Teppei Shibata

FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

Transforming growth factor (TGF) β2 and fibroblast growth factor (FGF) 2 are involved in regulation of posterior capsule opacification (PCO) and other processes of epithelial–mesenchymal transition (EMT) such as cancer progression, wound healing and tissue fibrosis as well as normal embryonic development. We previously used an in vivo rodent PCO model to show the expression of tropomyosin (Tpm) 1/2 was aberrantly up‐regulated in remodelling the actin cytoskeleton during EMT. In this in vitro study, we show the Tpms family of cytoskeleton proteins are involved in regulating and stabilizing actin microfilaments (F‐actin) and are induced by TGFβ2 during EMT in lens epithelial cells (LECs). Importantly, we found TGFβ2 and FGF2 played contrasting roles. Stress fibre formation and up‐regulation of α‐smooth muscle actin (αSMA) induced by TGFβ2 could be reversed by Tpm1/2 knock‐down by siRNA. Expression of Tpm1/2 and stress fibre formation induced by TGFβ2 could be reversed by FGF2. Furthermore, FGF2 delivery to TGFβ‐treated LECs perturbed EMT by reactivating the mitogen‐activated protein kinase (MAPK)/ extracellular signal‐regulated kinase (ERK) pathway and subsequently enhanced EMT. Conversely, MEK inhibitor (PD98059) abated the FGF2‐mediated Tpm1/2 and αSMA suppression. However, we found that normal LECs which underwent EMT showed enhanced migration in response to combined TGFβ and FGF2 stimulation. These findings may help clarify the mechanism reprogramming the actin cytoskeleton during morphogenetic EMT cell proliferation and fibre regeneration in PCO. We propose that understanding the physiological link between levels of FGF2, Tpm1/2 expression and TGFβs‐driven EMT orchestration may provide clue(s) to develop therapeutic strategies to treat PCO based on Tpm1/2.

The role of Prdx6 in the protection of cells of the crystalline lens from oxidative stress induced by UV exposure

PurposeThe immediate aim of this study was to investigate alterations in peroxiredoxin (Prdx) 6 at posttranslational levels, and the levels of protein oxidation, lipid peroxidation, and reactive oxygen species (ROS) in lens epithelial cells (LECs) after exposure to severe oxidative stress, such as ultraviolet-B (UV-B). Our ultimate aim was to provide new information on antioxidant defenses in the lens and their regulation, thereby broadening existing knowledge of the role of Prdx6 in lens physiology and pathophysiology.MethodsThe expression of the hyperoxidized form of Prdx6 and oxidation of protein were analyzed by western blotting and the OxyBlot assay in human LECs (hLECs). ROS levels were quantified using DCFH-DA dye, and cell viability was quantified by the MTS and TUNEL assays. To evaluate the protective effect of Prdx6, we cultured lenses with or without the TAT transduction domain (TAT-HA-Prdx6) and observed (and photographed) the cultures at specified time-points after the exposure to UV-B for the development of opacity.ResultsPrdx6 in hLECs was hyperoxidized after exposure to high amounts of UV-B. UV-B treatment of hLECs increased the levels of cell death, protein oxidation, and ROS. hLECs exposed to UV-B showed higher levels of ROS, which could be reduced by the application of extrinsic TAT-HA-Prdx6, attenuating UV-B-induced lens opacity and apoptotic cell death.ConclusionExcessive oxidative stress induces the hyperoxidation of Prdx6 and may reduce the ability of Prdx6 to protect LECs against ROS or stresses. Because extrinsic Prdx6 could attenuate UV-B-induced abuse, this molecule may have a potential in preventing cataractogenesis.

A positive feedback loop between nitric oxide and amyloid β (1-42) accelerates mitochondrial damage in human lens epithelial cells

We have reported that excessive nitric oxide (NO), like other reactive oxygen species (ROS), causes a decrease in cytochrome c oxidase (CCO) activity and ATP levels (mitochondrial damage) resulting in lens opacity. In addition, previous reports have shown that oxidative stress caused by ROS enhances amyloid β (Aβ) production in mammalian lenses, and that Aβ1-42 stimulates inducible nitric oxide synthase (iNOS) promoter activity. Based on these reports, we investigated the relationship between NO and Aβ1-42 production in human lens epithelial (HLE) cells. iNOS was induced by the co-incubation of HLE cells with 1000 IU interferon-γ (IFN-γ) and 100ng/ml lipopolysaccharide (LPS) for 48h. This led to enhanced NO release, an increase in the gene expression levels of proteins related to Aβ production, and the cellular accumulation of Aβ1-42. Moreover, both aminoguanidine (AG, a selective inhibitor of iNOS) and diethyldithiocarbamate (DDC, a nuclear factor-kappa B (NFκB) inhibitor) attenuated these changes in IFN-γ and LPS stimulated HLE cells. Based on our finding that Aβ1-42 accumulation is induced by co-incubation of HLE cells with both IFN-γ and LPS, we prepared a HLE cell model with Aβ1-42 accumulation (Aβ-accumulated-HLE cell model) by pre-stimulating cells with IFN-γ and LPS for 48h. Aβ1-42 accumulation caused NO production via iNOS, resulting in an enhancement in the mRNA levels for enzymes necessary for the proteolysis of amyloid precursor protein (APP) to Aβ in HLE cells. In addition, excessive NO produced in response to Aβ1-42 accumulation led to a decrease in CCO activity and ATP levels. Taken together, we hypothesize that excessive NO production in the lens epithelium enhances Aβ1-42 production, and that this enhancement accelerates NO release. The enhancement in NO production in the lens epithelium based on positive feedback (NO-Aβ positive feedback loop, a vicious cycle) may promote the onset of cataracts (lens opacification) via the decrease in CCO activity and ATP levels. These findings provide significant information that can be used to design further studies aimed at developing anti-cataract drugs.

In vitro and in vivo corneal effects of latanoprost combined with brimonidine, timolol, dorzolamide, or brinzolamide

To examine the relevance of concentration of benzalkonium chloride (BAK) on the cornea, we investigated the effects of latanoprost containing BAK alone and in combination with other antiglaucoma drug classes on corneal epithelium in vitro in a cultured rabbit corneal cell line (SIRC) and in vivo, using a corneal resistance device (CRD). [In vitro] statens seruminstitut rabbit corneal cells were exposed to 0.005% latanoprost for 30s, followed by either phosphate buffered saline (control), 0.1% brimonidine, 0.5% timolol, 1% dorzolamide, or 1% brinzolamide. The number of viable cells was counted at 8, 15, and 30min. [In vivo] Albino rabbits were administered one drop of 0.005% latanoprost, followed 5min later by one drop of an agent from the in vitro trial. This was repeated every 15min for a total of three times. The change in corneal barrier function was assessed by measuring the corneal resistance at 2 and 30min after the final administration. [In vitro] At 8min, the viable cell count in the latanoprost+dorzolamide group was significantly lower than in the control group. At 15 and 30min, all treatment groups, except the latanoprost+brimonidine group, demonstrated significantly lower viable cell counts than the control group. [In vivo] At 2min after the final eye drop, the latanoprost+timolol group and the latanoprost+brinzolamide group demonstrated significantly lower corneal resistance than did the latanoprost+brimonidine group. No significant difference was observed between the agents at 30min. In conclusion, when combining latanoprost containing benzalkonium chloride with other classes of antiglaucoma drugs, brimonidine may cause the least corneal damage, and the number of drug administrations may be an important factor.

Tropomyosin 2 heterozygous knockout in mice using CRISPR-Cas9 system displays the inhibition of injury-induced epithelial-mesenchymal transition, and lens opacity

The process of epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) after cataract surgery contributes to tissue fibrosis, wound healing and lens regeneration via a mechanism not yet fully understood. Here, we show that tropomyosin 2 (Tpm2) plays a critical role in wound healing and lens aging. Posterior capsular opacification (PCO) after lens extraction surgery was accompanied by elevated expression of Tpm2. Tpm2 heterozygous knockout mice, generated via the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system showed promoted progression of cataract with age. Further, injury-induced EMT of the mouse lens epithelium, as evaluated histologically and by the expression patterns of Tpm1 and Tpm2, was attenuated in the absence of Tpm2. In conclusion, Tpm2 may be important in maintaining lens physiology and morphology. However, Tpm2 is involved in the progression of EMT during the wound healing process of mouse LECs, suggesting that inhibition of Tpm2 may suppress PCO.

An Adult Case of Fulminant Mumps Keratitis With Positive Viral RNA in Aqueous Humor Detected by RT-PCR.

Purpose: To report an adult case of mumps keratitis with mumps virus in aqueous humor and decreased corneal endothelial cell density. Methods: Case report. Results: A 60-year-old female with a 39°C fever and bilateral parotid swelling diagnosed with mumps and treated for photophobia, pain, redness, and decreased vision in 1 eye, was referred to our hospital when her condition deteriorated despite receiving betamethasone phosphate instillation and antiglaucoma agents for elevated intraocular pressure (52 mm Hg) and iritis. Her right eye was normal, whereas her left eye showed 20/400 visual acuity, 21 mm Hg intraocular pressure, ciliary injection and edema, opacity, and Descemet folds in the entire cornea. Round white keratic precipitates were present on the posterior corneal surface, whereas anterior chamber cells could not be examined in detail because of corneal edema. Mumps virus was detected by reverse transcriptase polymerase chain reaction in an aqueous humor sample taken at the time of admission. Following diagnosis of keratitis, administration of 30 mg oral prednisolone daily and frequent instillation of betamethasone phosphate steadily improved her corneal edema and opacity. In her left eye, visual acuity recovered to 20/16 and keratitis was resolved at 4 weeks; however, corneal endothelial cell density was significantly decreased to less than 400 per square millimeter. Conclusions: Mumps keratitis may cause severe corneal endothelial cell loss.

Roles of TGF β and FGF Signals in the Lens: Tropomyosin Regulation for Posterior Capsule Opacity

Transforming growth factor (TGF) β and fibroblast growth factor (FGF) 2 are related to the development of posterior capsule opacification (PCO) after lens extraction surgery and other processes of epithelial–mesenchymal transition (EMT). Oxidative stress seems to activate TGF β1 largely through reactive oxygen species (ROS) production, which in turn alters the transcription of several survival genes, including lens epithelium-cell derived growth factor (LEDGF). Higher ROS levels attenuate LEDGF function, leading to down-regulation of peroxiredoxin 6 (Prdx6). TGF β is regulated by ROS in Prdx6 knock-out lens epithelial cells (LECs) and induces the up-regulation of tropomyosins (Tpms) 1/2, and EMT of LECs. Mouse and rat PCO are accompanied by elevated expression of Tpm2. Further, the expression of Tpm1/2 is induced by TGF β2 in LECs. Importantly, we previously showed that TGF β2 and FGF2 play regulatory roles in LECs in a contrasting manner. An injury-induced EMT of a mouse lens as a PCO model was attenuated in the absence of Tpm2. In this review, we present findings regarding the roles of TGF β and FGF2 in the differential regulation of EMT in the lens. Tpms may be associated with TGF β2- and FGF2-related EMT and PCO development.

Amyloid β1–43 Accumulates in the Lens Epithelium of Cortical Opacification in Japanese Patients

Purpose We investigated the accumulation of amyloid β (Aβ1-40, Aβ1-42, Aβ1-43) in the lens epithelium of patients with opacification of five different types (cortical cataract [COR]; nuclear cataract [NUC]; posterior subcapsular cataract [PSC]; retrodots [RD]; and water clefts [WC]). Methods Samples were collected from Japanese patients taken during cataract surgery; Aβ levels and mRNA expression were determined by ELISA and a real-time RT-PCR method, respectively. Results Levels of Aβ1-40 and Aβ1-42 in the lens epithelium of patients with COR, NUC, PSC, RD, and WC showed no significant differences in comparison with transparent lens epithelium. Levels of Aβ1-43 in the lens epithelium of patients with PSC and WC were not detected, and NUC and RD were slightly elevated. In contrast to the results in these cataract types, high Aβ1-43 levels were observed in the lens epithelium of patients with COR, and a close relationship was observed between Aβ1-43 levels and the degree of lens opacification (R = 0.8229, n = 6). The levels of Aβ1-43 were also higher in the lens epithelium of patients with mixed-cataract showing cortical opacification, and the Aβ1-43 levels in the lens epithelium of mixed-cataract patients with cortical opacification was significantly higher than in that of mixed-cataract patients without cortical opacification. In addition, the level of an amyloid precursor protein mRNA in the lens epithelium of mixed-cataract patients with cortical opacification was significantly higher than in transparent lens and mixed-cataract patients without cortical opacification. Conclusions We found high levels of Aβ1-43 accumulation in the lens epithelium of Japanese patients with cortical opacification.