CNV formation was significantly inhibited in LOX-1-deficient mice., Conclusions: These results suggest that LOX-1 plays an important role in the formation of CNV. This scavenging system might thus be a novel therapeutic target for CNV.

Purpose: To examine the role of thioredoxin-1 (TRX-1), an endogenous protein with a variety of redox-related roles, in the formation of choroidal neovascularization (CNV)., Methods: CNV was induced by laser photocoagulation of the ocular fundus in wild-type and transgenic mice overexpressing human TRX-1 (TRX-1 Tg). Mice were injected intraperitoneally with TRX-1, mutant TRX, or vehicle. The incidence of CNV was evaluated by lectin staining. Leukocyte recruitment and C3b deposition after laser injury were determined by immunohistochemistry and Western blotting. Moreover, TRX-1-associated proteins from human plasma were isolated by two-dimensional gel electrophoresis with the use of a column coupled with a mutant TRX-1 and were identified by mass spectrometry and proteomics analysis. Complement activation was determined by a fluid-phase, Results: The incidence of laser-induced CNV was reduced in TRX-1 Tg mice (56.1%) and in C57B/6 mice treated with TRX-1 (46.7%) but not in mutant TRX-1 (79.2%) compared with wild-type mice (85.7%). Furthermore, leukocyte recruitment was prevented in TRX-1-treated mice; C3b deposition was decreased in these and TRX-1 Tg mice. In human plasma, five proteins associated with TRX-1 were identified as apolipoprotein A-I, the CD5 antigen-like member of the scavenger receptor, cysteine-rich superfamily fibrinogen, albumin, and complement factor H (CFH). TRX-1 inhibited the alternative pathway C3 convertase, and its effect was additive with CFH., Conclusions: These findings show that TRX-1 interacts with CFH, regulates complement activity, and inhibits CNV, suggesting novel preventive and interventional therapeutic strategies for AMD.

Purpose: Toll-like receptor 4 (TLR4) is a transmembrane receptor that mediates immune responses to exogenous and endogenous ligands and interacts with heat shock proteins, which are reportedly involved in normal tension glaucoma (NTG). This study was undertaken to investigate whether TLR4 polymorphisms are associated with NTG., Methods: Two hundred fifty Japanese patients with NTG and 318 Japanese healthy control subjects were recruited. Eight single-nucleotide polymorphisms (SNPs) in the TLR4 gene were genotyped, and allelic and phenotypic diversity was assessed between cases and control subjects., Results: Strong linkage disequilibrium was observed among all SNPs (D' >or= 0.85), which were located in one haplotype block. With respect to allelic diversity, the minor allele of three SNPs (rs10759930, rs1927914, and rs7037117) carried a significantly increased risk of NTG. With regard to genotypic diversity, individuals with the minor allele of six SNPs (rs10759930, rs1927914, rs1927911, rs12377632, rs2149356, and rs7037117) had a 1.47- to 1.65-fold increased risk of NTG. rs7037117, located in the 3'-untranslated region of TLR4, was most strongly associated with NTG, and when incorporated into a haplotype with rs10759930, the strongest association was detected (P = 0.0038, P(c) = 0.0095)., Conclusions: Multiple SNPs in the TLR4 gene are associated with the risk of NTG. This finding suggests that the ligands and/or cytokines involved in the TLR4 signaling network may be risk factors for the development of NTG.

Purpose: To investigate the role of Y-27632, a specific inhibitor of Rho-associated protein kinase (ROCK) in regulating human Tenon fibroblast (HTF) activities including proliferation, adhesion, contraction, migratory response, and myofibroblast transdifferentiation. Effects of Y-27632 on prevention of postoperative scar formation were also examined in a rabbit model of glaucoma filtration surgery., Methods: After treatment of HTFs with Y-27632, cell toxicity, proliferation, migration, adhesion, and contraction were studied. The cytoskeleton and alpha-smooth muscle actin (alpha-SMA) expression were examined via immunohistochemistry. In vivo studies in Japanese white rabbits consisted of a full-thickness sclerostomy followed in the 7-day postoperative period by topical application of Y-27632. Intraocular pressure, morphologic changes in bleb features, and histology of surgical sites were evaluated., Results: Y-27632 had no direct toxicity or significant effects on cell proliferation of HTF. The cell adhesion assay showed that Y-27632 promoted adhesiveness to both fibronectin and collagen type I. Use of Y-27632 significantly inhibited collagen gel contraction and alpha-SMA expression in HTFs. Y-27632 also increased HTF motility. In vivo, Y-27632 inhibited wound healing and fibroproliferation after filtration surgery and significantly improved surgical outcome compared with the vehicle. Histologic examination revealed that blebs in the Y-27632-treated group differed from those in the vehicle-treated group in that they lacked significant collagen deposition in the sclerostomy area., Conclusions: Y-27632 had profound effects on activities of HTFs and was effective in preventing fibroproliferation and scar formation in a rabbit model of glaucoma surgery. A ROCK inhibitor may be an effective anti-scarring agent after glaucoma filtering surgery.

Purpose: To elucidate the intraocular pressure (IOP)-lowering effects and associated characteristics of Y-39983, a selective Rho-associated coiled coil-forming protein kinase (ROCK) inhibitor derived from Y-27632, in animal eyes., Methods: Y-39983 was compared with Y-27632 for selectivity of ROCK inhibition by biochemical assay. The IOP was monitored by pneumatonometer in albino rabbits and cynomolgus monkeys that were given topically administered Y-39983. The total outflow facility and uveoscleral outflow were measured by two-level constant-pressure perfusion and perfusion technique using fluorescein isothiocyanate-dextran, respectively, at 2 hours after topical administration of Y-39983 in albino rabbits. The ocular toxicologic effects of topical administration of Y-39983 were observed in albino rabbits and cynomolgus monkeys., Results: A biochemical assay showed that Y-39983 inhibited ROCK more potently than Y-27632. In rabbits, topical administration of Y-39983 significantly increased conventional outflow by 65.5%, followed by significant, dose-dependent reduction in IOP. Maximum IOP reduction was 13.2 +/- 0.6 mm Hg (mean +/- SE) at 0.1% Y-39983 in rabbits. In monkeys, at 3 hours after topical administration of 0.05% Y-39983, maximum reduction of IOP was 2.5 +/- 0.8 mm Hg. No serious side effects were observed in ocular tissues except sporadic punctate subconjunctival hemorrhage during long-term topical administration of Y-39983 four times a day (at 2-hour intervals) in rabbits or monkeys. However, punctate subconjunctival hemorrhage was not observed with administration twice daily (at a 6-hour interval) or three times a day (at 5-hour intervals)., Conclusions: Y-39983 causes increased outflow facility followed by IOP reduction. Y-39983 ophthalmic solution may be a candidate drug for lowering of IOP, since it increases conventional outflow and produces relatively few side effects.

Purpose: To investigate the effects of intravitreal injection of nattokinase (subtilisin NAT), a serine protease that is produced by Bacillus subtilis (natto), for induction of posterior vitreous detachment (PVD)., Methods: Different doses of nattokinase (1, 0.1, or 0.01 fibrin-degradation units [FU]) or physiologic saline as a control were injected into the vitreous cavity of rabbit eyes. Scanning electron microscopy was used to observe the retinal surfaces of four rabbit eyes per concentration. Histologic alterations were assessed by light microscopy, using four eyes from each group. Electroretinography (ERG) was performed to observe retinal function, ranging from 1 hour to 1 week after the nattokinase (1 or 0.1 FU) or saline solution administration, using four eyes from each group at each time point. Also, findings in all rabbits were monitored by slit lamp examination and by indirect ophthalmoscopy with a 20-D lens., Results: Scanning electron microscopy showed smooth retinal surfaces, indicating the occurrence of PVD at 30 minutes after intervention in all the experimental eyes injected with 0.1 or 1.0 FU nattokinase, but none of the control eyes. Light microscopy and ERG analysis showed no critical change even after the use of 0.1 FU nattokinase, an amount sufficient to induce PVD. However, toxicity in the forms of preretinal hemorrhage and ERG changes was noted with the higher dose (1 FU) of nattokinase., Conclusions: The results suggested that nattokinase is a useful enzyme for pharmacologic vitreolysis because of its efficacy in inducing PVD.

Purpose: To investigate sequence variations in the optineurin (OPTN) gene and their association with TNF-alpha polymorphisms in Japanese patients with glaucoma., Methods: The OPTN gene was analyzed in blood samples from 629 Japanese subjects. There were 194 patients with primary open-angle glaucoma (POAG), 217 with normal-tension glaucoma (NTG), and 218 with no eye disease (control subjects). The gene was screened for mutations by denaturing high-performance liquid chromatography. Genotyping of three polymorphisms of -308G-->A, -857C-->T, and -863C-->A in the TNF-alpha promoter region was performed. The associations between the genotypes and age, intraocular pressure (IOP), and visual field defects at the time of diagnosis were examined., Results: A possible glaucoma-causing mutation, His26Asp, was identified in 1 of the 411 Japanese patients with glaucoma. A c.412G-->A (Thr34Thr) polymorphism in the OPTN gene was significantly associated with POAG (genotype frequency, P = 0.011; allele frequency, P = 0.003). The frequency of TNF-alpha/-857T and optineurin/412A carriers was significantly higher (P = 0.006) in patients with POAG than in control subjects. Among the patients with POAG who were carriers of TNF-alpha/-857T, the optineurin/412A carriers had significantly worse (P = 0.020) visual field scores than the non-optineurin/412A ones. The frequency of TNF-alpha/-863A and optineurin/603A (or Lys98) carriers was significantly higher in patients with POAG (P = 0.008) or NTG (P = 0.027) than in control subjects. Among the patients with POAG who were carriers of TNF-alpha/-863A, the ones with optineurin/603A (or Lys98) had significantly worse (P = 0.026) visual field scores than did those with non-optineurin/603A (or Lys98)., Conclusions: These findings demonstrated that the OPTN gene is associated with POAG rather than NTG in the Japanese. Statistical analysis showed a possible interaction between polymorphisms in the OPTN and the TNF-alpha genes that would increase the risk for glaucoma.

Purpose: Recent evidence suggests that platelets play a major role in ischemia-reperfusion injury, not only through thrombus formation but also through participation in inflammatory reactions with leukocytes. This study was designed to investigate the contribution of platelets in leukocyte recruitment to inflamed regions in vivo., Methods: Thrombocytopenia was produced in male Long-Evans rats by intravenous injection of anti-platelet serum at 4 hours before ischemia-reperfusion. Leukocyte behavior in retinal microcirculation was evaluated with acridine orange digital fluorography. Expression of P-selectin in the postischemia retina was investigated by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. After 14 days of reperfusion, ischemia-induced retinal damage was evaluated histologically., Results: Leukocyte rolling along major retinal veins of thrombocytopenic rats was dramatically suppressed, and subsequent leukocyte accumulation in the postischemia retina was also significantly reduced (72.3%; P < 0.001) at 24 hours after reperfusion. Although RT-PCR revealed no significant reduction of P-selectin mRNA in platelet-depleted rat retina after transient ischemia, immunohistologic examination showed suppression of P-selectin expression on the vascular wall. Another immunologic examination using anti-platelet antibody detected adherent platelets, which can also express P-selectin on their surfaces, on postischemic vascular endothelium in vehicle-treated retina. Moreover, blockage of platelet glycoprotein IIb/IIIa resulted in substantial inhibition of leukocyte rolling. In addition, histologic examination showed the participation of platelets in retinal ischemia-reperfusion injury., Conclusions: This study demonstrated that the expression of P-selectin on platelets may contribute to the recruitment of leukocytes to tissues after ischemia.

Purpose: Infiltrating leukocytes have long been widely thought to be key mediators of ischemia-reperfusion injury. Recently, however, evidence suggests that platelets accumulating in postischemic tissues also contribute to ischemia-reperfusion injury because of their inflammatory properties and promotion of formation of thrombi. This study was designed to evaluate quantitatively the inhibitory effects of antithrombin (AT)-III on the interactions between blood cells and retinal endothelial cells in vivo after transient retinal ischemia., Methods: Transient retinal ischemia was induced for 60 minutes in male Long-Evans rats by ligation of the optic nerve. AT III (250 U/kg) was administered intravenously just after induction of ischemia. Leukocyte and platelet behavior in the retina was evaluated in vivo with a scanning laser ophthalmoscope. Expression of P-selectin and intracellular adhesion molecule (ICAM)-1 in the postischemic retina was investigated by reverse transcription-polymerase chain reaction and immunohistochemistry. After 14 days of reperfusion, ischemia-induced retinal damage was evaluated histologically., Results: Administration of AT III significantly inhibited leukocyte rolling along the major retinal veins and subsequent accumulation of leukocytes in the postischemic retina. Furthermore, the maximum number of rolling and adherent platelets was reduced by 76% (P < 0.01) and 48% (P < 0.01), respectively, at 12 hours after reperfusion. Immunohistochemical studies also revealed the suppressive effect of AT III on expression of P-selectin and ICAM-1. Finally, histologic examination demonstrated the protective effects of AT III against retinal damage after transient retinal ischemia., Conclusions: This study demonstrates the inhibitory effects of AT III on leukocyte and platelet recruitment to the postischemic retina, which may account for the neuroprotective properties of this alpha-2 globulin against retinal ischemia-reperfusion injury.

Purpose: N-syndecan is a transmembrane heparan sulfate proteoglycan, that is highly expressed in neural tissues. In the current study, changes in N-syndecan expression during retinal development were examined., Methods: Localization of N-syndecan in developing rat retina was examined by immunohistochemistry and in situ hybridization. The amount of the core protein was evaluated by immunoblot analysis, using retinal homogenates at various developmental stages. In addition, mRNA expression was semiquantified by reverse transcription-polymerase chain reaction (RT-PCR). To understand better the localization of N-syndecan in retinal neuronal cells, we performed immunocytochemistry using retinal ganglion cells in culture., Results: N-syndecan is highly expressed in nerve fiber-rich layers of the retina at early postnatal stages (between postnatal day [P]0 and P14). In contrast, immunoreactivity was faint during embryonic stages and late postnatal stages. In addition, in retinal flatmounted sections, N-syndecan immunoreactivity was observed on the axons of retinal ganglion cells. Intense signals were observed in the ganglion cell layer during in situ hybridization. Immunoblot analyses demonstrated that the amount of N-syndecan core protein reached a peak at approximately P14. The RT-PCR analyses using N-syndecan primers showed that an intense amplified band was observed in the cDNA derived from P14 retinas, whereas only faint bands were detected in the embryonic day (E)16 and P42 retinas. In retinal ganglion cells in culture, N-syndecan was located on the long, extended neurites., Conclusions: The data show that N-syndecan is transiently expressed, primarily in retinal neural fibers, during retinal development, indicating that it may be involved in formation of the retinal neural network.

Purpose: To investigate CYP1B1 gene mutations in Japanese patients with primary congenital glaucoma (PCG)., Methods: Sixty-five unrelated Japanese patients with PCG were screened by PCR-single-strand conformational polymorphism (SSCP) analysis followed by direct sequencing. No patients were offspring of consanguineous marriages, a common occurrence among patients in previous reports. PCG haplotypes were constructed with intragenic polymorphisms in affected individuals. Three-dimensional atomic structures of human CYP1B1 and four mutant CYP1B1 sequences representing missense mutations were assembled using homology modeling and were regularized by an energy-minimization procedure., Results: Eleven novel mutations, including seven definite and four probable mutations, were detected in 13 (20%) of the 65 unrelated patients. Of the seven definite mutations, three were predicted to truncate the CYP1B1 open reading frame. The other four were missense mutations (Asp192Val, Ala330Phe, Val364Met, and Arg444Gln), all located in conserved core structures determining proper folding and heme-binding ability of cytochrome P450 molecules. Molecular modeling demonstrated that two of four mutations in positions 330 and 364 were structurally neutral, but Arg444Gln caused significant structural change. Of the four probable mutations, three were missense (Val198Ile, Val320Leu, and Glu499Gly); the other was a base substitution in the noncoding region of exon 1., Conclusions: The 11 varied CYP1B1 mutations found in 13 unrelated Japanese patients with sporadic occurrence of PCG represent an allelic heterogeneity and may be unique to a specific population.

Purpose: Accumulating evidence suggests that platelets play an important role in ischemia-reperfusion injury. To fulfill that role, platelets flowing in the bloodstream would have to interact with retinal endothelial cells and to accumulate in the postischemic retina. This study was designed to investigate quantitatively platelet-endothelial interactions in postischemic retina after transient retinal ischemia., Methods: Transient retinal ischemia was induced in Long-Evans rats for 60 minutes by temporal ligation of the optic nerve. Isolated platelet samples labeled with carboxyfluorescein diacetate succinimidyl ester were administered intravenously to recipient rats after various reperfusion periods. Platelet-endothelial interactions in postischemic retina were evaluated in vivo with a scanning laser ophthalmoscope. Anti-P-selectin monoclonal antibody (mAb) was administered 5 minutes before the injection of labeled platelets. P-selectin gene expression in the postischemic retina was studied by semiquantitative polymerase chain reaction., Results: Under basal conditions, infused platelets showed minimal interactions with retinal endothelial cells. In contrast, postischemic retinas showed active platelet-endothelial interactions. Many platelets were observed rolling along and adhering to the major retinal veins. The number of rolling and adhering platelets reached a peak (555 +/- 65/mm per min and 25.8 +/- 3.2/mm(2)) 12 hours after reperfusion. However, the interactions between platelets and postischemic retinal endothelial cells were substantially inhibited by neutralizing P-selectin expressed on endothelial cells. In addition, P-selectin gene expression in postischemic retina corresponded with the time course of platelet-endothelial interactions during the reperfusion period., Conclusions: This study demonstrated that platelets actively interacted with retinal endothelial cells in the postischemic retina through P-selectin expressed on the retinal endothelial cells.

Purpose: Neurocan and phosphacan are nervous tissue-specific chondroitin sulfate proteoglycans (CSPGs) that are highly expressed in postnatal rat retina. To elucidate potential roles of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells (RGCs), in vitro experiments were conducted with purified RGCs., Methods: Neurocan and phosphacan were purified from postnatal rat brain by DEAE-column chromatography and subsequent gel chromatography. RGCs were obtained from postnatal rat retinas by a two-step immunopanning procedure using an anti-Thy 1,1 antibody and an anti-macrophage antibody. Neurite outgrowth from RGCs was examined on poly-L-lysine (PLL)-conditioned plates, and PLL-conditioned plates treated with neurocan or phosphacan., Results: Compared with PLL-conditioned plates, neurocan and phosphacan inhibited neurite outgrowth from RGCs at 48 and 72 hours after seeding. When chondroitin sulfate side chains linked to the core proteins were digested by chondroitinase ABC, the inhibitory effect remained, indicating that the core proteins are related to the effect. Furthermore, the digestion of chondroitin sulfate side chains linked to phosphacan core protein significantly promoted the inhibitory effect of phosphacan on neurite outgrowth from RGCs., Conclusions: Neurocan and phosphacan, which are highly expressed in postnatal rat retina, inhibit neurite outgrowth from postnatal rat RGCs, indicating that these proteoglycans may be inhibitory factors against neurite outgrowth from RGCs during retinal development.

Purpose: This study was designed to investigate the suppressive effects of antithrombin (AT)III on inflammatory reactions during endotoxin-induced uveitis (EIU) in rats by studying leukocyte-endothelium interactions., Methods: EIU was induced in Lewis rats by footpad injection of lipopolysaccharide (LPS). ATIII was administered immediately after or at 6 hours after LPS injection. Its suppressive effects on inflammatory leukocyte behavior were evaluated in vivo with acridine orange digital fluorography. Clinical signs of inflammation were also examined, and aqueous humor (AH) was collected to evaluate leukocyte infiltration and protein leakage. In a separate experiment, P-selectin mRNA expression was studied in the iris-ciliary body (ICB) and the retina., Results: After treatment with ATIII, leukocyte rolling was substantially inhibited along the retinal veins, suppressing subsequent leukocyte infiltration into the vitreous cavity. Similarly, leukocyte infiltration and protein leakage into the AH were significantly reduced with ATIII treatment. The clinical grade of EIU was substantially lower in ATIII-treated rats. In addition, delayed administration of ATIII after EIU induction significantly attenuated these inflammatory reactions. The levels of P-selectin mRNA expression in both ICB and retina, which were upregulated after LPS injection, were substantially lower in the ATIII-treated rats., Conclusions: ATIII treatment significantly inhibited inflammatory reactions induced with LPS. Its suppressive effects on P-selectin expression could contribute to the attenuation of leukocyte infiltration, possibly by inhibiting leukocyte rolling. The current findings suggest that ATIII may have a role in the management of patients with uveitis.

Purpose: To elucidate the roles of Rho-associated protein kinase (ROCK) in regulating intraocular pressure (IOP) and outflow facility in the rabbit eye., Methods: A specific ROCK inhibitor Y-27632 was used. The IOP, the outflow facility, and the pupil diameter were determined before and after the topical, intracameral, or intravitreal administration of Y-27632 in rabbits. Western blot analysis was used to identify specific ROCK isoform in human trabecular meshwork (TM) cells and bovine ciliary muscle (CM) tissues. The cell morphology and distribution of actin filaments and vinculin in TM cells were studied by cell biology techniques. Carbachol (Cch)-induced contraction of isolated bovine CM strips after administration of Y-27632 was measured in a perfusion chamber., Results: In rabbit eyes, administration of Y-27632 resulted in a significant decrease in IOP in a dose-dependent manner. An increase of the outflow facility and pupil size dilation was also observed in Y-27632-treated eyes. Western blot analysis revealed the presence of p160ROCK in human TM cells and bovine CM tissues. In cultured human TM cells, exposure to Y-27632 caused retraction and rounding of cell bodies as well as disruption of actin bundles and impairment of focal adhesion formation. Y-27632 in addition inhibited Cch-induced contraction of isolated bovine CM strips., Conclusions: Administration of Y-27632 caused a reduction in IOP and an increase in the outflow facility. The in vitro experiments suggest that the IOP-lowering effects of Y-27632 may be related to the altered cellular behavior of TM cells and relaxation of CM contraction. These studies suggest that ROCK inhibitors may have great potential to be developed for treatment of glaucoma and other ocular diseases.

Purpose: In a previous study it has been shown that adult rat hippocampus-derived neural stem cells can be successfully transplanted into neonatal retinas, where they differentiate into neurons and glia, but they cannot be transplanted into adult retinas. In the current study, the effect of mechanical injury to the adult retina on the survival and differentiation of the grafted hippocampal stem cells was determined., Methods: Mechanical injury was induced in the adult rat retina by a hooked needle. A cell suspension (containing 90,000 neural stem cells) was slowly injected into the vitreous space. The specimens were processed for immunohistochemical studies at 1, 2, and 4 weeks after the transplantation., Results: In the best case, incorporation of grafted stem cells was seen in 50% of the injured retinas. Most of these cells located from the ganglion cell layer through the inner nuclear layer close to the injury site. Immunohistochemically, at 1 week, more than half of the grafted cells expressed nestin. At 4 weeks, some grafted cells showed immunoreactivity for microtubule-associated protein (MAP) 2ab, MAP5, and glial fibrillary acidic protein (GFAP), suggesting progress in differentiation into cells of neuronal and astroglial lineages. However, they showed no immunoreactivity for HPC-1, calbindin, and rhodopsin, which suggests that they did not differentiate into mature retinal neurons. Immunoelectron microscopy revealed the formation of synapse-like structures between graft and host cells., Conclusions: By the manipulation of mechanical injury, the incorporation and subsequent differentiation of the grafted stem cells into neuronal and glial lineage, including the formation of synapse-like structures, can be achieved, even in the adult rat retina.

Purpose: Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan present exclusively in central nervous system tissues. In the current study the expression pattern and characterization of NGC during the development of the retina were investigated., Methods: Expressional changes of NGC mRNAs during rat retinal development were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The localization and characterization of NGC core proteins were investigated by immunoblot analysis and immunohistochemistry using an anti-NGC antibody., Results: Immunohistochemical analysis revealed that NGC was highly expressed in the nerve fiber layer (NFL) and inner plexiform layer (IPL) in rat postnatal developing retina. At embryonal stages, NGC immunoreactivities were faint. In contrast, at postnatal developmental stages (approximately postnatal day [P]7), intense immunoreactivity was observed in the NFL and IPL, where active dendrite branching was observed, and conventional synapses began to be formed. As retinal layer differentiation proceeded (from P14 to P42), immunoreactivities in the inner retinal layers gradually became fainter. Immunoblot and semiquantitative RT-PCR analyses showed that the peak level of NGC expression occurred on approximately P7 and P14. Glycosylation of the NGC core protein changed as the retinal layers matured. In immunoelectron microscopic analysis, NGC immunoreactivity was located on the axonal membranes of neuronal cells in the postnatal retina, whereas immunoreactivity was reduced on membranes at the adult stage. In retinal ganglion cells in vitro, NGC was highly localized in their spiny budding neurites., Conclusions: The results show spatiotemporal expression patterns of NGC, and suggest that it plays a role in the formation of neural networks in retinal development.

Purpose: Neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan synthesized primarily by neurons, is expressed abundantly in developing rat retina, whereas it is rarely expressed in adult rat retinas. This study investigated the reexpression of neurocan in a pathologic condition of adult rat retina., Methods: Transient retinal ischemia was produced by occlusion of the retinal artery for 60 minutes. After transient retinal ischemia, neurocan expression was investigated by reverse transcription-initiated polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot analysis., Results: Semiquantitative analysis using RT-PCR revealed that mRNA expression for neurocan increased at 24 hours after reperfusion. Furthermore, on immunoblot analysis using an anti-neurocan antibody, MAb 1G2, the intensity of the 220-kDa band as well as the 150-kDa band increased markedly at 24 and 72 hours after reperfusion. The 220-kDa band was predominant at 24 hours after reperfusion, whereas the intensity of the 150-kDa band became almost the same as that of the 220-kDa band at 72 hours after reperfusion. Immunohistochemical analysis revealed that upregulated neurocan immunoreactivity was associated with glial Müller cells., Conclusions: Thus, upregulated expression of neurocan in transient retinal ischemia was demonstrated. Furthermore, the immunohistochemical analysis revealed that the upregulated expression of neurocan is derived from Müller cells, although it has been thought that neurocan is synthesized by neurons so far. The neurocan expression by Müller cells suggests that this proteoglycan plays a role in the damage and repair processes in diseased retina.

Purpose: To investigate expression of 6B4 proteoglycan/phosphacan, the major constituent of chondroitin sulfate proteoglycan and a possible modulator of neural network formation in the developing central nervous system, in developing rat retina., Methods: Changes in expression and localization of 6B4 proteoglycan in developing rat retina were investigated by reverse transcription-initiated polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot analysis., Results: Semiquantitative RT-PCR revealed that mRNA expression of 6B4 proteoglycan in retinas peaked at postnatal day 14 (P14) and then decreased at P42. Immunohistochemical analyses using MAb 6B4, a monoclonal antibody against 6B4 proteoglycan, revealed faint immunoreactivity in the inner aspects of the retina at embryonal day 16 (E16). At birth, weak immunoreactivity was present in the nerve fiber layer (NFL) and inner plexiform layer (IPL). At P7 and P14, the NFL, IPL, and outer plexiform layer (OPL) stained intensely, but the ganglion cell layer (GCL) remained unstained. Between P21 and P42, immunoreactivity in the NFL and IPL weakened slightly. Immunoblot analyses showed a MAb 6B4 immunopositive band in the retinal soluble fraction treated with chondroitinase ABC. The amount of the immunopositive band increased rapidly as retinal development proceeded. Surprisingly, a significant amount of the immunopositive band was present in the retina even before digestion with chondroitinase ABC, indicating that at least part of 6B4 proteoglycan in rat retina exists in a non-proteoglycan form., Conclusions: The existence of 6B4 proteoglycan/phosphacan was thus demonstrated in rat retina, although some biochemical parameters were different from those of the 6B4 proteoglycan seen in brain.

Purpose: To elucidate the role of retinal Muller cells in N-methyl-D-aspartate (NMDA)- or kainic acid (KA)induced retinal damage., Methods: In experimental eyes, NMDA or KA was injected into the vitreous of rat eyes. Immunohistochemistry and western blot analysis were conducted to elucidate expression and localization of glial fibrillary acidic protein (GFAP) and ciliary neurotrophic factor (CNTF). In addition, the neuroprotective effects of CNTF were calculated by counting cells in the ganglion cell layer (GCL) and by measuring the thickness of the various retinal layers., Results: Morphometric analysis of retinal damage in NMDA- and KA-injected eyes showed significant cell loss in the GCL and thinning of the inner plexiform layer (IPL) of the retina, but not of other retinal layers. Immunohistochemistry demonstrated disappearance and/or decrease in immunoreactivities of calbindin- and calretinin- positive cells and their neurites and upregulated expression of both GFAP and CNTF in experimental eyes. Western blot analysis showed an increase in protein expression for CNTF in retinas of experimental eyes. Confocal images and sequential localization demonstrated colocalization of CNTF and GFAP in the inner retinal layer and possibly in Muller cells. In addition, pretreatment with CNTF (1 microg) before the intravitreal injection of NMDA (or KA) demonstrated that CNTF has neuroprotective effects against NMDA- or KA-induced neuronal death in the retina., Conclusions: These studies revealed the upregulated expression of CNTF and GFAP in Muller cells in response to NMDA- and KA-induced neuronal death, suggesting that production of CNTF in Muller cells may be a part of the endogenous neuroprotective system in the retina.

Purpose: To determine the expression pattern of multiple subtypes of cadherin adhesion receptor in postnatal mouse neural retina., Methods: The expression of N-cadherin, R-cadherin, cadherin-6, cadherin-8, and cadherin-11 in retinas at postnatal days 0 to 42 was analyzed by in situ hybridization of mRNA as well as by immunohistochemistry., Results: Each cadherin was expressed by different cell populations of the retina, and the following expression patterns were established by postnatal day 14: in the ganglion cell layer, all these molecules were expressed, but each occurred only in a subset of the cells. Likewise, in the inner nuclear layer, R-cadherin and cadherin-6 and -8 were expressed by a restricted population of amacrine cells, and cadherin-8 also by a subpopulation of bipolar cells. All horizontal cells expressed R-cadherin, and Muller cells expressed N-cadherin and cadherin-11. Proteins of R-cadherin and cadherin-6 were concentrated in neuropil layers., Conclusions: The pattern of differential expression of the five cadherins supports the idea that these molecules may play a role in selective cell interactions within the heterogeneous cell pool of the neural retina.

Purpose: To identify the expression of neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan, in retina and to elucidate its changes during development., Methods: Expressional changes of neurocan mRNAs in developing rat retinas were investigated by a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The localization and characterization of neurocan core proteins were also investigated with the use of Western blot analysis and immunohistochemistry., Results: Gene expression of neurocan was identified in retinas by RT-PCR. Semiquantitative analysis using Southern blot analysis revealed that mRNA expression for neurocan increased at increasing postnatal stages and that it reached its peak around postnatal day 7 (P7). Immunohistochemical studies demonstrated that in differentiating rat retinal (neuroblast) cells weak neurocan immunoreactivities were observed throughout the retina on embryonal days 14 (E14) and E16. During the early postnatal period, the immunoreactivities became most conspicuous in the inner and outer plexiform layers on P7 through P14. In adult retinas, only faint immunostaining was detected. Immunoblot analysis showed two positive bands of 220- and 150-kDa core glycoproteins after treatment with chondroitinase ABC. Further immunoblot analysis revealed that the expression of these two immunolabeled variants was regulated differently during retinal development., Conclusions: The temporal and spatial regulation of expression of neurocan and its proteolytic variant during retinal development suggest that it may play a role in differentiation and neural network formation.

Purpose: To investigate the neuroprotective effects of brain-derived neurotrophic factor (BDNF) against potassium cyanide (KCN)-induced retinal damage., Methods: Rats were injected intravitreally with iodinated BDNF. Two days later, eyeballs were dissected into various parts, and the level of radioactivity in each part was measured. Retinal damage was induced by incubating rat eyeballs with 5 mM KCN. BDNF was injected intravitreally 2 days before KCN treatment, and subsequent morphometric analysis was carried out to evaluate the retinal cell damage. To elucidate the mechanisms of BDNF's neuroprotective effects, the intravitreal concentrations of amino acids and the expression of calretinin were investigated., Results: Intravitreally injected BDNF was distributed evenly throughout the eyes, and the incorporation of iodinated BDNF into the retina was three times higher than in other ocular tissues. Immunohistochemical analysis demonstrated that exogenous BDNF diffused throughout the retina and was especially concentrated in the inner (INL) and outer nuclear layer. Morphometric analysis showed that the number of INL cells of the posterior area, 880 microm from the optic nerve head, was 190 +/- 4 with KCN treatment and 284 +/- 9 in control animals. Cell death appeared to be necrotic. When eyes injected with either phosphate-buffered saline (PBS) or BDNF were subjected to treatment with KCN, the number of INL cells was 186 +/- 5 in the PBS-treated controls and 253 +/- 8 in eyes treated with BDNF. Also, BDNF increased the number of calretinin-positive cells in the INL and reduced the KCN-induced elevation of intravitreal glutamate levels., Conclusions: BDNF injected intravitreally reaches the retina and attenuates the INL cell death caused by KCN-induced metabolic insult. The neuroprotective effects of BDNF are partly ascribed to the upregulation of a calcium-binding protein and the attenuation of glutamate release into the vitreous body.

Purpose: To identify the expression of chondroitin/dermatan sulfate proteoglycan decorin in retina and to elucidate its changes during development and ischemia-reperfusion., Methods: Expression of decorin in rat retina was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Distributional changes during development and transient ischemia in model eyes also were investigated by immunohistochemical experiments., Results: Gene expression of decorin core protein was identified in rat retina by RT-PCR. Decorin immunoreactivities were shown throughout the retina, especially in the ganglion cell layer. In developing rat retinas, at embryonic stages (embryonic day 16), decorin was distributed uniformly throughout the retina. As retina matured, the intensity of decorin immunostaining in retinal inner layers and retinal pigment epithelium increased. Furthermore, in experimental transient retinal ischemia, after transient downregulation of the decorin core protein gene between 24 and 48 hours after the ischemia, recovered (or increased) expression was shown by semiquantitative RT-PCR experiments. Immunohistochemical studies revealed strong decorin immunoreactivities in the damaged inner layers 1 week later., Conclusions: The expression of decorin was identified in adult and developing rat retina. The distributional changes of decorin during the retinal development suggest that this proteoglycan may play a role in the differentiation of retinal ganglion cells. Moreover, in rat ischemia-reperfusion models, the alterations in gene expression and immunohistochemical localization showed the contribution of this proteoglycan to the damage and repair processes in diseased retina.

Purpose: To evaluate the feasibility of introducing exogenous genes and phosphorothioate oligonucleotides into the anterior chamber tissues of rats and monkeys using the authors' fusogenic liposomes., Methods: Hemagglutinating virus of Japan liposomes containing LacZ DNA-high-mobility group 1 complexes or fluorescein isothiocyanate (FITC)-labeled phosphorothioate oligonucleotides were prepared and injected into the anterior chambers of rats (3 microliters) and rhesus monkeys (30 microliters). The expression of LacZ DNA was visualized histochemically by beta-Galactosidase assay and was followed for as long as 60 days in rats and 30 days in monkeys. FITC-labeled phosphorothioate oligonucleotides were observed by fluorescence microscopy for as long as 14 days in rats and 7 days in monkeys., Results: Injection of LacZ DNA-high-mobility group 1 complexes encapsulated in hemagglutinating virus of Japan liposomes resulted in blue staining in the trabecular meshwork and iris-ciliary body of rats and selectively in the trabecular meshwork of monkeys at the concentrations used. This LacZ expression lasted for as long as 14 days after injection in both animals. Phosphorothioate oligonucleotides (3 microM) also were introduced into the rat trabecular meshwork and iris-ciliary body and into the primate trabecular meshwork when encapsulated in hemagglutinating virus of Japan liposomes, although the injection of naked FITC-labeled phosphorothioate oligonucleotides at the same concentration resulted in little fluorescence in any anterior chamber tissue., Conclusions: This study shows that the use of hemagglutinating virus of Japan liposomes can transfer LacZ DNA and phosphorothioate oligonucleotides to adult rat and primate trabecular meshwork. This system may enable progress in glaucoma research and in the development of nonviral somatic gene therapy of the trabecular meshwork to treat glaucoma.

Purpose: To determine whether a reporter gene can be introduced into rat and mouse retinas in vivo using the authors' novel liposome system., Methods: Cytomegalovirus-promoted LacZ genes and nonhistone nuclear protein, high mobility group 1 (HMG1), were coencapsulated in liposomes by agitation and sonication. The liposomes were then coated with the envelope of inactivated hemagglutinating virus of Japan (HVJ; Sendai virus) by fusion (HVJ liposome). The HVJ liposome solution was injected into the vitreous cavity of adult Sprague-Dawley rats (n = 30) or the subretinal space of adult CD-1 mice (n = 42). LacZ expression was assessed by beta-galactosidase assay., Results: LacZ expression was demonstrated in the photoreceptors as long as 30 days after intravitreal and subretinal injections. beta-Gal activity was observed also in neurons and glial cells in the ganglion cell layer of the intravitreally injected rats and, although at lower intensity, in the retinal pigment epithelium of both animals. No inflammation or toxic effects secondary to the HVJ liposomes were detected on histologic examination., Conclusions: In vivo transfer and expression of a reporter gene into adult mammalian retina can be achieved using HVJ liposomes. This method offers a promise as a nonviral system for in vivo gene transfer into adult mammalian neural retina.

Purpose: To localize and determine the levels of interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), and tumor necrosis factor (TNF) gene expression in the process of endotoxin-induced uveitis (EIU) in rats., Method: EIU was induced by lipopolysaccharide (LPS) injection in male Lewis rats weighing 150 to 200 g. The levels of IL-1 alpha, IL-1 beta, and TNF gene expression in the iris-ciliary body (ICB) were quantified by a semiquantitative polymerase chain reaction (PCR) method; in situ hybridization histochemistry was carried out to localize the gene transcripts., Results: Little expression of IL-1 alpha, IL-1 beta, and TNF genes was observed in normal ICB. IL-1 alpha gene expression began to increase (about 10-fold greater than that of the control) as early as 1 hour after the LPS treatment, reached a peak (about 100-fold) at 3 to 6 hours. A second peak (60-fold) was observed at 24 hours, and the expression returned to near basal levels (3-fold) at 48 hours. Expression of IL-1 beta and TNF genes showed a pattern similar to that of IL-1 alpha. Three hours after LPS treatment, IL-1 beta and TNF genes were found by in situ hybridization histochemistry to be expressed by "histiocyte-like" cells in the stroma of the ICB. None of these genes were detected in the control rats., Conclusions: Expression of IL-1 alpha, IL-1 beta, and TNF genes was dramatically up-regulated in the process of EIU. These genes were found to be expressed in "histiocyte-like" cells in the ICB, and may have an important role in EIU.

Purpose: It was determined in cultured human retinal pigment epithelial (HRPE) cells whether there is gene expression for insulin, insulin-like growth factors (IGFs), insulin and IGF receptors, and IGF-binding proteins (IGFBPs); if these peptides are secreted by the HRPE cells, and whether they have mitogenic effects on these cells; and if IGF-related peptides bind to HRPE cells., Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR), nucleotide sequencing, and Southern blot analyses were done to identify gene expression. The presence of IGFs in the tissue culture medium was detected by radioimmunoassay. For determination of mitogenic effects and receptor binding characterization, [3H]-thymidine incorporation and radioreceptor binding measurements were performed., Results: The genes for IGF-I and IGF-II, IGFBP-2, insulin receptor, and type I and II IGF receptors were detected. In the tissue culture supernatant, there was immunoreactivity for IGF-I and IGF-II. Insulin, IGF-I, and IGF-II stimulated DNA synthesis with EC50s of 3, 10, and 30 to 100 nM, respectively. Scatchard analyses of [125I]-IGF-I binding and [125I]-insulin binding to the cells showed that the cells have relatively abundant insulin and IGF-I binding sites., Conclusions: HRPE cells express genes for IGF-I and IGF-II, and conditioned medium from these cells is immunoreactive to their protein products. The cells express genes for insulin receptor, type I and II IGF receptors, and IGFBP-2. IGF-I, IGF-II, and insulin are all mitogenic, possibly as a result of their interactions with either the insulin or type I IGF receptor. The cells bind insulin and IGF-I with high affinity. These results suggest that IGF-I and IGF-II production by HRPE cells may be essential for autocrine/paracrine-mediated regulation of proliferation. The presence of insulin receptor suggests that insulin has a role in the regulation of HRPE function.

Purpose: To investigate gene and protein expression of glucose transporter isoforms in cultured human retinal pigment epithelial cells. To investigate growth factor-dependent stimulation of glucose transport and the effect of growth factors on the gene and protein expression in retinal pigment epithelial cells., Methods: Glucose transport activity was analyzed by [3H]2-deoxyglucose uptake studies. Gene and protein expression of glucose transporter isoforms were analyzed by polymerase chain reaction, Northern blot analysis, and Western blot analysis., Results: Polymerase chain reaction, nucleotide sequencing, and Southern blot analyses revealed that the retinal pigment epithelial cells express GLUT-1, -3, and -5 genes. Northern and Western blot analysis detected only GLUT-1 transcripts and protein. A 24-hour exposure to fetal bovine serum (15%), basic fibroblast growth factor (50 ng/ml), platelet-derived growth factor (10 ng/ml), epidermal growth factor (50 ng/ml), and insulin-like growth factor-1 (50 ng/ml) significantly stimulated [3H]2-deoxyglucose uptake in cultured human retinal pigment epithelial cells. Western blot analysis showed that serum and platelet-derived growth factor induced an increase of GLUT-1 protein in the membrane preparation in the cells. Serum, fibroblast growth factor, platelet-derived growth factor, and insulin-like growth factor-1 did not increase GLUT-1 gene expression to an appreciable level, as shown by Northern blot analysis., Conclusion: Cultured human retinal pigment epithelial cells dominantly express GLUT-1 gene and protein with minor expression of GLUT-3 and -5 genes. Fetal bovine serum, fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-1 significantly, although modestly, increase glucose transport activity of the cells.

Purpose: This study examined whether retinal pigment epithelial (RPE) cells have the capacity to express transforming growth factor-beta (TGF-beta). Also examined were TGF-beta isoform genes expressed in the RPE cells., Methods: Complementary DNA (cDNA) was generated from polyA+ RNA extracted from human RPE cells in culture, and polymerase chain reaction (PCR) using degenerate and specific primers of the known TGF-beta isoforms was carried out by using the cDNAs as templates. Sequencing and Southern blot analysis of the PCR products, and Northern blot analysis were performed to identify which isoforms are expressed in RPE cells., Results: PCR using degenerate primers showed that most of the amplified sequences were derived from TGF-beta 2, although TGF-beta 1 expression was shown by using specific primers. Northern blot analyses confirmed not only the expression of the TGF-beta gene, but also suggested that alternative splicing of TGF-beta mRNA in the RPE cells occurred., Conclusion: Cultured human RPE cells expressed TGF-beta 1 and beta 2 genes, whereas gene expression of TGF-beta 3 was not confirmed. Our data suggest that RPE cell are intraocular origin of TGF-beta production.

Various kinds of neuropeptides have been identified to be immunoreactive in the drainage angle of mammalian eyes. However, little is known about second messenger system involvement with these peptides. To determine whether some of these peptides are linked to a calcium signalling system in the trabecular meshwork (TM) cells, their effects on [Ca2+]i transients in fura-2 loaded cultured bovine TM cells were studied with a digital video-imaging system. The main findings of this study were: (1) The basal [Ca2+]i was 164.0 +/- 1.0 nM (mean +/- standard error of the mean, n = 668). (2) Of the neuropeptides examined, neuropeptide Y (NPY) (10(-6)M) is the most potent because it increased [Ca2+]i by about four-fold from the basal level. Other peptides--substance P, bombesin, calcitonin gene-related peptide, and vasoactive intestinal peptide induced smaller increases in [Ca2+]i. (3) We defined a response as positive if [Ca2+]i increased to a value that was 1.2-fold over the basal level. The majority of the TM cells reacted to NPY, whereas only 20-30% of the cells reacted to any of the other peptides. (4) The chelation of extracellular Ca2+ shortened the half-life of a NPY-induced response without affecting its latency. (5) NPY (10(-6)M) significantly increased the formation of inositol triphosphate following a 15 sec exposure. The same was the case for inositol monophosphate and inositol diphosphate. The results of this study suggest that in bovine TM cells, NPY stimulation is coupled to Ca2+ signalling through an increase in polyphosphoinositide turnover.