The effect of axial length on the short-term outcomes of cataract surgery combined with ab interno trabeculotomy.

PURPOSE: Minimally invasive glaucoma surgery is safer and effective surgical modality for patients with glaucoma. To compare the effect of axial length (AL) on the surgical outcomes of combined cataract surgery and ab interno trabeculotomy (phaco-LOT), a retrospective, non-randomized comparative study was performed. METHODS: In total, 458 eyes of 458 open-angle glaucoma patients who underwent phaco-LOT and were followed-up without any intervention for at least 6 months were enrolled. All were divided into a long-AL group (AL ≥ 26.0 mm, 123 eyes) and a not-long-AL group (AL < 26.0 mm, 335 eyes). The principal outcomes were the changes in intraocular pressure (IOP) and medication scores. We also sought a correlation between postoperative IOP spike and hyphema. RESULTS: Significant postoperative reductions in IOP and medication scores were apparent in all subjects. The IOP reductions were significant at all timepoints in the not-long-AL group, but not until 1 month postoperatively in the long-AL group, and the IOP change was significantly lower in the long-AL group from postoperative day 1 to 3 months. On subanalysis of subjects by age, the microhook used, the pre-operative IOP, and the medication score, a significantly higher incidence of IOP spike was observed in the long-AL group in weeks 1 and 2 (both p < 0.05), but this did not correlate with hyphema status, implying that a different mechanism was in play. CONCLUSION: Phaco-LOT was effective regardless of AL, but the postoperative IOP decrease was lower and the early postoperative incidence of IOP spike was higher in long-AL eyes.

Contributing factors for intraocular pressure control in patients with mostly normal-tension glaucoma after initial Ex-PRESS drainage device implantation.

PURPOSE: To investigate the postoperative intraocular pressure (IOP) control and identify the factors associated with failure of initial Ex-PRESS surgery in patients with open-angle glaucoma for 3 years. METHODS: A total of 79 patients with medically uncontrolled open-angle glaucoma (55 normal-tension glaucoma and 24 primary open-angle glaucoma) were enrolled. All patients underwent Ex-PRESS implantation (including combined cataract surgery). The outcome measure was the survival rate using life table analysis, the failure was defined as IOP of > 18 mmHg (criterion A), > 15 mmHg (criterion B) or > 12 mmHg (criterion C) and/or IOP reduction of < 20% from baseline (each criterion) without any glaucoma medications. The Cox proportional hazards model was used to identify risk factors for IOP management defined as the above criterion.  RESULTS: The mean preoperative IOP was 19.3 ± 5.8 mmHg. At 36 months, the mean IOP was 11.8 ± 3.6 mmHg with a mean IOP change of 7.5 mmHg (reduction rate 39.0%). The cumulative probability of success was 58% (95%CI: 42-64%) (criterion A), 48% (95%CI: 37-59%) (criterion B) and 30% (95%CI: 20-40%) (criterion C). In multivariate analyses, factors that predicted poor IOP control included the intervention of bleb needling after 6 months after the surgery (HR: 2.43; 95%CI: 1.35-4.37; P = 0.032). Transient hypotony was observed in 4 patients. CONCLUSION: The implementation of bleb needling after Ex-PRESS surgery in the late postoperative period was suggested to be the main risk factor for achieving lower IOP.

Apoptosis inhibitor of macrophages/CD5L enhances phagocytosis in the trabecular meshwork cells and regulates ocular hypertension.

The trabecular meshwork (TM) cells of the eye are important for controlling intraocular pressure (IOP) and regulating outflow resistance in the aqueous humor. TM cells can remove particles and cellular debris by phagocytosis, decreasing both outflow resistance and IOP. However, the underlying mechanisms remain unclear. Here, we investigate whether apoptosis inhibitor of macrophages (AIM), which mediates the removal of dead cells and debris in renal tubular epithelial cells, regulates the phagocytic capacity of TM cells. In vitro experiments revealed that CD36, the main receptor for AIM, colocalized with AIM in human TM cells; additionally, phagocytosis was stimulated when AIM was provided. Furthermore, in a mouse model with transient IOP elevation induced by laser iridotomy (LI), removal of accumulated iris pigment epithelial cells or debris in the TM and recovery of IOP to baseline levels were delayed in AIM-/- mice, compared with control mice. However, treatment with AIM eyedrops rescued AIM-/- mice from the elevated IOP after LI. Since AIM is a protein known to inhibit macrophage apoptosis, we additionally verified its involvement in macrophage removal of cellular debris and IOP. There were no statistically significant differences in the number of macrophages between control mice and AIM-/- mice in the TM. Additionally, we confirmed the rescue effect of the rAIM eyedrops after macrophages had been removed by clodronate liposomes. Therefore, AIM plays an important role in regulating the phagocytic capacity of TM cells, thereby affecting outflow resistance. Our results suggest that drugs targeting the phagocytic capacity of TM cells via the AIM-CD36 pathway may be used to treat glaucoma.

Synergic effects of EP2 and FP receptors co-activation on Blood-Retinal Barrier and Microglia.

Macular edema (ME) is caused with disruption of the blood-retinal barrier (BRB) followed by fluid accumulation in the subretinal space. Main components of the outer and inner BRB are retinal pigment epithelial (RPE) cells and retinal microvascular endothelial cells, respectively. In addition, glial cells also participate in the functional regulation of the BRB as the member of 'neurovascular unit'. Under various stresses, cells in neurovascular units secrete inflammatory cytokines. Neuroinflammation induced by these cytokines can cause BRB dysfunction by degrading barrier-related proteins and contribute to the pathophysiology of ME. Prostaglandins (PGs) are crucial lipid mediators involved in neuroinflammation. Among PGs, a novel EP2 agonist, omidenepag (OMD) acts on not only the uveoscleral pathway but also the conventional pathway, unlike F prostanoid (FP) receptor agonists. Moreover, the combination use of the EP and the FP agonist is not recommended because of the risk of inflammation. In this study, we investigated effects of OMD and latanoprost acid (LTA), a FP agonist, on BRB and microglia in vitro and in vivo. To investigate the function of outer/inner BRB and microglia, in vitro, ARPE-19 cells, human retinal microvascular endothelial cells (HRMECs), and MG5 cells were used. Cell viability, inflammatory cytokines mRNA and protein levels, barrier morphology/function, and microglial activation were evaluated using proliferation assays, qRT-PCR, ELISA, immunocytochemistry, trans-epithelial electrical resistance, and permeability assay. Moreover, after vitreous injection into the mouse, outer BRB morphology, glial activation, and cytokine expression were assessed. Each OMD and LTA alone did not affect the viability or cytokines expression of the three types of cells. In ARPE-19 cells, the co-stimulation of OMD and LTA increased the mRNA and protein levels of inflammatory cytokines (IL-6, TNF-α, and VEGF-A) and decreased the barrier function and the junction-related protein (ZO-1 and ß-catenin). By contrast in HRMECs, the co-stimulation affected significant differences in the mRNA levels of some cytokine (IL-6 and TNF-α) but enhanced the barrier function. In MG5 cells, the cytokines mRNA and size of Iba1-expressed cell were increased. A non-steroidal anti-inflammatory inhibited the barrier dysfunction and the junction-related protein downregulation in ARPE-19 cells and activation of MG5 cells. Also in vivo, the co-stimulation induced outer BRB disruption, cytokine increase, and retinal glial activation. Therefore, the co-stimulation of EP2 and FP induced the inflammatory cytokine-mediated outer BRB disruption, the enhanced inner BRB function, and the microglial activation. The BRB imbalance and the intrinsic prostaglandin production may be involved in OMD-related inflammation.

Role of Autotaxin in High Glucose-Induced Human ARPE-19 Cells.

Autotaxin (ATX) is an enzymatic with lysophospholipase D (lysoPLD) activity. We investigated the role of ATX in high glucose (HG)-induced human retinal pigment epithelial (ARPE-19) cells to explore the pathogenesis of diabetic retinopathy (DR). We performed a quantitative real-time polymerase chain reaction, Western blotting, immunocytochemistry, enzyme-linked immunosorbent assay, cell permeability assay, and transepithelial electrical resistance measurement in HG-induced ARPE-19 cells and compared their results with those of normal glucose and osmotic pressure controls. ATX expression and its lysoPLD activity, barrier function, and expression of vascular endothelial growth factor receptors VEGFR-1 and VEGFR-2 were downregulated, while fibrotic responses, cytoskeletal reorganization, and transforming growth factor-ß expression were upregulated, in the HG group. Our results suggest that HG induces intracellular ATX downregulation, barrier dysfunction, and fibrosis, which are involved in early DR and can be targeted for DR treatment.

Effects of topical TGF-ß1, TGF-ß2, ATX, and LPA on IOP elevation and regulation of the conventional aqueous humor outflow pathway.

PURPOSE: The effects of aqueous mediators possibly increasing the outflow resistance, transforming growth factor-ß1 (TGF-ß1), TGF-ß2, autotaxin (ATX), and lysophosphatidic acid (LPA) on human trabecular meshwork (hTM) cells and monkey Schlemm's canal endothelial (SCE) cells were characterized and compared, and the effects of intracameral application of these mediators on intraocular (IOP) elevation were also examined. METHODS: Cells were treated with TGF-ß1, TGF-ß2, ATX, LPA, or vehicle, and mRNA and protein expression levels of α-SMA, COL1A1, fibronectin, ß-catenin, and ZO-1 were examined with real-time quantitative PCR (RT-qPCR) or immunofluorescence analyses or both. The permeability of cell monolayers was measured by determining the transendothelial electrical resistance (TEER) or with the fluorescein isothiocyanate (FITC)-dextran permeability assay. IOP was evaluated in rabbit eyes after intracameral administration of the mediators. RESULTS: All mediators induced upregulation of α-SMA, COL1A1, and fibronectin in hTM cells. The effect of TGF-ß2 on mRNA expression of fibrotic markers was statistically significantly greater than that of TGF-ß1. The effects of ATX and LPA indicated the time-dependent difference in the upregulation of α-SMA, COL1A1, and fibronectin. The TEER and FITC-dextran permeability of the SCE cells was evaluated after treatment with TGF-ß1 and TGF-ß2, but no statistically significant change was observed within 24 h. ATX and LPA also reduced permeability statistically significantly after 3 h and 0.5 h, respectively, and the effect of LPA was more rapid compared to that of ATX. Statistically significant IOP elevation was observed in rabbit eyes as early as 0.5-2.0 h after ATX and LPA treatment and at 24 h after treatment with TGF-ß2. CONCLUSIONS: TGF-ß2 and ATX and LPA regulate aqueous outflow by modulation of hTM cells and SCE cells, and differences in timing between the effects of each mediator were observed. ATX and LPA showed more rapid effects on IOP elevation than TGF-ß2. It was suggested that TGF-ß2 and ATX/LPA are involved in increases of IOP, but the timing and sustainability differ between mediators, and they may play specific roles in different glaucoma subtypes.

Effects of mammalian target of rapamycin inhibitors on fibrosis after trabeculectomy.

Glaucoma, the second leading cause of blindness worldwide, is characterized by aberrant elevations of intraocular pressure (IOP), which can damage the optic nerve. IOP reduction is the only effective therapy for prevention of visual impairment and blindness in both hypertensive and normotensive individuals, and in some cases, trabeculectomy is a major surgical procedure that can lower IOP in patients with glaucoma. No matter how surgical technique and postoperative care advances, excessive scarring and tissue fibrosis could result from increased human conjunctival fibroblast (HCF) proliferation and extracellular matrix (ECM) deposition of the subconjunctival tissue and scleral flaps would persist after trabeculectomy. And these issues are major impediments to IOP reduction and filtering of bleb formations, so the modulation of the factors which can induce fibrosis could used as a novel strategy to control scarring after trabeculectomy. In this study, we examined the effects of mammalian target of rapamycin (mTOR) inhibitors (rapamycin or Torin1) on the fibrotic response induced by transforming growth factor-beta 1 (TGF-ß1) in cultured human conjunctival fibroblast (HCF) cells. The study also examined the effects of mTOR inhibitor on fibrosis after trabeculectomy in rabbit eyes. In in vitro studies, we stimulated HCFs with TGF-ß1, and confirmed that the expression levels of fibronectin, collagen type I alpha 1 chain (COL1A1), and α-smooth muscle actin (SMA) were significantly upregulated in HCFs with TGF-ß1, by means of quantitative real-time polymerase chain reaction and immunocytochemistry. And those TGF-ß1-induced changes were significantly attenuated with mTOR inhibitors, rapamycin or Torin1. Additionally the migration rate of HCFs was examined under conditions of TGF-ß1 induction, TGF-ß1-induced changes were significantly attenuated with mTOR inhibitors. A rabbit model of trabeculectomy was examined in vivo, and the effects of topical mTOR inhibitor were also examined, and found that topical treatment with mTOR inhibitor significantly suppressed collagen deposition in rabbit eyes after trabeculectomy. These results have demonstrated that mTOR inhibitors may provide a novel treatment modality for reducing the fibrotic response in HCFs and improving bleb scarring after filtration surgery.

Neuroprotective role of sphingolipid rheostat in excitotoxic retinal ganglion cell death.

The glutamate excitotoxicity has been suggested as a factor involved in the loss of retinal neuronal cells, including retinal ganglion cell (RGC), in various retinal degenerative diseases including ischemia-reperfusion injury, diabetic retinopathy, and glaucoma. Excitotoxic RGC death is caused not only by direct damage to RGCs but also by indirect damage due to the inflammation of retinal glial cells. Sphingosine 1-phosphate (S1P) and ceramides are bioactive sphingolipids which have been shown to possess important physiological roles in cellular survival and apoptosis, and the balance between S1P and ceramide, sphingolipid rheostat, has been suggested to be important for determining cellular fate. Therefore, we conducted the present study to clarify the neuroprotective role of sphingolipid rheostat in excitotoxic RGC death in vivo and in vitro. Acute RGC death was induced by intravitreal N-methyl-d-aspartate (NMDA) injection in the mouse. The mRNA expression of sphingosine kinase (SphK1/SphK2) was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The expressions of SphK1/2, S1P, S1P-receptor (S1PR), glial fibrillary acidic protein (GFAP), Iba1, and CD31 were examined by immunostaining. Retinal sphingolipids and ceramides were quantified by liquid chromatography with tandem mass spectrometry. The neuroprotective effect of the sphingosine kinase inhibitor (SKI) on RGC death was assessed by RGC count and Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Further, the in vitro effect of SKI was investigated using rat primary cultured RGCs and glial cells. In addition, MG5 cells and A1 cells, which were mouse microglia and astrocyte cell-line, were also used. The expression of cleaved-caspase-3, GFAP, and Iba1 in RGCs, primary glial cells, MG5 cells, and A1 cells was assessed by immunostaining. NMDA injection resulted in mRNA upregulation of SphK1; however, SphK2 was reduced in the mouse retina. SphKs, S1P, S1PR1, S1PR2, and GFAP expression increased in the early-stage NMDA group, whereas S1P and GFAP were higher in the late-stage NMDA + SKI group. In the NMDA group, S1P expression was lower whereas sphingosine, C20, C22, and C24 ceramides showed higher levels. The proportion of very-long-chain ceramide was elevated in the NMDA group but reduced in the NMDA + SKI group. SKI treatment significantly increased RGC survival in retinal wholemount analysis and decreased apoptosis in the ganglion cell layer and inner nuclear layer. In vitro, SKI suppressed excitotoxic RGC death, cleaved-caspase-3 expression, and activated glial cells. The findings in the present study provide the first evidence demonstrating the involvement of sphingolipid rheostat in the neuroprotection against excitotoxic RGC death. Therefore, regulation of sphingolipid rheostat might serve as a potential therapy for retinal degenerative disease.

Crosstalk between transforming growth factor ß-2 and Autotaxin in trabecular meshwork and different subtypes of glaucoma.

BACKGROUND: Elevated transforming growth factor (TGF)-ß2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-ß2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators. METHODS: The level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-ß levels were measured using the Bio-Plex Pro TGF-ß Assay. RNA scope was used to assess the expression of ATX and TGF-ß2 in human's eye specimen. And in vitro studies were performed using hTM cells to explore if trans-signaling of TGF-ß2 regulates ATX expressions. RESULTS: TGF-ß2/ATX ratio was significantly high in AH of control or POAG compared with SOAG, and negatively correlated with IOP. RNA scope revelated positive expressions of both TGF-ß2 and ATX in ciliary body (CB) and TM in control, but ATX expressions was significantly enhanced in SOAG. In hTM cells, ATX expressions were regulated by TGF-ß2 with concentration-dependent manner. In counter, ATX also induced TGF-ß1, TGF-ß2 and TGFBI upregulations and activation of the Smad-sensitive promoter, as well as upregulation of fibrotic markers, and these upregulation was significantly suppressed by both TGF-ß and ATX inhibition. CONCLUSIONS: Trans-signaling of TGF-ß2 regulates ATX expressions and thereby induced upregulations of TGF-ßs or fibrosis of hTM. TGF-ß2 trans-signaling potently regulate ATX transcription and signaling in hTM cells, which may reflect different profile of these mediators in glaucoma subtypes. Trial Registration This prospective observational study was approved by the Institutional Review Board of the University of Tokyo and was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan (ID: UMIN000027137). All study procedures conformed to the Declaration of Helsinki. Written informed consent was obtained from each patient.

Central visual field change after fornix-based trabeculectomy in Japanese normal-tension glaucoma patients managed under 15 mmHg.

PURPOSE: To investigate how the central visual field would be changed after fornix-based trabeculectomy with mitomycin C in Japanese normal-tension glaucoma (NTG) patients monitored for more than 10 years including before surgery. METHODS: This is a retrospective cohort study. We identified twenty-eight eyes of 28 NTG cases regularly monitored for more than 5 years and examined static visual field (VF) tests for more than five times before and after fornix-based trabeculectomy (including combined surgery). Based on preoperative data for 6.3 years, we evaluated postoperative changes for 6.0 years in 10-2 VF and 30-2 VF. RESULTS: Six patients were male and 22 females, the mean age was 57.9 years, and the mean deviation was - 13.7 decibels. After surgery, mean IOP decreased from 13.9 to 9.0 mmHg (P ≤ 0.01), and medication score also did. The rate of 10-2 VF deterioration was significantly suppressed from - 1.0 dB/year preoperatively to - 0.4 dB/year postoperatively (P ≤ 0.01). And when 30-2 VF was divided into six relevant sectors, the rate of deterioration at cecocentral and arcuate areas of the superior hemifield was suppressed postoperatively (P ≤ 0.01 and P = 0.042, respectively). CONCLUSION: Based on long-term preoperative data, a significant positive change in 10-2 VF was observed after fornix-based trabeculectomy with mitomycin C in Japanese NTG patients with a mean IOP of 13.9 mmHg. Central vision could be expected to be maintained for NTG patients even if its preoperative IOP is low after successful trabeculectomy.

Structural Changes and Astrocyte Response of the Lateral Geniculate Nucleus in a Ferret Model of Ocular Hypertension.

We investigated structural changes and astrocyte responses of the lateral geniculate nucleus (LGN) in a ferret model of ocular hypertension (OH). In 10 ferrets, OH was induced via the injection of cultured conjunctival cells into the anterior chamber of the right eye; six normal ferrets were used as controls. Anterograde axonal tracing with cholera toxin B revealed that atrophic damage was evident in the LGN layers receiving projections from OH eyes. Immunohistochemical analysis with antibodies against NeuN, glial fibrillary acidic protein (GFAP), and Iba-1 was performed to specifically label neurons, astrocytes, and microglia in the LGN. Significantly decreased NeuN immunoreactivity and increased GFAP and Iba-1 immunoreactivities were observed in the LGN layers receiving projections from OH eyes. Interestingly, the changes in the immunoreactivities were significantly different among the LGN layers. The C layers showed more severe damage than the A and A1 layers. Secondary degenerative changes in the LGN were also observed, including neuronal damage and astrocyte reactions in each LGN layer. These results suggest that our ferret model of OH is valuable for investigating damages during the retina-brain transmission of the visual pathway in glaucoma. The vulnerability of the C layers was revealed for the first time.

Oxidative stress induces ferroptotic cell death in retinal pigment epithelial cells.

The dysfunction and cell death of retinal pigment epithelial (RPE) cells are hallmarks of late-stage dry (atrophic) age-related macular degeneration (AMD), for which no effective therapy has yet been developed. Previous studies have indicated that iron accumulation is a source of excess free radical production in RPE, and age-dependent iron accumulation in RPE is accelerated in patients with dry AMD. Although the pathogenic role of oxidative stress in RPE in the development of dry AMD is widely accepted, the mechanisms of oxidative stress-induced RPE cell death remain elusive. Here, we show that ferroptotic cell death, a mode of regulated necrosis mediated by iron and lipid peroxidation, is implicated in oxidative stress-induced RPE cell death in vitro. In ARPE-19 cells we observed that the ferroptosis inhibitors ferrostatin-1 and deferoxamine (DFO) rescued tert-butyl hydroperoxide (tBH)-induced RPE cell death more effectively than inhibitors of apoptosis or necroptosis. tBH-induced RPE cell death was accompanied by the three characteristics of ferroptotic cell death: lipid peroxidation, glutathione depletion, and ferrous iron accumulation, which were all significantly attenuated by ferrostatin-1 and DFO. Exogenous iron overload enhanced tBH-induced RPE cell death, but this effect was also attenuated by ferrostatin-1 and DFO. Furthermore, mRNA levels of numerous genes known to regulate iron metabolism were observed to be influenced by oxidative stress. Taken together, our observations suggest that multiple modes of cell death are involved in oxidative stress-induced RPE cell death, with ferroptosis playing a particularly important role.

The role of sphingosine 1-phosphate receptors on retinal pigment epithelial cells barrier function and angiogenic effects.

Sphingosine-1-phosphate (S1P) is a lysophospholipid mediator, promoting angiogenesis and inflammation via interactions with its receptors (S1P1-5), but the receptors and signaling pathways responsible for the progression of choroidal neovascularization (CNV) remain unknown. We investigated the roles of S1P/S1P receptors in RPE cells. ARPE-19 cells were treated with S1P dissolved in carrier proteins of albumin or apolipoprotein M (ApoM). The mRNA expression levels of interleukin-8 (IL-8), C-C motif chemokine ligand 2 (CCL2), and vascular endothelial growth factor (VEGF) were evaluated using quantitative real-time polymerase chain reaction. The protein level of hypoxia-inducible factor (HIF)-1α was assessed via enzyme-linked immunosorbent assay. HIF transcriptional activity was evaluated with a dual-reporter luciferase assay. Cellular barrier integrity was evaluated using transepithelial electrical resistance and the FITC-dextran permeability assay. The suppressive effect of an S1P antagonist on CNV progression was investigated with a laser-induced CNV model in mice. The increase in expression of IL-8, CCL2, and VEGF due to albumin-bound S1P was significantly mitigated by an S1P2 antagonist. The expression of HIF-1α significantly decreased with inhibition of S1P2 and S1P3. In addition, albumin-bound S1P disrupted the barrier integrity of retinal pigment epithelial cells via S1P2, whereas integrity was strengthened by ApoM-bound S1P. CNV lesions were significantly reduced in the mouse model with intravitreal injection of S1P2 antagonist. This study demonstrated that S1P significantly promotes angiogenesis, inflammation, and barrier integrity, which was attenuated by inhibition of S1P2 or S1P3, suggesting that regulation of S1P2 and S1P3 is a novel therapeutic target for CNV.

Development of a Novel Intraocular-Pressure-Lowering Therapy Targeting ATX.

Elevated intraocular pressure (IOP) is the major cause of glaucoma, which is the second leading cause of blindness. However, current glaucoma treatments cannot completely regulate IOP and progression of glaucoma. Our group recently found that autotaxin (ATX) activity in human aqueous humor (AH) was positively correlated with increased IOP in various subtypes of glaucoma. To develop new IOP-lowering treatments, we generated a novel ATX inhibitor as an ophthalmic drug by high-throughput screening, followed by inhibitor optimization. Administration of the optimized ATX inhibitor (Aiprenon) reduced IOP in laser-treated mice exhibiting elevated IOP and higher level of ATX activity in AH and normal mice in vivo. The stimulation of ATX induced outflow resistance in the trabecular pathway; however, administration of Aiprenon recovered the outflow resistance in vitro. The in vitro experiments implied that the IOP-lowering effect of Aiprenon could be correlated with the altered cellular behavior of trabecular meshwork (TM) and Schlemm's canal endothelial (SC) cells. Overall, our findings showed that ATX had major impact in regulating IOP as a target molecule, and potent ATX inhibitors such as Aiprenon could be a promising therapeutic approach for lowering IOP.

Light Stress-Induced Increase of Sphingosine 1-Phosphate in Photoreceptors and Its Relevance to Retinal Degeneration.

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammation and angiogenesis. In this study, we investigated the possible involvement of S1P in the pathology of light-induced retinal degeneration in vivo and in vitro. The intracellular S1P and sphingosine kinase (SphK) activity in a photoreceptor cell line (661W cells) was significantly increased by exposure to light. The enhancement of SphK1 expression was dependent on illumination, and all-trans-retinal significantly promoted SphK1 expression. S1P treatment reduced protein kinase B (Akt) phosphorylation and increased the protein expression of cleaved caspase-3, and induced photoreceptor cell apoptosis. In vivo, light exposure enhanced the expression of SphK1 in the outer segments of photoreceptors. Intravitreal injection of a SphK inhibitor significantly suppressed the thinning of the outer nuclear layer and ameliorated the attenuation of the amplitudes of a-waves and b-waves of electroretinograms during light-induced retinal degeneration. These findings imply that light exposure induces the synthesis of S1P in photoreceptors by upregulating SphK1, which is facilitated by all-trans-retinal, causing retinal degeneration. Inhibition of this enhancement may be a therapeutic target of outer retinal degeneration, including age-related macular degeneration.

Activation of the Sphingosine 1 Phosphate-Rho Pathway in Pterygium and in Ultraviolet-Irradiated Normal Conjunctiva.

Sphingosine 1 phosphate (S1P) is a bioactive lipid that regulates cellular activity, including proliferation, cytoskeletal organization, migration, and fibrosis. In this study, the potential relevance of S1P-Rho signaling in pterygium formation and the effects of ultraviolet (UV) irradiation on activation of the S1P/S1P receptor axis and fibrotic responses were investigated in vitro. Expressions of the S1P2, S1P4, and S1P5 receptors were significantly higher in pterygium tissue than in normal conjunctiva, and the concentration of S1P was significantly elevated in the lysate of normal conjunctival fibroblast cell (NCFC) irradiated with UV (UV-NCFCs). RhoA activity was significantly upregulated in pterygium fibroblast cells (PFCs) and UV-NCFCs, and myosin phosphatase-Rho interacting protein (MRIP) was upregulated, and myosin phosphatase target subunit 1 (MYPT1) was downregulated in PFCs. Fibrogenic changes were significantly upregulated in both PFCs and UV-NCFCs compared to NCFCs. We found that the activation of the S1P receptor-Rho cascade was observed in pterygium tissue. Additionally, in vitro examination showed S1P-rho activation and fibrogenic changes in PFCs and UV-NCFCs. S1P elevation and the resulting upregulation of the downstream Rho signaling pathway may be important in pterygium formation; this pathway offers a potential therapeutic target for suppressing pterygium generation.

Apolipoprotein M Inhibits Angiogenic and Inflammatory Response by Sphingosine 1-Phosphate on Retinal Pigment Epithelium Cells.

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammatory responses and proangiogenic factors. It has been suggested that S1P upregulates choroidal neovascularization (CNV) and may be deeply involved in the pathogenesis of exudative age-related macular degeneration (AMD). Recent studies have suggested that apolipoprotein M (ApoM), a carrier protein for S1P, modulates the biological properties of S1P in the pathogenesis of atherosclerosis. However, the role of ApoM/S1P in AMD has not been explored. We investigated the effect of S1P on proangiogenic factors in human retinal pigment epithelium (RPE) cell lines in vitro. S1P promoted the expression of vascular endothelial growth factor in RPE cells. Hypoxia inducible factor-1α expression was also upregulated. These S1P-induced enhancements in growth factors and chemotactic cytokines in RPE cells were significantly inhibited by ApoM treatment. Additionally, in vivo experiments using a laser-induced CNV murine model demonstrated that intravitreal ApoM injection significantly reduced the progression of CNV formation. Although the detailed mechanisms remain to be elucidated, the present results provide a novel potential therapeutic target for AMD, and demonstrate a suppressive role for ApoM and S1P in the pathology of CNV progression.

[Foreign Body in the Anterior Chamber 10 Months after Uneventful Cataract Surgery].

PURPOSE: A foreign body detected in the anterior chamber 10 months after uneventful sutureless cataract surgery. CASE REPORT: Clear corneal phacoemulsification and intraocular lens placement (PEA + IOL) were performed on a 74-year-old man suffering from vision loss caused by senile cataracts. After uneventful surgery, the postoperative course appeared to be problem-free. OBSERVATION: However, 10 months after the operation, the patient reported blurred vision. Slit-lamp examination revealed an oil droplet, reminiscent of a foreign body, about 1 mm in diameter, on the IOL surface. The visual acuity problem did not become exacerbated and the anterior segment of the eye remained intact. Although we removed the foreign body, qualitative testing was impossible because the body disintegrated. CONCLUSIONS: A rare case of a foreign body in the anterior chamber 10 months after uneventful PEA + IOL. We are of the view that the body may have been composed of ophthalmic ointment delivered from the conjunctival sac at the end of surgery.

A MEK inhibitor arrests the cell cycle of human conjunctival fibroblasts and improves the outcome of glaucoma filtration surgery.

Better agents are needed to improve glaucoma filtration surgery outcomes compared to current ones. The purpose of this study is to determine whether mitogen-activated protein kinase kinase (MEK) inhibitors can effectively arrest the cell cycle of human conjunctival fibroblasts (HCFs) and inhibit the formation of fibrosis and scarring following glaucoma filtration surgery. A cell counting kit­8 assay revealed that the MEK inhibitor PD0325901 exhibited concentration-dependent growth inhibition of HCFs. Quantitative PCR, immunocytochemistry, and western blotting demonstrated decreased expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 and increased expression of p27 in HCFs treated with PD0325901. Flow cytometry indicated that PD0325901 arrested the cell cycle of HCFs in the G0/1 phase. The cell-migration assay showed that HCF migration rate was significantly suppressed by PD0325901 exposure. Rabbits were divided into PD0325901-treatment and control groups, and glaucoma filtration surgery was performed. Although intraocular pressure did not differ between PD0325901-treatment and control groups, bleb height was greater in the treatment group. Histopathological evaluation revealed that fibrotic changes were significantly attenuated in the PD0325901-treatment group compared to the control group. In conclusion, the MEK inhibitor impedes HCF proliferation via cell-cycle arrest and may be beneficial for glaucoma filtration surgery by reducing bleb scarring.

An Autotaxin-Induced Ocular Hypertension Mouse Model Reflecting Physiological Aqueous Biomarker.

Purpose: Animal models of ocular hypertension (OH) have been developed to understand the pathogenesis of glaucoma and facilitate drug discovery. However, many of these models are fraught with issues, including severe intraocular inflammation and technical challenges. Lysophosphatidic acid (LPA) is implicated in trabecular meshwork fibrosis and increased resistance of aqueous outflow, factors that contribute to high intraocular pressure (IOP) in human open-angle glaucoma. We aimed to elevate IOP by increasing expression of the LPA-producing enzyme autotaxin (ATX) in mouse eyes. Methods: Tamoxifen-inducible ATX transgenic mice were developed. Tamoxifen was administered to six- to eight-week-old mice via eye drops to achieve ATX overexpression in the eye. IOP and retinal thickness were measured over time, and retinal flat-mount were evaluated to count retinal ganglion cells (RGCs) loss after three months. Results: Persistent elevation of ATX expression in mouse eyes was confirmed through immunohistochemistry and LysoPLD activity measurement. ATX Tg mice exhibited significantly increased IOP for nearly two months following tamoxifen treatment, with no anterior segment changes or inflammation. Immunohistochemical analysis revealed enhanced expression of extracellular matrix near the angle after two weeks and three months of ATX induction. This correlated with reduced outflow facility, indicating that sustained ATX overexpression induces angle fibrosis, elevating IOP. Although inner retinal layer thickness remained stable, peripheral retina showed a notable reduction in RGC cell count. Conclusions: These findings confirm the successful creation of an open-angle OH mouse model, in which ATX expression in the eye prompts fibrosis near the angle and maintains elevated IOP over extended periods.