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T Hiramitsu

Publications and source records attributed to T Hiramitsu.

At least 19 recordsLinked to original sources
PubMed2006

Intercellular adhesion molecule-1 mediates the inhibitory effects of hyaluronan on interleukin-1beta-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts via down-regulation of NF-kappaB and p38.

OBJECTIVE: In rheumatoid arthritis (RA), it is well known that rheumatoid synovial fibroblasts (RSF) produce matrix metalloproteinases (MMPs) when stimulated with proinflammatory cytokines such as interleukin-1beta (IL-1beta), which causes joint destruction. We have previously shown that hyaluronan (HA) inhibits IL-1beta actions in RSF via CD44, the principal HA receptor. However, CD44 mediates HA effects only partially, and intracellular events after the HA binding to its receptors remain unclear. We investigated the role of intercellular adhesion molecule-1 (ICAM-1), another cell surface receptor for HA, and the intracellular signalling pathways in the actions of HA. METHODS: RSF were isolated from rheumatoid synovial tissues by enzymatic digestion and cultured in monolayers. The confluent cells were incubated for 48 h with IL-1beta, IL-1beta in the presence of HA, or IL-1beta in the presence of HA with pretreatment with anti-ICAM-1 antibody. Secretion of MMP-1 and MMP-3 was analysed by immunoblotting and immunofluorescence cytochemistry. Immunofluorescence cytochemistry was also performed to evaluate binding of HA to ICAM-1. The phosphorylation of nuclear factor (NF)-kappaB and mitogen-activated protein kinases (MAPKs) was analysed by immunoblotting. RESULTS: Production of MMP-1 and MMP-3 by RSF was stimulated by IL-1beta. HA at > or =2 mg/ml significantly inhibited MMP production induced by IL-1beta in a dose-dependent manner. Moreover, pretreatment with anti-ICAM-1 antibody at 50 mug/ml significantly blocked the effects of HA on the actions of IL-1beta on RSF, as shown by immunoblotting and immunofluorescence cytochemistry. Another immunofluorescence cytochemistry study demonstrated that HA bound RSF via ICAM-1. Inhibition studies revealed the requirement of NF-kappaB, p38 and c-jun NH2-terminal kinase (JNK) for IL-1beta-induced MMP production. IL-1beta activated all three pathways, whereas HA down-regulated their phosphorylation. Pretreatment with anti-ICAM-1 antibody reversed the inhibitory effects of HA on the activation of NF-kappaB and p38 without affecting JNK. CONCLUSION: HA suppresses IL-1beta-enhanced MMP-1 and MMP-3 synthesis in RSF via ICAM-1 through down-regulation of NF-kappaB and p38. Intra-articular injection of HA of high molecular weight may work through such a mechanism in RA joints.

Arthritis, Rheumatoid↗
PubMed2004

COOH-terminal heparin-binding fibronectin fragment induces nitric oxide production in rheumatoid cartilage through CD44.

OBJECTIVES: To examine the mechanism of nitric oxide (NO) production by a COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in rheumatoid arthritis (RA) cartilage. METHODS: Articular cartilage slices from RA knee joints and normal hip joints were cultured with HBFN-f. Secreted NO levels in conditioned media were determined. Cultures were pretreated with anti-CD44 antibody or HBFN-f-derived synthetic peptide (peptide V; WQPPRARI) to evaluate the role of CD44 in HBFN-f action. Immunofluorescence histochemistry was performed using fluorescein isothiocyanate-conjugated anti-CD44 antibody. RESULTS: HBFN-f stimulated NO production in a dose-dependent manner. Whereas CD44 expression was up-regulated in RA cartilage, anti-CD44 antibody blocked HBFN-f-stimulated NO production. Peptide V with heparin-binding ability significantly reduced NO levels elevated by HBFN-f. Compared with normal cartilage, cartilage response to HBFN-f and the blocking effects of anti-CD44 antibody on HBFN-f action were stronger in RA cartilage. CONCLUSIONS: The present study clearly demonstrated that HBFN-f stimulated NO production through CD44 in RA cartilage. Increased expression of CD44 in RA cartilage may play a pathological role in joint destruction through enhanced NO production by binding to fibronectin fragments such as HBFN-f.

Aged↗
PubMed2001

Microdialysis measurement of ascorbic acid in rabbit vitreous after photodynamic reaction.

A method for long-term intravitreous microdialysis was used to measure endogenous reduced ascorbic acid in the vitreous of rabbits by HPLC-ECD before and after exposure to intense visible light in the presence of fluorescein. Cellulose microdialysis probes were implanted into the vitreous humor of each eye and after stabilization ascorbic acid measurements were recorded over a 14 day period. Under this experimental condition, normal ascorbic acid concentrations in vitreous varied from 98.0 +/- 9.8 to 106.9 +/- 20.3 microM(mean +/- S.D.). The eyes received light irradiation (25 000 lux) for 2 hr and fluorescein was used as the photosensitizer once or twice. No immediate effects on ascorbic acid concentrations could be observed in the eyes irradiated twice without fluorescein i.v. injections and in the twice fluorescein injected without irradiation. However, in the eyes irradiated once with fluorescein (30 mg kg(-1)), ascorbic acid concentration after irradiation significantly decreased from day 2 and continued over a period of 10 days compared with that before irradiation and maximal reduction was 32.6% (P < 0.005) on day 6 after irradiation. By day 13, the ascorbic acid concentration returned to control levels (P > 0.01). In the eyes irradiated twice with fluorescein injections, ascorbic acid concentration after irradiation decreased even more over the experimental period and the maximal reduction was 65.5% (P < 0.005) on day 5 after irradiation and did not recover over the next 9 days. In the eyes irradiated twice with fluorescein injections plus administration of ascorbic acid (150 mg kg(-1)) 30 min before irradiation, a significant increase (52.5%) of ascorbic acid (P < 0.005) was found on day 1 and control levels of ascorbic acid were maintained from day 2 onward. The protective role of ascorbic acid in the vitreous humor against photodynamic reaction is suggested.

Animals↗
PubMed2001

L-DOPA produced nitric oxide in the vitreous and caused greater vasodilation in the choroid and the ciliary body of melanotic rats than in those of amelanotic rats.

The nitrogen cycle initiates direct reduction of N2 to NH3 by enzymatic reactions. We hypothesize that L-dihydroxyphenylalanine (L-DOPA), a catecholamine, could be a source of nitric oxide (NO). In order to determine whether L-DOPA generates NO and induces any biological change in the eye, we measured the generation of NO in vitro and in vivo, and investigated the histopathological changes caused by injection of L-DOPA into the vitreous of rats. We also hypothesized that melanin granules may affect the generation of NO during the metabolism of L-DOPA, since L-DOPA is a precursor of melanin in the brain and the eye. Therefore, we compared the effects of L-DOPA on the generation of NO between amelanotic and melanotic rats. NO was measured as diffusion currents by NO electrodes. In vitro, various concentrations of L-DOPA (5, 29.9, 79.4, 152.7, and 249 microM) were added to the medium. The inhibition of NO generation by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) was tested. In vivo, NO generation in the vitreous of rats was measured and the eyes were enucleated under anesthesia after L-DOPA injection. The ocular tissues were subjected to histological examination. NO was produced from L-DOPA in a dose-dependent manner and was scavenged by carboxy-PTIO in vitro. NO in the vitreous of melanotic rats was generated from L-DOPA. Histological examination with hematoxylin-eosin staining revealed vasodilation in the ciliary vessels and the choroid after L-DOPA injection. Both effects were greater in melanotic rats than in amelanotic rats. The vasodilation may be attributable to NO as well as to superoxides, which can be regulated by the existence of melanin.

Animals↗
PubMed2000

Melanin granules prevent the cytotoxic effects of L-DOPA on retinal pigment epithelial cells in vitro by regulation of NO and superoxide radicals.

Inasmuch as the nitrogen cycle elicits the direct reduction of N2 to NH3 through enzymatic reactions, and inasmuch as L-DOPA (L-dihydroxyphentlalamine), a catecholamine, can be a source of nitric oxide (NO), it is possible that melanin granules in the eye affect the generation of NO, which causes damage to the retinal pigment epithelial (RPE) cells during the oxidation of L-DOPA. In order to confirm this possibility, we analyzed the correlations of NO generation, cell growth, and superoxide dismutase (SOD) activities in two types (melanotic and amelanotic) of bovine RPE cells following exposure to L-DOPA. NO generation from L-DOPA was determined using an NO detector that is reliant on redox currents. The concentration of NO was measured in terms of diffusion currents run between a working electrode and a counter electrode, both being set in culture medium placed in a Petri dish. For the assays, L-DOPA was added to the medium at various concentrations (5, 29.9, 79.4, 152.7 or 249 microM), and 6 min after addition, an NO-trapping agent 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) was also added. The melanotic and amelanotic types of RPE cells were cultured separately in medium with L-DOPA under an atmosphere containing 20, 10 or 5% oxygen. Cell numbers were counted using a Coulter counter, and SOD activities were determined following incubation for 24, 48 or 72 hr using a modification of the luminol assay. The results obtained indicated that: (a) NO was produced from L-DOPA in a concentration-dependent manner and was trapped quantitatively by carboxy-PTIO; (b) the generation of NO was inhibited more markedly in the melanotic cell line than in the amelanotic one, suggesting an increased tolerance to L-DOPA-derived cytotoxicity in the former; and (c) the SOD activities were more affected by oxygen concentration in the melanotic cells than in the amelanotic ones. From these results, it is concluded that melanin granules in RPE cells have a role in preventing the cytotoxicity derived from L-DOPA and in regulating the generation of NO and superoxide radicals.

Animals↗
PubMed1999

Effects of fluorescent light on growth of bovine retinal pigment epithelial cells in vitro incubated with linoleic acid or linoleic acid hydroperoxide.

Light-induced peroxidation of polyunsaturated fatty acids (PUFA) may generate lipid hydroperoxides, which may have toxic effects on retinal pigment epithelial (RPE) cells in vitro. We investigated the effects of cool-white fluorescent light on the RPE cells incubated with linoleic acids (LA) or linoleic acid hydroperoxides (LHP) and the influence of antioxidative enzymes. We measured the bovine RPE cell number after exposure to fluorescent light (610 and 1,200 lux) in the presence of LA or LHP. Furthermore, the effects of superoxide dismutase (SOD) and catalase on LA- or LHP-treated RPE cells were also examined. Both LA and LHP treatment increased RPE cell number under weak illumination (610 lux), but dose-dependently decreased the number of cells exposed to strong illumination (1,200 lux). With exposure to strong illumination, LA caused a greater reduction in RPE cell number than LHP. Multiple linear regression analysis showed that the number of RPE cells was significantly decreased in a manner dependent on the interactions of the illuminance of light and the concentrations of LA or LHP. The antioxidative enzymes significantly ameliorated the damage to RPE cells from LA or LHP and exposure to light. Therefore, the exposure to fluorescent light augmented the cytotoxic effects of LA and LHP on RPE cells, and this effect is likely to be mediated by reactive oxygen species.

Animals↗
PubMed1999

The localization of glutathione peroxidase in the photoreceptor cells and the retinal pigment epithelial cells of Wistar and Royal College of Surgeons dystrophic rats.

INTRODUCTION: If degenerating photoreceptor outer segments not phagocytized by RPE cells in the retina of Royal College Surgeons (RCS) rats were to undergo peroxidation, the distribution of glutathione peroxidase (GSH-PO) in the mitochondria or cytoplasm of the retina might be altered. We evaluated the immunocytochemical localization of GSH-PO to identify subcellular organelles in sections of the retinas of RCS rats. METHODS: Immunoblot analysis confirmed the presence of GSH-PO molecules in the retinas of RCS and Wistar rats aged 3 weeks. Sections were reacted with the F(ab) fragment of anti-rat alphaGSH-PO and then examined by laser scanning microscopy (LSM) and transmission electron microscopy (TEM). RESULTS: The size of the GSH-PO molecule in the retina was about 21 KD in the mitochondria and 23 KD in the cytosol in both strains of rats. LSM revealed fluorescent granules in the photoreceptor inner segments of the Wistar rats, and immunohistochemical TEM revealed GSH-PO in the mitochondria of their photoreceptor inner segments and retinal pigment epithelial (RPE) cells. In the RCS rats, the degenerating photoreceptor outer segments were clearly seen to be positive for anti-GSH-PO by conventional light microscopy (CLM). However, the photoreceptor inner segments of the RCS rats were negative for staining with anti-GSH-PO by LSM, and no GSH-PO could be detected in the mitochondria of the photoreceptor inner segments or RPE cells by immuno-TEM. CONCLUSION: Degeneration of the photoreceptor outer segments induced mitochondrial damage in the photoreceptor inner segments, and as a result GSH-PO shifted from the photoreceptor inner segments to the degenerating outer segments.

Animals↗
PubMed1998

Changes in lipid peroxide level in retinal pigment epithelial cells in vitro upon addition of linoleic acids or linoleic acid hydroperoxides under varying concentrations of oxygen.

We previously observed the presence of autofluorescent lipofuscin or its like in retinal pigment epithelial (RPE) cells, which were incubated with linoleic acid hydroperoxides (LHP). We studied the effect of oxygen on the level of lipid peroxides in RPE cells in the presence of linoleic acids (LA) or LHP. The level of lipid peroxides in these cells was determined by use of the thiobarbituric acid-reactive substance (TBARS), which responded to oxygen concentrations qualitatively, and a linear regression analysis. Multiple linear regression analysis disclosed that treatment with LA for 24 hr resulted in detectable increase in the level of TBARS in the cells, whereas treatment with LA or LHP for 48 hr caused detectable decrease. Stepwise linear regression analysis showed that the level of TBARS decreased in an oxygen-tension dependent manner in the cells incubated with LA for 48 hr. Thus, it was shown that short-term incubation with LA increased the level of TBARS in the cells and that LA decreased its level in an oxygen-tension dependent manner. For these results, the postulation was made that the prolonged auto-oxidation of LA caused production of lipofuscin-like materials, a complex of lipid peroxides and proteins that were insoluble in SDS and acetic acid solution.

Animals↗
PubMed1996

Effects of superoxide dismutase and catalase on growth of retinal pigment epithelial cells in vitro following addition of linoleic acid or linoleic acid hydroperoxide.

The rod outer segments of the retina that are phagocytized by retinal pigment epithelial (RPE) cells are susceptible to lipid peroxidation because of their high content of polyunsaturated fatty acids. Linoleic hydroperoxides (LHP), synthesized by peroxidation of linoleic acids (LA), produce greater damage to retinal function than does LA. We compared the effects of LHP and LA on the growth of cultured chick embryonic RPE cells and analyzed a model of data sets using multiple linear regression for the number of cells as a function of time. The spectrum of LA had a sharp peak at 205 nm and a broad spectrum at 235 nm, while LHP had only a broad spectrum at 235 nm. Exposure to LA and LHP caused dose-dependent damage of chick embryonic RPE cells: they were significantly more affected by the addition of LHP than LA. The antioxidative enzymes catalase and superoxide dismutase minimized damage to the RPE cells caused by LHP in proportion to the enzyme concentration. However, RPE cells incubated with LA were more affected by the enzymes than when no enzymes were added.

Animals↗
PubMed1996

Comparative effects of linoleic acid and linoleic acid hydroperoxide on growth and morphology of bovine retinal pigment epithelial cells in vitro.

PURPOSE: Outer segments of the photoreceptor rods that are phagocytized by the retinal pigment epithelial (RPE) cells contain a high proportion of polyunsaturated fatty acids (PUFA). PUFA are susceptible to lipid peroxidation. We hypothesized that the resulting peroxides could injure RPE cells leading to retinal degeneration. Accordingly, we compared the effects of linoleic acid (LA) and its hydroperoxide (LHP) on the growth and morphology of RPE cells using laser scanning microscopy and transmission microscopy. METHODS: We counted the number of RPE cells after incubation for 24 and 48 hrs with concentrations of LA or LHP of 0.035, 0.175, and 0.35 mM. To observe the actin filaments, cultured RPE cells were stained with rhodamine phalloidin. The cells were prefixed with 2% glutaraldehyde and postfixed in 1% osmium tetroxide. Specimens were embedded in Epon 812 after dehydration, and the ultrathin sections were doubly stained with 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy. RESULTS: Exposure to LA or LHP produced dose-dependent damage to RPE cells with a significantly greater effects of LHP than LA. After incubation for 24 hrs with 0.35 mM LA, the number of vacuoles in RPE cells exceeded that observed in control RPE cells by 365 nm laser microscopy. Exposure to 0.35 mM LHP for 24 hrs produced a pycnotic nucleus, with diffuse and granular autofluorescences observed in and around it. Exposure of RPE cells to 0.35 mM LA for 24 hrs showed that the LA incorporated into the lysosomes was digested and released extracellularly from lysosomes via exocytotic vesicles. However, such exposure to LHP damaged the RPE cells, including the membranes in the pinocytotic vesicles. The packed membranes resembled myelin. CONCLUSIONS: While the LA incorporated into the lysosomes was released extracellularly, LHP persisted in the RPE cells, being observed as autofluorescent lipofuscin-like materials. LHP was cytotoxic, and caused damage to the membranes of pinocytotic vesicles and lysosomes.

Animals↗
PubMed1995

Hemoglobin-induced lipid peroxidation in the retina: a possible mechanism for macular degeneration.

To investigate a possible link between subretinal hemorrhage and macular degeneration, oxyhemoglobin (HbO2) or methemoglobin (metHb) was incubated with retinal homogenate and unsaturated phospholipid peroxidation was monitored by (a) assay of thiobarbituric acid-reactive substances (TBARS), (b) luminescence originating from an energy transfer of lipid-degraded products to rose bengal, and (c) the decrease in composition of highly unsaturated fatty acids of phospholipids. TBARS formation and rose bengal luminescence in the case of metHb-induced lipid peroxidation were about 1.5 times greater than those in HbO2-induced lipid peroxidation. alpha-Tocopherol, a lipid-soluble antioxidant, and docosahexaenoic acid, a major unsaturated fatty acid, were slightly more rapidly decomposed after a 60-min incubation with metHb than with HbO2 at the same concentration. Atomic absorption analysis revealed that an equal concentration of iron was released from both HbO2 and metHb during incubation with retinal homogenates. The released iron may promote microsomal phospholipid peroxidation in the presence of endogenous ascorbate or NADPH-dependent cytochrome P-450 reductase because ascorbate oxidase and p-chloromercuribenzoic acid (an inhibitor of sulfhydryl enzymes) inhibited metHb- or HbO2-induced lipid peroxidation. MetHb-induced lipid peroxidation in retina was inhibited by KCN or NaN3, which binds to FeIII of metHb. KCN or NaN3 had no effect on HbO2-induced lipid peroxidation, because conversion of HbO2 to metHb, which can proceed in HbO2 incubated with phospholipid liposome, did not occur in retinal homogenates. It is concluded that metHb induces peroxidation of retinal unsaturated phospholipids (1) directly and (2) by releasing iron.

Animals↗
PubMed1995

A model of rod outer segment derived lipofuscin.

The rod outer segments (ROS) from pig eyes were incubated with ferrous iron and albumin. The fluorescent lipid peroxides formed in the reaction were extracted with chloroform-methanol, ethanol-ether or water. After centrifugation, the fluorescence intensities of the supernatant and the aqueous solution of the sediments were measured with a Fluorospectrophotometer. The sediments were observed with a fluorescence microscope. The fluorescence spectra has an excitation maximum at 365 nm and an emission maximum at 430-440 nm. The fluorescence intensities were greater in the water soluble fraction than in the organic solevent fraction. The fluorescence microscopy showed the blue emitting fluorophores. The fluorophores from ROS peroxidized with ferrous iron have different properties from the lipofuscin in human RPE extracted with the organic solvents.

Albumins↗
PubMed1991

Preventive effect of antioxidants on lipid peroxidation in the retina.

Lipid peroxidation was induced with porcine retinal homogenates using ferric iron or UV light. A number of lipid and aqueous-soluble antioxidants were tested and their protective effects measured after 60 min of exposure. The iron system produced up to 5 times more lipid hydroperoxides as quantified by the fluorescence thiobarbituric acid (TBA) assay. Of the three substituted dialkyl phenols evaluated, the most efficient was compound S-17224 which was able to afford greater than 90% protection at 10(-4) M concentration in the iron-induced system. At 10(-5) M, inhibition was 78% and at 10(-6) M 33%. By comparison, 10(-4) M gamma-oryzanol inhibited the reaction by 61% and alpha-tocopherol by only 14%. The combination of 10(-6) M S-17224 and 10(-4) M gamma-oryzanol potentiated the effect by another 23%. In the light-catalyzed system. S-17224 and alpha-tocopherol were more efficient (71%). The model system described here is simple and can be employed to study additional antioxidants as well as in the retinotoxic evaluation of ophthalmic drugs.

Animals↗
PubMed1990

Studies of experimentally induced retinal degeneration: 2. Early morphological changes produced by lipid peroxides in the albino rabbit.

When pure, synthetic lipid hydroperoxides (LHP) were injected into the vitreous body of albino rabbits, electrical activity was decreased in all components of the electroretinogram (ERG) in a progressive and time-related manner. In the early phase morphological changes occurred at the interface between the photoreceptor outer segments and the retinal pigment epithelium (RPE) in response to LHP. These findings commenced within a few hours after injection and continued during the following 2-3 weeks, when the ERG was completely extinguished. Two days after injection, the rod outer segments (ROS) were swollen and damage of the RPE apical villi was observed. This initial event was followed by additional changes in the RPE which embraced swelling, accumulation of residual bodies, complete loss of ROS and enlargement and disruption of Bruch's membrane. The precursors of residual bodies appeared to result from focal, peroxidative damage to ROS discs which apparently rendered these materials undegradable by the RPE. As ROS degeneration continued, the RPE showed hypertrophy and modification. These studies provided evidence for a sequential destruction of the neural retina and RPE during oxidative damage involving lipid peroxidation. The mechanism appeared to differ from that produced by other toxic compounds or those which resulted from vitamin E or A deficiency. This new model system is thought to be useful in 1) explaining differences in susceptibility of inner ROS disks versus other membranes, 2) determining how the RPE metabolizes abnormal ROS, 3) studying RPE reactivity following trauma and/or retinal detachment, and 4) determining factors which produce degeneration of Bruch's membrane.

Animals↗