TRPV4 is activated by mechanical stimulation to induce prostaglandins release in trabecular meshwork, lowering intraocular pressure.

Trabecular meshwork constitutes the conventional outflow pathway and controls intraocular pressure by regulating aqueous outflow. Mechanical stimulation has been studied as one of the triggers to regulate aqueous outflow in trabecular meshwork, but it is not well understood. We investigated that how transient receptor potential cation channel subfamily V member 4 (TRPV4) functions in human trabecular meshwork cells (HTMC) and affects intraocular pressure (IOP). HTMC were treated with TRPV4 siRNA, followed by incubation for 24 hours. We confirmed the suppression of TRPV4 mRNA expression and the reduction of Ca2+ influx by the TRPV4 agonist GSK1016790A in TRPV4 siRNA-treated HTMC. TRPV4 siRNA-treated HTMC exhibited a significant reduction in Ca2+ influx and production of arachidonic acid and prostaglandin (PG) E2 induced by mechanical stretch, and direct activation of TRPV4 by GSK1016790A increased production of arachidonic acid, PGE2, and PGD2 and inhibited gel contraction. Furthermore, TRPV4-deficient mice had higher IOP than wild-type mice, and GSK1016790A administration lowered IOP. These results suggest that TRPV4 mediates the cellular response induced by trabecular meshwork stretch, leading to IOP reduction through the production of prostaglandins and inhibition of cell contraction. Targeting TRPV4 may have therapeutic benefits that lead to lowering IOP in glaucoma patients.

Mechanical stretch induces Ca2+ influx and extracellular release of PGE2 through Piezo1 activation in trabecular meshwork cells.

The trabecular meshwork (TM) constitutes the main pathway for aqueous humor drainage and is exposed to complex intraocular pressure fluctuations. The mechanism of homeostasis in which TM senses changes in intraocular pressure and leads to normal levels of outflow resistance is not yet well understood. Previous reports have shown that Piezo1, a mechanically-activated cation channel, is expressed in TM and isolated TM cells. Therefore, we tested hypothesis that Piezo1 may function in response to membrane tension and stretch in TM. In human trabecular meshwork (hTM) cells, PIEZO1 was showed to be abundantly expressed, and Piezo1 agonist Yoda1 and mechanical stretch caused a Piezo1-dependent Ca2+ influx and release of arachidonic acid and PGE2. Treatment with Yoda1 or PGE2 significantly inhibited hTM cell contraction. These results suggest that mechanical stretch stimuli in TM activates Piezo1 and subsequently regulates TM cell contraction by triggering Ca2+ influx and release of arachidonic acid and PGE2. Thus, Piezo1 could acts as a regulator of intraocular pressure (IOP) within the conventional outflow pathway and could be a novel therapeutic strategy to modulate IOP in glaucoma patients.

The Neuroprotective Effect of the Adiponectin Receptor Agonist AdipoRon on Glutamate-Induced Cell Death in Rat Primary Retinal Ganglion Cells.

Purpose: To determine whether the adiponectin receptor (AdipoR) agonist AdipoRon inhibits glutamate-induced neuronal cell death and to investigate the neuroprotective mechanism of AdipoRon in rat primary retinal ganglion cells (RGCs). Methods: The expression pattern of AdipoR1 and AdipoR2 in rat retina and primary RGCs was examined by immunostaining. The neuroprotective effect of AdipoRon on glutamate-induced cell death was evaluated in rat primary RGCs. Cellular levels of reactive oxygen species (ROS) were also measured. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), estrogen-related receptor-α (Esrra), mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor α (PPARα), and catalase mRNA levels were examined. Results: The expression of AdipoR1 and AdipoR2 was confirmed in rat retina and primary RGCs. AdipoRon significantly increased the survival rate of glutamate-induced cell death and decreased ROS production. Additionally, the mRNA levels of PGC-1α, Esrra, and TFAM were upregulated by AdipoRon. Conclusions: These results suggest that AdipoRon has a neuroprotective effect by inhibiting ROS production via upregulation of PGC-1α, Esrra, and TFAM against glutamate-induced RGC death.

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.

Interaction Between Pilocarpine and Ripasudil on Intraocular Pressure, Pupil Diameter, and the Aqueous-Outflow Pathway.

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, TGFß2-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 TGFß2 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.

The Anti-Inflammatory Effect of Ripasudil (K-115), a Rho Kinase (ROCK) Inhibitor, on Endotoxin-Induced Uveitis in Rats.

Purpose: To investigate the anti-inflammatory properties of ripasudil, a Rho kinase (ROCK) inhibitor, using endotoxin-induced uveitis (EIU) in rats. Methods: Endotoxin-induced uveitis was induced by footpad injection of lipopolysaccharide (LPS). Ripasudil was administered intraperitoneally 1 hour before and after LPS injection. The aqueous humor was collected 24 hours after injection, and the infiltrating cells, protein concentration, and levels of monocyte chemotactic protein-1 (MCP-1) were determined. Infiltrating cells in the iris ciliary body (ICB) and adherent leukocytes in retinal vessels were evaluated. The mRNA levels of IL-1ß, IL-6, TNF-α, and MCP-1 in the retina and ICB were determined. A mouse macrophage cell line, RAW264.7, was stimulated with LPS in the presence or absence of ripasudil, and the expression of MCP-1 and nuclear translocation of nuclear factor (NF)-κB was analyzed. Results: Ripasudil significantly reduced infiltrating cells and protein exudation in the aqueous humor, as well as the number of infiltrating cells in the ICB and adherent leukocytes in retinal vessels in EIU. Additionally, the protein level of MCP-1 in the aqueous humor and mRNA levels of IL-1ß, IL-6, TNF-α, MCP-1, and intercellular adhesion molecule-1 in the ICB and retina were suppressed by ripasudil. The production of MCP-1 and nuclear translocation of NF-κB in RAW264.7 cells were also suppressed by ripasudil. Conclusions: The Rho/ROCK pathway plays a role in adhesion molecule expression and inflammatory cell infiltration in EIU, and ripasudil is a potent anti-inflammatory agent against ocular inflammatory diseases, including acute uveitis and possibly uveitic glaucoma.

Sphingosine-1-Phosphate (S1P)-Related Response of Human Conjunctival Fibroblasts After Filtration Surgery for Glaucoma.

Purpose: To investigate levels of sphingosine-1-phosphate (S1P) in aqueous fluid samples taken before and after filtration surgery and S1P-induced human conjunctival fibroblast (HCF) responses. Methods: Levels of S1P and its related sphingophospholipids in aqueous fluid obtained immediately before and after filtration surgery were determined by liquid chromatography-tandem mass spectrometry. HCFs were used for all in vitro experiments. The expression of five S1P receptor subtypes in HCFs was examined by quantitative real-time PCR. The effect of S1P and receptor-specific antagonists on HCF viability and cell migration was assessed by WST-1 assay and scratch migration assay, respectively. Differentiation to myofibroblasts and extracellular matrix production was evaluated by examining changes in F-actin, α-smooth muscle actin (αSMA), and collagen expression with immunocytochemistry, Western blotting, and collagen accumulation assay, respectively. Results: No significant S1P levels in the aqueous fluid samples were detectable immediately before surgery, but postoperative levels of several lysophospholipids, including S1P, dehydro-S1P, and sphingosine, were significantly increased to bioactive concentrations in aqueous fluid in the blebs (P < 0.0001). mRNA expression of the three main S1P receptor subtypes was detected in HCFs. Although S1P levels did not influence HCF proliferation, S1P enhanced cell migration, which could be inhibited by the S1P2 antagonist JTE 013. F-actin, αSMA, and collagen expression was significantly increased by S1P stimulation and was reduced by JTE 013. Conclusions: Bioactive S1P concentrations were present in the aqueous fluid at the end of filtration surgery. S1P activated HCFs via S1P2 receptors. These results revealed the potential of S1P2 antagonists in preventing scarring after glaucoma filtration surgery.

Role of Glutathione Peroxidase 4 in Corneal Endothelial Cells.

PURPOSE: To investigate the role of glutathione peroxidase 4 (GPx4) in corneal endothelial cells. MATERIALS AND METHODS: An immortalized human corneal endothelial cell line was used. Cells were transfected with either siRNA specifically silencing GPx4 or scrambled control siRNA. Knockdown was confirmed by Real-time RT-PCR and immunoblotting. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal immunostaining. Cytotoxicity, cell death, and cell proliferation were evaluated using a lactate dehydrogenase (LDH) activity assay, Annexin V staining, and WST-8, respectively. Furthermore, cells transfected with GPx4 siRNA or control siRNA were treated with hydrogen peroxide or ferrous sulfate, and cytotoxicity was evaluated using the LDH activity assay. RESULTS: The treatment of siRNA decreased the expression of GPx4 at both mRNA and protein levels. The knockdown of GPx4 significantly increased the levels of lipid oxidation and LDH activity. Annexin V-positive cells increased in GPx4 siRNA-treated cells. The proliferation of GPx4 siRNA-treated cells was downregulated compared with that of control siRNA-treated cells. GPx4 knockdown enhanced hydrogen peroxide- and ferrous sulfate-induced cytotoxicity. CONCLUSION: These results suggest that GPx4 is an important antioxidant enzyme for maintaining redox status and protecting corneal endothelial cells from oxidative stress.

Glutathione peroxidase 4 plays an important role in oxidative homeostasis and wound repair in corneal epithelial cells.

Oxidative stress is involved in the pathologies of corneal epithelial cells. However, the importance of specific antioxidant enzymes in corneal epithelial cells is not fully understood. The purpose of this study is to elucidate the role of glutathione peroxidase 4 (GPx4) in corneal epithelial cells. For in vitro experiments, an immortalized human corneal epithelial cell line was used. Cytotoxicity measured through LDH activity, lipid peroxidation immunostained for 4-hydroxynonenal, cell viability, and cell death were compared between cells transfected with either GPx4 siRNA or scrambled control siRNA. In addition, the rescue effects of α-tocopherol and ferrostatin-1, a ferroptosis inhibitor, were examined in the cells with deficient GPx4 expression. For in vivo experiments, we applied n-heptanol on the cornea of GPx4+/+ and GPx4+/- mice to create corneal epithelial wound. The epithelial defect area size was measured up to 48 h after epithelial wound creation. Knockdown of GPx4 strongly induced cytotoxicity and cell death in human corneal epithelial cells. Cell death induced by GPx4 knockdown was characterized by positive staining for both annexin V and propidium iodide, nuclear translocation of AIF, and without activation of caspase 3, and was rescued by α-tocopherol and ferrostatin-1. The delayed wound healing of GPx4 siRNA-transfected cells were ameliorated by α-tocopherol in vitro. In addition, loss of one GPx4 allele was sufficient to significantly delay the healing of experimental corneal epithelial wounds in vivo. Our results suggest that the antioxidant enzyme GPx4 plays an important role in oxidative homeostasis, cell survival, and wound healing in corneal epithelial cells.

Role of Glutathione Peroxidase 4 in Glutamate-Induced Oxytosis in the Retina.

PURPOSE: The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in glutamate-induced oxytosis in the retina. METHODS: For in vitro studies, an immortalized rat retinal precursor cell line R28 was used. Cells were transfected with siRNA specifically silencing GPx4 or with scrambled control siRNA. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal (4-HNE) immunostaining. Cytotoxicity and cell death were evaluated using an LDH activity assay and annexin V staining, respectively. Cells transfected with GPx4 siRNA or control siRNA were treated with glutamate (1 or 2 mM), and the cytotoxicity was evaluated using the LDH activity assay. For in vivo studies, retinal ganglion cell damage was induced by intravitreal injection of 25-mM N-methyl-D-aspartate (NMDA, 2 µL/eye) in GPx4+/+ and GPx4+/- mice. The evaluation of lipid peroxidation (4-HNE immunostaining), apoptosis (TUNEL staining), and cell density in the ganglion cell layer (GCL) were performed at 12 h, 1 day, and 7 days after the NMDA injection. RESULTS: GPx4 knockdown significantly increased LDH activity by 13.9-fold (P < 0.01) and increased peroxidized lipid levels by 3.2-fold in R28 cells (P < 0.01). In cells transfected with scrambled control siRNA, treatment with glutamate at 1 or 2 mM did not increase LDH activity; whereas, in cells transfected with GPx4 siRNA, glutamate treatment significantly increased LDH activity (1.52-fold, P < 0.01). GPx4+/- mice exhibited higher levels of lipid peroxidation in retinas treated with NMDA than GPx4+/+ mice (1.26-fold, P < 0.05). GPx4+/- mice had more TUNEL-positive cells induced by NMDA in GCL (1.45-fold, P < 0.05). In addition, the cell density in GCL of GPx4+/- mice was 19% lower than that in GPx4+/+ mice after treatment with NMDA (P < 0.05). CONCLUSION: These results suggest that defective GPx4 expression is associated with enhanced cytotoxicity by glutamate-induced oxytosis in the retina.

Role of glutathione peroxidase 4 in conjunctival epithelial cells.

PURPOSE: The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in conjunctival epithelial cells. METHODS: An immortalized human conjunctival epithelial cell line was used. Cells were transfected with catalase, GPx1, GPx4, SOD1, SOD2, or control siRNA. Knockdown was confirmed by RT-PCR and immunoblotting. The cytotoxicity induced by knockdown of these antioxidant enzymes was examined by assay of LDH activity. Furthermore, evaluations of lipid peroxidation, cellular levels of reactive oxygen species, cell proliferation, and apoptosis were conducted in cells treated with GPx4 or control siRNA. In oxidative stress study, cells treated with GPx4 or control siRNA were applied with hydrogen peroxide or ferric sulfide, and their cytotoxicity was evaluated by assay of LDH activity. RESULTS: Small interfering RNA of catalase, GPx1, GPx4, SOD1, and SOD2 siRNA remarkably inhibited the mRNA and protein expression of each gene. Knockdown of GPx4 and SOD1 but not catalase, GPx1, and SOD2 significantly induced cytotoxicity. Glutathione peroxidase 4 knockdown increased lipid oxidation and reactive oxygen species. The proliferation of GPx4 siRNA-treated cells was reduced compared with control siRNA-treated cells. Moreover, cell death in GPx4 siRNA-treated cells was characterized by positive staining for annexin V. In an oxidation stress study, GPx4 siRNA knockdown enhanced the cytotoxicity induced by hydrogen peroxide or ferric sulfide. CONCLUSION: These results suggest that GPx4 is essential for maintaining oxidative homeostasis and keeping defense against oxidative stress in conjunctival epithelial cells.

Evaluation of zinc (II) chelators for inhibiting p53-mediated apoptosis.

In a previous study, we reported that sodium orthovanadate (vanadate) is the first known inhibitor that is capable of protecting mice from death from the radiation-induced gastrointestinal syndrome via its ability to block both transcription-dependent and transcription-independent p53 apoptotic pathways. In this paper, we report that vanadate has a unique activity for inducing the denaturation of p53 relative to other known radioprotective p53 inhibitors, pifithrin-α (PFTα) and pifithrin-µ (PFTµ). This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation. Based on the fact that p53 denaturation can be induced by the dissociation of a zinc ion, which is used as a structural factor of p53, we screened some zinc (II) chelators for the suppression of the DNA binding activity of p53 in vitro and the inhibition of radiation-induced p53-dependent apoptosis in MOLT-4 cells. The findings indicate that two of five zinc (II) chelators also suppressed apoptosis. Among the inhibitors tested, Bispicen (N,N'-Bis(2-pyridylmethyl)-1,2-ethanediamine) had the highest inhibition activity. A mechanistic study using cells bearing different p53 status or functions (i.e., p53-knockdown MOLT-4 transformant and its revertants, p53 mutant cells, p53-null cells), and p53-independent apoptotic stimuli revealed that the suppressive effect of Bispicen on apoptosis is specifically mediated through p53. Moreover, Bispicen, similar to vanadate, induces the denaturation of p53 as well as the blocking of both transcription-dependent and -independent apoptotic pathways. Our findings indicate that the use of zinc (II) chelators represent a new approach for protecting against radiation-induced p53-dependent apoptosis through the inhibition of p53-dependent apoptotic pathways.

A case with tracheo-innominate artery fistula. Successful management of endovascular embolization of innominate artery.

Tracheo-innominate artery fistula (TIF) is known as a fatal complication after tracheostomy. We report a 9-year-old girl with early hypoxic encephalopathy who had a tracheo-innominate artery fistula with exsanguinating hemorrhage from her tracheostoma 10 months after tracheostomy. After temporary control of bleeding, embolization of the innominate artery was performed. The patient has remained well 1 year after the procedure. We reviewed the aetiology, diagnosis and management of the tracheo-innominate fistula, and findings suggest that endovascular embolization of the innominate artery may be an appropriate treatment for patients with tracheo-innominate artery fistula.