N6-methyladenosine (m6A) is an endogenous A3 adenosine receptor ligand.

About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.

Long-term intraocular pressure-lowering efficacy and safety of ripasudil-brimonidine fixed-dose combination for glaucoma and ocular hypertension: a multicentre, open-label, phase 3 study.

PURPOSE: To evaluate the long-term efficacy and safety of ripasudil-brimonidine fixed-dose combination (RBFC), a new intraocular pressure (IOP)-lowering medication for glaucoma and ocular hypertension (OHT). METHODS: This prospective, multicentre (23 sites in Japan), open-label study enrolled patients with primary open-angle glaucoma (POAG), OHT or exfoliative glaucoma and assigned them to one of four combination therapy cohorts, based on previous treatment(s) received: prostaglandin (PG) analogue (Cohort 1); PG analogue and beta-adrenoceptor blocker (ß-blocker) (Cohort 2); PG analogue, ß-blocker and carbonic anhydrase inhibitor (Cohort 3); or other/no treatment (Cohort 4). After a ≥ 4-week screening period, eligible patients received twice-daily RBFC for 52 weeks in addition to the treatments they were already receiving. Efficacy was assessed by change in IOP from baseline through week 52. Adverse events and adverse drug reactions (ADRs) were monitored throughout. RESULTS: In total, 179 patients from Cohort 1 (n = 48), Cohort 2 (n = 44), Cohort 3 (n = 41) and Cohort 4 (n = 46) entered the RBFC treatment period. For all cohorts, mean IOP was significantly reduced at 11:00 (2 h after instillation of RBFC) through week 52 with the changes from baseline at week 52 of - 2.7 to - 4.1 mmHg across cohorts; all p < 0.001. Common ADRs were conjunctival hyperaemia (58%), allergic conjunctivitis (18%) and blepharitis (17%), most of which were mild in severity. CONCLUSION: These data demonstrated the long-term efficacy and safety of RBFC, both alone and in combination with other anti-glaucoma agents. RBFC may offer a new treatment option for the long-term management of glaucoma and OHT. TRIAL REGISTRATION: Japan Registry of Clinical Trials Identifier: jRCT2080225063. DATE OF REGISTRATION: 17 February 2020.

Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance.

Transforming growth factor-beta 2 (TGF-ß2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-ß2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-ß2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-ß2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-ß2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm's canal (MSC) cell monolayers was decreased by TGF-ß2 treatment. SAHA inhibited the effects of TGF-ß2 on the permeability of these cells. TGF-ß2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-ß2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-ß2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-ß signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-ß. These results imply that SAHA prevents TGF-ß2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-ß signaling in TM and MSC cells.

Elevated soluble vascular endothelial growth factor receptor levels in aqueous humor from patients with different types of glaucoma.

We investigated the aqueous humor levels of vascular endothelial growth factor (VEGF), soluble VEGF receptor (sVEGFR)1, and sVEGFR2 in glaucoma patients and the correlations among them. Aqueous humor was collected from the anterior chamber at the start of glaucoma or cataract surgery. The levels of VEGF and its receptors, sVEGFR1 and sVEGFR2, were measured using multiplex bead-based immunoassays. Aqueous humor samples were obtained from 79 participants: 21 with primary open angle glaucoma (POAG), 22 with uveitic glaucoma (UG), 19 with neovascular glaucoma (NVG), and 17 with cataracts as controls. sVEGFR1 levels were significantly higher in NVG than in the other cases (NVG, 2839.8 pg/mL, P < 0.001). The sVEGFR2 levels of glaucoma patients were significantly higher than those of the controls (POAG, 699.0 pg/mL; UG, 866.2 pg/mL; NVG, 1198.1 pg/mL; P < 0.001). In the aqueous humor of glaucoma patients, sVEGFR1 and sVEGFR2 levels were positively correlated (POAG, P = 0.0196; UG, P = 0.0047; NVG, P = 0.0050). VEGF levels were negatively correlated with both sVEGFR1 (P = 0.0197) and sVEGFR2 (P = 0.0015) in POAG patients. In UG patients, the correlation between VEGF and sVEGFR1 levels was negative (P = 0.0144). sVEGFR2 levels were increased in various glaucomatous eyes. sVEGFR levels were negatively correlated with VEGF levels in some glaucoma types, implying that sVEGFRs may modulate the effects of aqueous VEGF in glaucoma pathogenesis.

Potential roles of the IL-6 family in conjunctival fibrosis.

Elevated intraocular pressure (IOP) is a significant risk factor for vision loss due to glaucoma, which is a major cause of blindness worldwide. Glaucoma filtration surgery (GFS) is an important method to reduce IOP by guidance of aqueous humor into a newly built filtration bleb in the conjunctiva; management of the wound healing mechanism is essential for the success of GFS. Here, we investigated the roles of interleukin (IL)-6 family members during the wound healing process after GFS. At the surgical site, the expression levels of genes encoding IL-6, oncostatin M (OSM), their receptors, and collagen I were elevated at 3 h after GFS, whereas the levels of genes encoding transforming growth factor (TGF)-ß, α-smooth muscle actin (SMA), type IV collagen, and fibronectin were elevated at 3 days after GFS. IL-6 trans-signaling and OSM signaling suppressed TGF-ß-induced expression of α-SMA and collagen IV, as well as activation of the non-canonical TGF-ß pathway, suggesting that IL-6 and OSM may aid in controlling the phase transition from inflammation to proliferation and remodeling. The suppressive effects of OSM were accompanied by STAT3 activation, such that STAT1 function was complementary to STAT3. Taken together, these observations indicated that IL-6 family members constitute early response genes after GFS, which can suppress TGF-ß-induced expression of late response genes at the surgical site after GFS.

Safety and efficacy of ripasudil in Japanese patients with glaucoma or ocular hypertension: 12-month interim analysis of ROCK-J, a post-marketing surveillance study.

BACKGROUND: Ripasudil is approved in Japan for glaucoma or ocular hypertension (OH) when other treatments are ineffective or cannot be administered. Its long-term safety and efficacy are being examined in a post-marketing surveillance study; 12-month data are described here. METHODS: This prospective, open-label, observational study enrolled patients with glaucoma or OH who started ripasudil during routine care. The key safety outcome was the incidence of adverse drug reactions (ADRs), focusing on allergy and/or inflammation-related ADRs such as blepharitis (including allergic) or conjunctivitis (including allergic). The primary efficacy endpoint was least squares mean (LSM) ± standard error (SE) change in intraocular pressure (IOP) from baseline to 12 months in all patients and in diagnostic groups. Secondary endpoints were change in IOP in groups stratified by treatment initiation pattern, number of concomitant drugs, and baseline IOP. RESULTS: Overall, 3359 patients (48% male, mean age ± standard deviation [SD] 69.1 ± 12.7 years) were evaluated for safety and 3323 for efficacy. Diagnoses were primary open-angle glaucoma (43.9%), normal-tension glaucoma (36.6%), secondary glaucoma (8.7%), OH (4.2%), and primary closed-angle glaucoma (2.4%). Mean ± SD observation period was 300.1 ± 122.4 days; 1010 patients (30.1%) discontinued ripasudil by 12 months. ADRs occurred in 626 patients (18.6%); the most common were conjunctival hyperemia and blepharitis. Allergy and/or inflammation-related ADRs occurred in 388 patients (11.6%), most commonly blepharitis (5.6%) and conjunctivitis (4.2%). IOP decreased significantly from a mean ± SD 18.1 ± 6.1 mmHg at baseline; the LSM ± SE IOP change throughout 12 months of ripasudil treatment was - 2.6 ± 0.1 mmHg (- 14.0 ± 0.4%; p < 0.001). A significant decrease in IOP at 12 months was seen in all categories of baseline IOP (p < 0.001), and all types of glaucoma (p < 0.001), except neovascular glaucoma. Ripasudil was associated with a significant reduction in IOP at 12 months whether initiated as monotherapy or in combination with ≤4 concomitant glaucoma therapies (p < 0.001). CONCLUSIONS: Ripasudil was safe and effective in patients with glaucoma or OH during routine care. No new safety signals were identified, and significant reductions in IOP were maintained over 12 months.

Correction to: Safety and efficacy of ripasudil in Japanese patients with glaucoma or ocular hypertension: 12-month interim analysis of ROCK-J, a post-marketing surveillance study.

An amendment to this paper has been published and can be accessed via the original article.

Clinical Features of Disaster-Associated Direct Deaths during Recent Inland Earthquakes in Japan.

Natural disasters, including earthquakes, cause disaster-associated direct deaths due to hazards and disaster-related deaths. This study was a retrospective and observational study that explored the effect of natural disasters on direct death. Although research reports on disaster-related deaths are common, there are few reports of disaster-associated direct death caused by events, such as house collapses, fires, and sediment-related factors. The amendment of the Building Standards Law in 1981 has made Japanese building standards more stringent. We sought to examine the determinants of the number of disaster-associated direct deaths during recent inland earthquakes in Japan. Following 2016 Kumamoto earthquakes (April 14, 21:26 [magnitude (M) 6.5], April 15, 0:03 [M 6.4], and April 16, 1:25 [M 7.3] and the subsequent numerous aftershocks), police necropsies confirmed 50 disaster-associated direct deaths (28 women [56%]). Thirty-four victims (68%) were elderly people 65 years of age or older, and 38 victims (76%) died as a result of a collapsed house. These percentages are consistent with those associated with recent inland earthquake disasters in Japan. The main finding was a linear correlation between the number of completely collapsed houses and the number of deaths due to house collapse during recent inland earthquakes in Japan (P = 0.02). It is suggested that the maintenance of houses may be important in reducing the number of disaster-associated direct deaths during inland earthquakes. The amendment of the Building Standards Law might reduce the number of disaster-associated direct deaths during inland earthquakes.

Changes in corneal endothelial cell shape after treatment with one drop of ROCK inhibitor.

PURPOSE: We performed an investigation of the temporal impact on corneal endothelial cell (CEC) shape after ophthalmic instillation of one drop of the ROCK inhibitor, ripasudil. METHODS: Subjects comprised 32 healthy adult volunteers, each of whom instilled ripasudil in the right eye. CEC shape [defined as CEC density (CECD), coefficient of variation (CV), and hexagonal cell rate (6A)], central corneal thickness (CCT), intraocular pressure (IOP), and conjunctival hyperemia were evaluated before instillation (baseline) and at 15 min, 1 h, 2 h, and 4-6 h after instillation by non-contact specular microscopy and slit-lamp microscopy. The incidence of pseudo gutta was also assessed. RESULTS: The CECD (cells/mm2) significantly decreased after instillation as follows: 2661 cells/mm2 at baseline, 2578 cells/mm2 at 15 min, 2527 cells/mm2 at 1 h, 2575 cells/mm2 at 2 h, and 2613 cells/mm2 at 4-6 h. After instillation, CV significantly increased and 6A significantly decreased. Pseudo gutta appeared in 18 subjects (56.3%) after instillation. There was no difference in CCT before and after instillation. IOP significantly decreased after instillation. The most severe conjunctival hyperemia was observed at 15 min after instillation, but had nearly disappeared within 2 h. CONCLUSIONS: Pseudo gutta appeared in more than half of the subjects, and the CEC shape changed upon instillation of one drop of ripasudil; however, it recovered at 4-6 h after instillation. Therefore, these changes would not greatly impact clinical treatment. Similar to the presence of conjunctival hyperemia, changes in CEC shape occur at early stages after instillation of ripasudil.

Interleukin-6-mediated trans-signaling inhibits transforming growth factor-ß signaling in trabecular meshwork cells.

Glaucoma is one of the major causes of blindness, and transforming growth factor-ß2 (TGF-ß2) has been found to be elevated in the aqueous humor of eyes with primary open-angle glaucoma (POAG). TGF-ß2 in aqueous humor causes the glaucoma-related fibrosis of human trabecular meshwork (HTM), suggesting an important role of TGF-ß in POAG pathogenesis. Here, we sought to elucidate the effects of IL-6 trans-signaling on TGF-ß signaling in HTM cells. Using a multiplex immunoassay, POAG patients decreased IL-6 levels and increased soluble IL-6 receptor (sIL-6R) levels compared with the controls. In in vitro experiments, we observed that the IL-6 level was increased in the conditioned medium of HTM cells after TGF-ß2 stimulation. To elucidate the relationship between TGF-ß2 and IL-6 in HTM cells, we conducted Western blotting and immunohistochemical analyses, and we noted that the combination of IL-6 and sIL-6R (IL6/sIL-6R) suppressed TGF-ß-induced up-regulation of α-smooth muscle actin in HTM cells, whereas IL-6 alone did not. This suggests that trans-signaling, not classic signaling, of IL-6 suppresses TGF-ß-induced fibrosis of HTM. IL6/sIL-6R also suppressed TGF-ß-mediated activation of myosin light chain 2 (MLC2), Smad2, and p38. Of note, these inhibitory effects of IL6/sIL-6R on TGF-ß were partly reduced by siRNA-mediated knockdown of STAT3. Moreover, IL-6/sIL-6R partly inhibited TGF-ß-induced activation of the Smad-sensitive promoter detected with luciferase reporter gene assays and up-regulation of TGFRI and TGFRII, evaluated by quantitative real-time RT-PCR. Strikingly, overexpression of TGFRI and TGFRII diminished these inhibitory effects of IL-6/sIL-6R. We conclude that of IL-6-mediated trans-signaling potently represses TGF-ß signaling in HTM cells.

DNA methyltransferase inhibitor suppresses fibrogenetic changes in human conjunctival fibroblasts.

Purpose: This study aimed to clarify the effects of a DNA methyltransferase inhibitor on fibrogenetic changes in human conjunctival fibroblasts (HConF). Methods: HConF were pretreated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-dC) for 48 h. After one passage, the cells were treated with 5 ng/ml of transforming growth factor (TGF)-ß2 for 48 h, and the expression levels of α-smooth muscle actin (α-SMA), extracellular matrix proteins, and phosphorylated Smad3 were evaluated with western blotting. A fusion construct between the COL1A2 promoter and the luciferase gene was introduced into the HConF after the first passage, and the construct's activity was detected via a luciferase reporter gene assay. Results: TGF-ß2-induced upregulation of α-SMA was suppressed by pretreatment with 5-Aza-dC (0.1, 1.0, and 10 µM) in a dose-dependent manner. Upregulation of type I collagen was also suppressed by 10 µM 5-Aza-dC pretreatment. In contrast, 5-Aza-dC had no inhibitory effect on the expression of fibronectin or phosphorylated Smad3. However, COL1A2 promoter activity was suppressed with 5-Aza-dC pretreatment. Conclusions: In HConF, fibrogenetic changes were partly suppressed with a DNA methyltransferase inhibitor, suggesting an indirect inhibitory effect of the inhibitor on the COL1A2 promoter in HConF.

Efficacy and safety of Ex-PRESS® mini shunt surgery versus trabeculectomy for neovascular glaucoma: a retrospective comparative study.

BACKGROUND: The objective of this study is to evaluate and compare the short-term efficacy and safety of Ex-PRESS® mini shunt surgery and trabeculectomy for neovascular glaucoma (NVG). METHODS: Patients with NVG who underwent Ex-PRESS® mini shunt surgery or trabeculectomy as a primary glaucoma surgery between March 2013 and October 2015 were included in the study, and their medical charts were retrospectively reviewed. The Ex-PRESS® and trabeculectomy groups included 14 eyes and 30 eyes, respectively. Surgical failure was defined by an intraocular pressure (IOP) of ≥21 mmHg (condition A) or ≥ 18 mmHg (condition B); Kaplan-Meier survival analyses and the multivariable Cox proportional hazards model were used to assess efficacies. RESULTS: Kaplan-Meier survival analyses indicated that the probabilities of success at 1 year for the Ex-PRESS® group were 25.7 and 31.8% based on complete and qualified success under condition A, respectively. The corresponding values for the trabeculectomy group were 47.8 and 69.3%, and there was a significant difference in qualified success with condition A (Fig. 1; P = 0.018), while there were no significant differences in the other criteria. Ex-PRESS® mini shunt surgery and higher intraocular pressure were independent prognostic factors using Cox proportional hazards model analyses in qualified success as in condition A (P = 0.012 and 0.0495, respectively). The occurrences of postsurgical hyphema and bleb leaks were significantly higher in the trabeculectomy group (P = 0.005 and 0.008, respectively). CONCLUSION: During a 1 year follow-up, Ex-PRESS® mini shunt surgery was a less effective, but safer treatment for NVG compared with trabeculectomy.

Early bleb parameters as long-term prognostic factors for surgical success: a retrospective observational study using three-dimensional anterior-segment optical coherence tomography.

BACKGROUND: The object of this study is to investigate the effect of early bleb parameters measured by three-dimensional anterior-segment optical coherence tomography on the surgical success of trabeculectomy. METHODS: This retrospective study included 45 patients with 19 of exfoliation glaucoma, 17 of primary open angle glaucoma, 4 of neovascular glaucoma, 4 of uveitic glaucoma and 1 of glaucoma caused from familial amyloid polyneuropathy who underwent trabeculectomy. Bleb parameters, such as total bleb height, the position and the width of filtration openings on the scleral flap, bleb wall thickness, fluid-filled cavity height, and bleb wall intensity were assessed by three-dimensional anterior-segment optical coherence tomography 0.5 months after trabeculectomy, and were subjected to a Cox proportional hazard model as potential prognostic factors. Surgical success was defined as: IOP < 21 mmHg (A), < 18 mmHg (B), < 15 mmHg (C) with (qualified success) or without medication (complete success). Complete failure was defined as hypotony and additional glaucoma surgeries required. RESULTS: The width of filtration openings was identified as a prognostic factor for all criteria. By multivariable analysis, the width of the filtration openings was a prognostic factor in all criteria tested, and the preoperative IOP were significant prognostic factors for surgical success in qualified success in criteria B and C. Separate from the median widths of filtration openings, wide filtration opening showed significant survival ratio for qualified success in criteria A and B and for complete success in all criteria, respectively. CONCLUSIONS: The width of filtration opening at an early stage is a prognostic factor for surgical success of trabeculectomy.

Stimulation of the adenosine A3 receptor, not the A1 or A2 receptors, promote neurite outgrowth of retinal ganglion cells.

Among candidate neuroprotective agents, adenosine is thought to be a possible treatment for central nervous system disorders. Adenosine elicits biological effects through four G protein-coupled receptors (A1, A2A, A2B, and A3). The A2A and A2B receptors stimulate adenylyl cyclase (AC) and increase cyclic adenosine monophosphate (cAMP) levels, whereas A1 and A3 receptors inhibit AC and decrease cAMP levels. Several studies have investigated the effects of adenosine receptors (AdoRs) in glaucoma, because modulation of A1, A2A, or A3 receptor regulates intraocular pressure. In addition, AdoR-related phenomena may induce neuroprotective effects in retinal neurons. Notably, A1, A2A, and A3 receptor agonists reportedly inhibit retinal ganglion cell (RGC) death in in vitro and in vivo glaucoma models. However, there is limited knowledge of the effects of AdoR activation on neurite outgrowth or the regeneration of RGCs. In this report, we described the role of an AdoR subtype in neurite outgrowth and RGC axonal regeneration. The distribution of AdoRs in the retina was evaluated by immunohistochemical analysis. Using primary cultured rat RGCs in vitro and an optic nerve crush model in vivo, neurite elongation was evaluated after stimulation by the following AdoR agonists: CHA, an A1 receptor agonist; CGS21680, an A2A receptor agonist; BAY60-6583, an A2B receptor agonist; and 2-Cl-IB-MECA, an A3 receptor agonist. To determine the mechanism of neurite promotion, the candidate molecules of signal transduction associated with the neurite elongation of AdoRs were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. All four AdoRs (A1, A2A, A2B, and A3) were present in the inner retinal layers. Among the agonists for AdoR, only 2-Cl-IB-MECA significantly promoted neurite outgrowth in primary cultured RGCs. Signaling pathway analyses showed that 2-Cl-IB-MECA caused upregulated phosphorylation of Akt in cultured RGCs. Additionally, LY294002, an inhibitor of Akt, suppressed the neurite-promoting effects of the A3 receptor agonist in RGCs. Moreover, 2-Cl-IB-MECA increased the number of regenerating axons in the optic nerve crush model. Taken together, these data indicate that activation of the A3 receptor, not the A1 or A2 receptors, promotes in vitro and in vivo neurite outgrowth during the regeneration of rat RGCs, which is caused by the activation of an Akt-dependent signaling pathway. Therefore, AdoR activation may be a promising candidate for the development of novel regenerative modalities for glaucoma and other optic neuropathies.

Decreased MCP-1/CCR2 axis-mediated chemotactic effect of conjunctival fibroblasts after transdifferentiation into myofibroblasts.

The purpose of this study is to investigate the change in chemotactic effects of human conjunctival fibroblasts (HConFs) after transdifferentiation into myofibroblasts, and to explore related molecular mechanisms. HConFs were treated with 5 ng/mL transforming growth factor (TGF)-ß2 for 48 h to induce transdifferentiation into myofibroblasts. The cytokine concentrations in the conditioned media of HConFs were measured by multiplex bead-based immunoassays. The Boyden chamber assay was used to assess the chemotactic effects using the monocyte cell line, THP-1 cells. The concentration of monocyte chemoattractant protein (MCP)-1 in the conditioned media was decreased after transdifferentiation into myofibroblasts (P < 0.001). The conditioned media of HConFs exerted a chemotactic effect on THP-1 cells, but this effect decreased after transdifferentiation into myofibroblasts (P = 0.032). The number of migrated THP-1 cells decreased significantly upon treatment with neutralizing anti-MCP-1 antibodies (P = 0.006) and tended to decrease upon treatment with C-C chemokine receptor (CCR) 2 antagonist. The chemotactic effect of HConFs mediated by the MCP-1/CCR2 axis was decreased after transdifferentiation into myofibroblasts.

In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS.

The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23-25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.

Effects of mechanical stress and vitreous samples in retinal pigment epithelial cells.

In rhegmatogenous retinal detachment (RRD), scattered RPE cells from the basement membrane into the vitreous cavity undergo an epithelial mesenchymal transition (EMT) and form the intraocular fibrous membrane in response to vitreous fluid. We investigated whether exposure to vitreous samples was associated with EMT-associated signals and mesenchymal characters. Human vitreous samples were collected from patients with RRD, epiretinal membrane (ERM), or macular hole (MH). We evaluated the effects of vitreous on ARPE-19 cells in suspension cultures using poly 2-hydroxyethyl methacrylate-coated dishes and three-dimensional (3D) Matrigel cultures. We found that exposure to vitreous samples did not induce morphological changes or accelerate wound closure in monolayers. Several samples showed increased phosphorylation of Smad2 and nuclear translocation of nuclear factor-κB. Mechanical stress triggered an elevation of phosphorylation levels in Smad2. In addition, exposure to vitreous fluid increased the phosphorylation of p38 mitogen-activated protein kinase in cell suspension cultures after mechanical stress. Moreover, ARPE-19 cells showed a stellate invasive phenotype in 3D Matrigel cultures with vitreous samples. In this study, we demonstrated that mechanical stress and vitreous were associated with EMT-associated signals and invasive phenotypes in 3D cultures but not in monolayers. These results have important implications for the role of vitreous humor in the induction of EMT and intraocular fibrosis.

Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 µM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM cells.

The effects of ripasudil (K-115), a Rho kinase inhibitor, on activation of human conjunctival fibroblasts.

The most common cause of glaucoma surgery failure is scar formation induced by activation of wound-healing responses and resultant fibrosis at the surgical site. We investigated the effects of ripasudil, a Rho kinase inhibitor, on activation of human conjunctival fibroblasts (HConF). HConF were pretreated with different concentrations of ripasudil for 1 h before addition of transforming growth factor (TGF)-ß2, followed by incubation for 48 h. TGF-ß2-treated fibroblasts exhibited a significant increase in expression of α-smooth muscle actin (α-SMA), a marker of fibroblast-to-myofibroblast differentiation, and this increase was significantly suppressed, in a dose-dependent manner, by pretreatment with ripasudil. Ripasudil pretreatment also significantly attenuated TGF-ß2-induced fibronectin production and collagen gel contraction. TGF-ß2 increased both the number of viable cells and the number of cells in the G2/M phase of the cell cycle; these effects were attenuated by pretreatment with ripasudil. In addition, we explored the effects of ripasudil on stimulation of HConF by activated macrophages. Human monocytic cell line THP-1 cells were differentiated into M1 or M2 macrophage-like cells, and HConF were treated with conditioned media derived from these macrophages in the presence or absence of ripasudil. Conditioned medium from M2 macrophage-like cells induced a significant increase in α-SMA expression, viable cell numbers, and gel contraction, all of which were significantly suppressed by ripasudil. Thus, overall, ripasudil attenuated activation of human conjunctival fibroblasts. Ripasudil may be of therapeutic utility, preventing excessive scarring after glaucoma filtration surgery.

Angle closure caused by a plateau-like iris associated with an enlarged Soemmering's ring: a case report.

BACKGROUND: This study evaluated the anterior ocular segment in a pseudophakic eye with angle closure due to a plateau-like iris associated with Soemmering's ring, using ultrasound biomicroscopy (UBM) and anterior segment optical coherence tomography (AS-OCT). CASE PRESENTATION: A 60-year-old woman was referred from a local clinic due to sudden-onset ocular pain and uncontrolled intraocular pressure (IOP) in the left eye, which was 56 mmHg after treatment with latanoprost, timolol, and dorzolamide eye drops. Fourteen years earlier, she developed acute primary angle closure. At that time, because the IOP remained elevated after a peripheral iridectomy, cataract extraction combined with goniosynechialysis was added. After the IOP decreased to within the normal range, a secondary intraocular lens was implanted outside the bag. On this admission, UBM and AS-OCT images showed angle closure caused by the combination of a plateau-like iris and contact between the mydriatic pupillary margin and enlarged Soemmering's ring. After adding 2 % pilocarpine four times a day, the mydriasis resolved slightly, and the IOP decreased to the normal range between 8 and 18 mmHg. AS-OCT images showed re-opening of the angle structure after treatment with 2 % pilocarpine. CONCLUSION: The intraocular pressure and angle structure in eyes with a plateau iris after cataract extraction should be followed carefully.