Masaki Tanito

Redox-Sensing Release of Human Thioredoxin from T Lymphocytes with Negative Feedback Loops

Thioredoxin (TRX) is released from various types of mammalian cells despite no typical secretory signal sequence. We show here that a redox-active site in TRX is essential for its release from T lymphocytes in response to H2O2 and extracellular TRX regulates its own H2O2-induced release. Human T cell leukemia virus type I-transformed T lymphocytes constitutively release a large amount of TRX. The level of TRX release is augmented upon the addition of H2O2, but suppressed upon the addition of N-acetylcysteine. In the culture supernatant of a Jurkat transfectant expressing the tagged TRX-wild type (WT), the tagged TRX protein is rapidly released at 1 h and kept at a constant level until 6 h after the addition of H2O2. In contrast, another type of transfectant expressing the tagged TRX mutant (C32S/C35S; CS) fails to release the protein. H2O2-induced release of TRX from the transfectant is inhibited by the presence of rTRX-WT in a dose-dependent manner. Preincubation of the transfectant with rTRX-WT for 1 h at 37°C, but not 0°C, results in a significant suppression of the TRX release, reactive oxygen species, and caspase-3 activity induced by H2O2, respectively. Confocal microscopy and Western blot analysis show that extracellular rTRX-WT added to the culture does not obviously enter T lymphocytes until 24 h. These results collectively suggest that the oxidative stress-induced TRX release from T lymphocytes depends on a redox-sensitive event and may be regulated by negative feedback loops using reactive oxygen species-mediated signal transductions.

Multiplex Cytokine Analysis of Aqueous Humor in Eyes with Primary Open-Angle Glaucoma, Exfoliation Glaucoma, and Cataract

PURPOSE To measure levels of various inflammatory cytokines in the aqueous humor of patients with primary open-angle glaucoma (POAG), exfoliation glaucoma (EXG), and senile cataract. METHODS Aqueous humor samples were obtained from 64 eyes of 64 Japanese subjects (POAG, 20 eyes; EXG, 23 eyes; and cataract, 21 control eyes). The levels of eight cytokines including interleukin (IL)1-β, IL-6, IL-8, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, serum amyloid A (SAA), migration inhibitory factor (MIF), and vascular endothelial growth factor (VEGF)-A were estimated using the multiplex bead immunoassay technique. RESULTS Compared with the cataract group, the levels of TGF-β1, IL-8, and SAA were significantly higher in aqueous humor samples from the POAG (5.0-fold, 2.3-fold, and 11.9-fold, respectively) and EXG (12.5-fold, 4.0-fold, and 18.3-fold, respectively) groups. Except for a significant decrease in the IL-6 level in the POAG (0.23-fold) group, no other cytokine levels differed in the POAG and EXG groups compared with the cataract group. The levels of TGF-β1, IL-8, and SAA were positively correlated with each other (ρ = 0.723-0.786; P < 0.0001), the intraocular pressure (IOP) (ρ = 0.392-0.662; P < 0.0001-0.0019), and the number of glaucoma medications (ρ = 0.478-0.659; P < 0.0001-0.0001). CONCLUSIONS Cytokine networks including TGF-β1, IL-8, and SAA in aqueous humor may have critical roles in IOP elevations in patients with open-angle glaucoma.

Cytoprotective Effects of Geranylgeranylacetone against Retinal Photooxidative Damage

Exposure to excessive light induces retinal photoreceptor cell damage, leading to development and progression of various retinal diseases. We tested the effect of geranylgeranylacetone (GGA), an acyclic polyisoprenoid, on light-induced retinal damage in mice. Oral treatment with GGA (1.0 mg/d) for 5 d induced thioredoxin (Trx) and heat shock protein 72 (Hsp72) predominantly in the retinal pigment epithelium (RPE). After white light exposure (8000 lux for 2 h), the percentage of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive photoreceptor cells decreased significantly at 24 and 96 h, and the number of photoreceptor cell nuclei at 96 h and the electroretinographic amplitudes of the a- and b-waves at 4 and 10 d increased significantly in GGA-pretreated mice compared with saline-pretreated mice. Light-induced upregulations of 8-hydroxy-2-deoxyguanosine and 4-hydroxy-2-nonenal-modified protein, markers of oxidative stress, were inhibited by GGA pretreatment. To elucidate the cytoprotective mechanism of GGA and Trx, we used human K-1034 RPE cells and mouse photoreceptor-derived 661W cells. In K-1034 cells, GGA (10 μm) induced intracellular Trx, Hsp72, and extracellular Trx but not extracellular Hsp72. Extracellular Trx (0.75 nm) attenuated H2O2 (200 μm)-induced cell damage in 661W cells. Pretreatment with GGA and overexpression of Trx in K-1034 cells counteracted H2O2 (50 μm)-induced attenuation of cellular latex bead incorporation. Protection of phagocytotic activity through induction of Trx and possibly Hsp72 in RPE cells and elimination of oxidative stress in the photoreceptor layer through release of Trx from RPE cells may be mechanisms of GGA-mediated cytoprotection. Therefore, Trx is a neurotrophic factor released from RPE cells and plays a crucial role in maintaining photoreceptor cell integrity.

Thioredoxin-1 Ameliorates Myosin-Induced Autoimmune Myocarditis by Suppressing Chemokine Expressions and Leukocyte Chemotaxis in Mice

Background—Cardiac myosin–induced myocarditis is an experimental autoimmune myocarditis (EAM) model used to investigate autoimmunological mechanisms in inflammatory heart diseases and resembles fulminant myocarditis in humans. We investigated the therapeutic role of thioredoxin-1 (TRX-1), a redox-regulatory protein with antioxidant and antiinflammatory effects, in murine EAM. Methods and Results—EAM was generated in 5-week-old male BALB/c mice by immunization with porcine cardiac myosin at days 0 and 7. Recombinant human TRX-1 (rhTRX-1), C32S/C35S mutant rhTRX-1, or saline was administered intraperitoneally every second day from day 0 to 20. In addition, rabbit anti-mouse TRX-1 serum or normal rabbit serum was administered intraperitoneally on days −1, 2, and 6. Animals were euthanized on day 21. Histological analysis of the heart showed that TRX-1 significantly reduced the severity of EAM, whereas mutant TRX-1 failed to have such an effect, and anti–TRX-1 antibody enhanced the disease markedly. Immunohistochemical analysis showed that TRX-1 significantly suppressed cardiac macrophage inflammatory protein (MIP)-1&agr;, MIP-2, and 8-hydroxydeoxyguanosine expression and macrophage infiltration into the heart in EAM. Although serum levels of MIP-1&agr; were not suppressed by TRX-1 until day 21, both an in vitro chemotaxis chamber assay and an in vivo air pouch model showed that TRX-1 significantly suppressed MIP-1&agr;– or MIP-2–induced leukocyte chemotaxis. However, real-time reverse transcription–polymerase chain reaction showed that TRX-1 failed to decrease chemokine receptor expression increased in the bone marrow cells of EAM mice. Conclusions—TRX-1 attenuates EAM by suppressing chemokine expressions and leukocyte chemotaxis in mice.

Macular Carotenoid Levels of Normal Subjects and Age-Related Maculopathy Patients in a Japanese Population

PURPOSE Macular carotenoid pigments composed of lutein and zeaxanthin are thought to affect the development of age-related maculopathy (ARM). Macular carotenoid levels were measured in normal Japanese subjects and Japanese patients with ARM. DESIGN Observational case-control series. PARTICIPANTS One hundred normal eyes of 100 normal subjects and 187 eyes of 97 patients with ARM; all were Japanese. The definitions of early ARM and late ARM (exudative age-related macular degeneration [AMD] and dry AMD) were used according to an accepted international classification system. METHODS Macular carotenoid levels were measured using resonance Raman spectroscopy. MAIN OUTCOME MEASURE Raman signal intensity generated from carbon-carbon double bond vibrations of lutein and zeaxanthin. RESULTS The mean (+/-standard deviation [SD]) macular carotenoid level in normal subjects was 1471+/-540 Raman counts. The macular carotenoid levels in normal subjects declined with age. The mean macular carotenoid level was 620+/-204 (+/-SD) in eyes with early ARM and 427+/-283 (+/-SD) in eyes with late ARM (equal to AMD). The macular carotenoid levels of early ARM and AMD were significantly lower than those in normal subjects older than 60 years (1100+/-340 [+/-SD]). No difference was revealed in carotenoid levels by the severity for ARM, type of AMD (exudative, atrophic, and disciform scar), or types of choroidal neovascularization (classic, minimally classic, occult, polypoidal choroidal vasculopathy), although small numbers in some groups weakened statistical power. Macular carotenoid levels were affected by the severity of macular disease in the opposing eye. The average for normal eyes where AMD was found in the opposite eye was significantly lower than that of normal eyes in the absence of AMD in the opposite eye (i.e., healthy volunteers older than 60 years). CONCLUSIONS Macular carotenoids decreased even in older healthy individuals. The ARM patients showed lower macular carotenoid levels than healthy people. Low macular carotenoid levels may be one of the risk factors of progression in ARM.

Thioredoxin suppresses 1-methyl-4-phenylpyridinium-induced neurotoxicity in rat PC12 cells

Thioredoxin (TRX) is a redox-active protein which plays a cytoprotective role against oxidative stress. Geranylgeranylacetone (GGA), used widely as an anti-ulcer drug, has been reported to induce TRX as well as heat shock protein 70 (HSP70) in hepatocytes and other cells. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes dopaminergic denervation and Parkinsonism in humans. The 1-methyl-4-phenylpyridinium ion (MPP(+)), an active metabolite of MPTP, induces cell death in a rat pheochromocytoma cell line (PC12 cells). We found that MPP(+) suppresses TRX expression in PC12 cells. Overexpression or administration of TRX attenuates MPP(+)-induced neurotoxicity on PC12 cells. Moreover, GGA induces expression of TRX and HSP70 and attenuates MPP(+)-induced toxicity in PC12 cells. These results indicate that TRX and GGA have a possible potential as new therapeutic agents for Parkinson disease.

Loss of Neuroprotective Survival Signal in Mice Lacking Insulin Receptor Gene in Rod Photoreceptor Cells

Insulin receptor (IR) signaling provides a trophic signal for transformed retinal neurons in culture, but the role of IR activity in vivo is unknown. We previously reported that light causes increased tyrosine phosphorylation of the IR in vivo, which leads to the downstream activation of the phosphoinositide 3-kinase and Akt pathway in rod photoreceptor cells. The functional role of IR in rod photoreceptor cells is not known. We observed that light stress induced tyrosine phosphorylation of the IR in rod photoreceptor cells, and we hypothesized that IR activation is neuroprotective. To determine whether IR has a neuroprotective role on rod photoreceptor cells, we used the Cre/lox system to specifically inactivate the IR gene in rod photoreceptors. Rod-specific IR knock-out mice have reduced the phosphoinositide 3-kinase and Akt survival signal in rod photoreceptors. The resultant mice exhibited no detectable phenotype when they were raised in dim cyclic light. However, reduced IR expression in rod photoreceptors significantly decreased retinal function and caused the loss of photoreceptors in mice exposed to bright light stress. These results indicate that reduced expression of IR in rod photoreceptor cells increases their susceptibility to light-induced photoreceptor degeneration. These data suggest that the IR pathway is important for photoreceptor survival and that activation of the IR may be an essential element of photoreceptor neuroprotection.

Attenuation of retinal photooxidative damage in thioredoxin transgenic mice

Thioredoxin (TRX) is an endogenous redox (reduction/oxidation) regulator that has cytoprotective effects against various types of oxidative stresses. Exposure to excessive levels of white light induces retinal photoreceptor damage. To test the cytoprotective effect of overexpressed TRX against retinal photooxidative damage, both TRX transgenic (trx-tg) mice and C57BL/6 (wild type) mice were exposed to intense white fluorescent light. The amounts of oxidized and tyrosine-phosphorylated proteins decreased in the neural retinas of the trx-tg mice compared to the wild type mice after light exposure. The electroretinographic amplitudes were higher and the formation of oxidized DNA was lower in trx-tg mice compared to wild type mice after light exposure. These results suggest that overexpression of TRX suppresses retinal photooxidative damage. TRX intensification may be a useful therapeutic strategy to prevent retinal photic injury.

Delay of photoreceptor degeneration in tubby mouse by sulforaphane

In this study, the homozygous tubby (tub/tub) mutant mouse, with an early progressive hearing loss and photoreceptor degeneration, was used as a model system to examine the effects of systemic administration of a naturally occurring isothiocyanate, sulforaphane (SF), on photoreceptor degeneration. Several novel observations have been made: (i) the mRNA and protein expression of thioredoxin (Trx), thioredoxin reductase (TrxR) and NF‐E2‐related factor‐2 (Nrf2) were significantly reduced even prior to photoreceptor cell degeneration in the retinas of tub/tub mice, suggesting that retinal expression of the Trx system is impaired and that Trx regulation is involved in the pathogenesis of retinal degeneration in this model, (ii) intraperitoneal injection with SF significantly up‐regulated retinal levels of Trx, TrxR, and Nrf2, and effectively protected photoreceptor cells in tub/tub mice as evaluated functionally by electroretinography and morphologically by quantitative histology, and (iii) treatment with PD98059, an inhibitor of extracellular signal‐regulated kinases (ERKs), blocked SF‐mediated ERKs activation and up‐regulation of Trx/TrxR/Nrf2 in the retinas of tub/tub mice. This suggests that ERKs and Nrf2 are involved in the mechanism of SF‐mediated up‐regulation of the Trx system to protect photoreceptor cells in this model. These novel findings are significant and could provide important information for the development of a unique strategy to prevent sensorineural deafness/retinal dystrophic syndromes and also other forms of inherited neurological disorders.

Topical dexamethasone-cyclodextrin microparticle eye drops for diabetic macular edema.

PURPOSE To test the safety and efficacy of topical 1.5% dexamethasone aqueous eye drops with cyclodextrin microparticles for diabetic macular edema (DME). METHODS Nineteen eyes of 19 consecutive patients with DME were administered dexamethasone-cyclodextrin eye drops three or six times a day for 4 weeks and then observed for 4 weeks without treatment. Visual acuity, intraocular pressure, and spectral domain optical coherent tomography-measured central macular thickness recordings at weeks 0 (baseline), 4, and 8. These parameters were compared using Bonferroni-corrected paired t-tests. RESULTS At weeks 0, 4, and 8, logMAR visual acuity (mean ± SD) was 0.52 ± 0.41, 0.37 ± 0.40 (P = 0.0025 vs. baseline), and 0.45 ± 0.41, respectively; central macular thickness (μm) was 512 ± 164, 399 ± 154 (P = 0.0016 vs. baseline), and 488 ± 172 (P = 0.0116 versus week 4), respectively; and intraocular pressure (mm Hg) was 15.2 ± 3.1, 17.4 ± 4.2 (P = 0.0015 vs. baseline) and 15.8 ± 4.0, respectively. At week 4, in 12 (63%) of 19 eyes, central macular thickness had decreased more than 10%, and the mean change was -20% (-65% to +10%). In 14 of 19 eyes (74%) visual acuity (logMAR) had improved more than 0.1 at week 4. No subjects showed severe adverse effects related to the eye drops. CONCLUSIONS Based on this short pilot study, topical dexamethasone-cyclodextrin eye drops are well tolerated, decrease central macular thickness, and improve visual acuity in DME. The results encourage comparative studies between dexamethasone cyclodextrin microparticle eye drops and other treatments for DME. (http://www.umin.ac.jp/ctr number, UMIN000001790.).