Kazuhiro Ohgami

Effects of fucoxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo

The aim of the present study was to investigate the efficacy of fucoxanthin on endotoxin-induced uveitis (EIU) in rats. The effects of fucoxanthin on endotoxin-induced leucocyte and protein infiltration, nitric oxide (NO), prostaglandin (PG)-E2 and tumour necrosis factor (TNF)-alpha concentrations in rat aqueous humour, as well as on the cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) protein expression in a mouse macrophage cell line (RAW 264.7 cells) were studied. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). Immediately after the LPS injection, either 0.1, 1 or 10mgkg(-1) of fucoxanthin was injected intravenously. The aqueous humour was collected 24hr later from both eyes, and both the number of cells infiltrating into the aqueous humour and the aqueous humour protein concentration were measured. The levels of PGE2, NO and TNF-alpha were determined by enzyme-linked immunosorbent assay. The RAW 264.7 cells were pretreated with various concentrations of fucoxanthin for 24hr and subsequently incubated with LPS for 24hr. COX-2 and iNOS protein expression was analysed by the Western blotting method. Levels of PGE2, NO and TNF-alpha production were determined. Fucoxanthin suppressed the development of EIU in a dose-dependent fashion. Treatment with fucoxanthin resulted in a reduction in PGE2, NO and TNF-alpha concentrations in the aqueous humour. The expression of COX and iNOS protein in the fucoxanthin treated RAW264.7 cells decreased significantly compared to that the LPS group. It also significantly reduced the concentration of PGE2, NO and TNF-alpha production in the medium of cells. The present result indicate fucoxanthin suppresses the inflammation of EIU by blocking the iNOS and COX-2 protein expression and its anti-inflammatory effect on eye is comparable with the effect of predinisolone used in similar doses.

Macular Pigment Lutein Is Antiinflammatory in Preventing Choroidal Neovascularization

Background—Choroidal neovascularization (CNV) is a critical pathogenesis in age-related macular degeneration, the most common cause of blindness in the developed countries. The aim of the current study was to investigate the effect of lutein supplementation on the development of the murine model of laser-induced CNV together with underlying molecular mechanisms. Methods and Results—Mice were orally pretreated with lutein daily from 3 days before laser photocoagulation untill the end of the study. The index of CNV volume was significantly suppressed by the treatment with lutein, compared with vehicle-treated animals. Lutein treatment led to significant inhibition of macrophage infiltration into CNV and of the in vivo and in vitro expression of inflammation-related molecules including vascular endothelial growth factor, monocyte chemotactic protein −1, and intercellular adhesion molecule-1. Importantly, lutein suppressed I&kgr;B-α degradation and nuclear translocation of nuclear factor (NF)-&kgr;B p65 both in vivo and in vitro. Additionally, the development of CNV was significantly suppressed by inhibiting NF-&kgr;B p65 nuclear translocation, to the levels seen in the lutein treatment. Conclusions—Lutein treatment led to significant suppression of CNV development together with inflammatory processes including NF-&kgr;B activation and subsequent upregulation of inflammatory molecules, providing molecular evidence of potential validity of lutein supplementation as a therapeutic strategy to suppress CNV.

Elevations of AGE and vascular endothelial growth factor with decreased total antioxidant status in the vitreous fluid of diabetic patients with retinopathy

Background/aims: Advanced glycation end product (AGE) induces vascular endothelial growth factor (VEGF) expression in cell culture and animal models, being considered to be involved in development of diabetic retinopathy; oxidative stress also has a part in diabetic retinopathy. However, the interrelations between AGE, VEGF, and oxidative stress remain to be elucidated. In this study, vitreous AGE, VEGF, and total antioxidant levels in were determined in diabetic patients with retinopathy, and the relations among them investigated. Methods: ELISA (enzyme linked immunosorbent assay) was used to determine the vitreous levels of AGE and VEGF in 41 patients with diabetic retinopathy and 28 non-diabetic control subjects. Total antioxidant levels in vitreous of 20 diabetic patients and 18 controls were also analysed by ELISA. Results: The vitreous levels of AGE and VEGF were significantly higher in diabetic patients than in control subjects (p<0.01 for both). There was a significant correlation between the vitreous AGE and VEGF levels (p<0.001). Total antioxidant status was decreased in vitreous in patients with diabetes compared with the controls (p<0.01). Furthermore, both AGE and VEGF levels were inversely correlated with the total antioxidant status (p<0.01 and p<0.05, respectively). Conclusion: This study suggests that AGE and decreased total antioxidant status may contribute to the pathogenesis of diabetic retinopathy via induction of VEGF.

Inhibition of choroidal neovascularization with an anti-inflammatory carotenoid astaxanthin

PURPOSE Astaxanthin (AST) is a carotenoid found in marine animals and vegetables. The purpose of the present study was to investigate the effect of AST on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms. METHODS Laser photocoagulation was used to induce CNV in C57BL/6J mice. Mice were pretreated with intraperitoneal injections of AST daily for 3 days before photocoagulation, and treatments were continued daily until the end of the study. CNV response was analyzed by volumetric measurements 1 week after laser injury. Retinal pigment epithelium-choroid levels of IkappaB-alpha, intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2 were examined by Western blotting or ELISA. AST was applied to capillary endothelial (b-End3) cells, macrophages, and RPE cells to analyze the activation of NF-kappaB and the expression of inflammatory molecules. RESULTS The index of CNV volume was significantly suppressed by treatment with AST compared with that in vehicle-treated animals. AST treatment led to significant inhibition of macrophage infiltration into CNV and of the in vivo and in vitro expression of inflammation-related molecules, including VEGF, IL-6, ICAM-1, MCP-1, VEGFR-1, and VEGFR-2. Importantly, AST suppressed the activation of the NF-kappaB pathway, including IkappaB-alpha degradation and p65 nuclear translocation. CONCLUSIONS AST treatment, together with inflammatory processes including NF-kappaB activation, subsequent upregulation of inflammatory molecules, and macrophage infiltration, led to significant suppression of CNV development. The present study suggests the possibility of AST supplementation as a therapeutic strategy to suppress CNV associated with AMD.

Five New Genome Types of Adenovirus Type 37 Caused Epidemic Keratoconjunctivitis in Sapporo, Japan, for More Than 10 Years

ABSTRACT Human adenovirus type 37 (HAdV-37) is a major cause of epidemic keratoconjunctivitis and has recently been the largest causative agent of keratoconjunctivitis in Japan. To investigate the genetic characteristics of HAdV-37 strains isolated in Sapporo, we analyzed the genome types and genetic relationships of 51 strains isolated there from 1990 through 2001. By using DNA restriction analysis, eight genome types (HAdV-37/D1, HAdV-37/D3, and HAdV-37/D6 to HAdV-37/D11) were identified, including five new ones. The restriction fragments of these genome types shared more than 95% identity with those of the prototype strain. By DNA sequence analysis, five and three single nucleotide substitutions, respectively, were found in partial sequences of the hexon and fiber genes. The combinations of mutations resulted in four hexon and fiber types (hx1 to hx4 and f1 to f4) and six hexon/fiber pairs (hx1/f1, hx2/f1, hx1/f2, hx1/f3, hx3/f4, and hx4/f4). The six pairs correlated well with certain genome types. In all three epidemics of keratoconjunctivitis to strike Sapporo in the past 12 years, specific genome types and fiber types were usually isolated: in the first epidemic, HAdV-37/D1 (f1) and HAdV-37/D3 (f1); in the second, HAdV-37/D6 (f2) and HAdV-37/D8 (f3); and in the third, HAdV-37/D10 (f4) and HAdV-37/D11 (f4). We conclude that mutations in the adenovirus genome occurred chronologically and that certain mutations were correlated with the epidemics of adenoviral keratoconjunctivitis.

New Genome Type of Adenovirus Serotype 4 Caused Nosocomial Infections Associated with Epidemic Conjunctivitis in Japan

ABSTRACT Human adenovirus type 4 is one of the major serotypes isolated from patients with adenoviral conjunctivitis. In 2001 we encountered nosocomial infections with epidemic conjunctivitis in the ophthalmology ward of one hospital in Sapporo, which is in the northern part of Japan. Adenoviruses were isolated from the patients with this nosocomial infection and identified as adenovirus type 4 (AdV-4) by a neutralization test with serotype-specific antiserum. When the cleavage patterns of the isolates were compared with the full viral genome with BamHI and SmaI, the cleavage patterns of the isolates were shown to be different from those of AdV-4p and other previously known AdV-4 variants. The nucleotide sequences of the fiber gene of the isolates showed the highest homologies (94.3%) with AdV-4 among the nucleotide sequences available from GenBank and formed a monophyletic cluster along with the prototype strain of AdV-4. The isolates, however, were located in a different lineage from those of AdV-4p and the AdV-4 variant from the sporadic infections. We conclude that the nosocomial infection that appeared in 2001 was caused by a new genome type of AdV-4, which was designated AdV-4c.

Increased Osteopontin Levels in the Vitreous of Patients with Diabetic Retinopathy

Purpose: Osteopontin (OPN) has diverse functions such as cell adhesion, chemoattraction, immunomodulation, and angiogenesis. The aim of this study is to analyze the OPN levels in vitreous fluid obtained from diabetic retinopathy (DR) and non-DR patients. Methods: Nineteen patients out of 11 with DR and 8 without DR underwent pars plana vitrectomy and vitreous fluid was obtained simultaneously. Two distinct sandwich enzyme-linked immunosorbent assay systems (systems 1 and 2) were applied, which have been developed in our laboratories to quantify the OPN concentrations in vitreous fluid. Results: The non-thrombin-cleaved full-length OPN levels in the vitreous fluid were 921.63 ± 45.38 ng/ml in DR and 632.80 ± 83.43 ng/ml in non-DR using system 1. Also, vitreous thrombin-cleaved and noncleaved OPN levels were increased to 2,109.22 ± 151.651 and 1,651.13 ± 229.82 ng/ml in patients with DR and non-DR using system 2. The vitreous OPN levels were significantly higher in DR than those in non-DR (p < 0.01 by system 1 and p < 0.05 by system 2). Conclusion: Thrombin-cleaved and noncleaved vitreous OPN levels in patients with DR were increased compared with control subjects, suggesting that OPN plays a potential role in the pathogenesis of diabetic retinal ischemia.

Expression of erythropoietin receptor in human epiretinal membrane of proliferative diabetic retinopathy

Purpose: It is widely accepted that intravitreous levels of erythropoietin (Epo) are elevated in patients with ischaemic retinal diseases such as proliferative diabetic retinopathy (PDR). The aim of this study was to examine the expression of Epo and the Epo receptor (EpoR) in epiretinal membranes with and without diabetes. Methods: Eighteen epiretinal membranes (PDR (n = 10), idiopathic epiretinal membranes (IERMs) without diabetes (n = 4) and inner limiting membranes (ILMs) (n = 4)) were obtained during pars plana vitrectomy. Formalin-fixed and paraffin-embedded tissues were examined by immunohistochemistry with anti-Epo and EpoR antibodies. Results: The histopathological findings demonstrated that PDR membranes consisted of a variety of endothelial cells forming a microvascular cavity with red blood cells and non-vascular stromal mononuclear cells. Membranous and cytoplasmic immunoreactivity for EpoR was strongly detected in endothelial cells and stromal cells in all PDR patients. Although microvessels were not observed in IERMs and ILMs, immunoreactivity for EpoR was noted in the cellular component of IERMs, and was weakly detected in ILMs. Epo was not expressed in any membrane. Conclusion: EpoR was strongly expressed in microvessels of all PDR membranes. The in vivo evidence in this study suggests that Epo in the vitreous binds to EpoR in PDR membranes, which subsequently leads to the proliferation of new retinal vessels. EpoR immunoreactivity in non-vascular stromal cells in PDR membranes, and IERMs and ILMs might be indirectly correlated with ischaemia.

Positive association of pigment epithelium-derived factor with total antioxidant capacity in the vitreous fluid of patients with proliferative diabetic retinopathy

Background: Pigment epithelium-derived factor (PEDF), a glycoprotein with potent neuronal differentiating activity, was recently found to inhibit advanced glycation end product (AGE)-induced retinal hyperpermeability and angiogenesis through its antioxidative properties, suggesting that it may exert beneficial effects on diabetic retinopathy by acting as an endogenous antioxidant. However, the inter-relationship between PEDF and total antioxidant capacity in the eye remains to be elucidated. Aims: To determine vitreous PEDF and total antioxidant levels in patients with proliferative diabetic retinopathy (PDR), and to investigate the relationship between them. Methods: Vitreous levels of PEDF and total antioxidant capacity were measured by an ELISA in 39 eyes of 36 patients with diabetes and PDR and in 29 eyes of 29 controls without diabetes. Results: Vitreous levels of total antioxidant capacity were significantly lower in patients with diabetes and PDR than in controls (mean (SD) 0.16 (0.05) vs 0.24 (0.09) mmol/l, respectively, p<0.001). PEDF levels correlated positively with total antioxidant status in the vitreous of patients with PDR (r = 0.37, p<0.05) and in controls (r = 0.41, p<0.05). Further, vitreous levels of PEDF in patients with PDR without vitreous haemorrhage (VH(−)) were significantly (p<0.05) decreased, compared with those in the controls or in patients with PDR with vitreous haemorrhage (VH(+); PDR VH(−), 4.5 (1.1) μg/ml; control, 7.4 (4.1) μg/ml; PDR VH(+) 8.5 (3.6) μg/ml). Conclusion: This study demonstrates that PEDF levels are associated with total antioxidant capacity of vitreous fluid in humans, and suggests that PEDF may act as an endogenous antioxidant in the eye and could play a protective role against PDR.

Macrophage migration inhibitory factor ameliorates UV-induced photokeratitis in mice.

Acute ultraviolet (UV) exposure causes photokeratitis, and induces apoptosis in corneal cells of the eye. Macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine. Today, MIF is considered as an integral component of the host antimicrobial alarm system and stress response that promotes the proinflammatory functions of immune cells. Also, MIF is considered to contribute the wound healing process. The aim of the present study is to determine the effects of MIF expression on UV irradiated corneal damage. MIF transgenic (MIF-Tg), wild type (WT), and MIF deficient (MIF KO) mice were UVB-irradiated of 400mJ/cm2 to induce acute UV-photokeratitis. MIF Tg mice constitutively produce high levels of MIF. Morphological changes were most severe in MIF KO mice, and WT and MIF Tg mice were following. Corneal basement membrane of MIF-Tg was well preserved. Prominent higher level of MIF was observed in MIF-Tg than WT after UVB irradiation in cornea. TUNEL staining showed a significantly smaller number of TUNEL positive nuclei in MIF-Tgm (6.2+/-4.3 cells/section, p<0.01 compared with WT) than WT (30.7+/-9.1) and MIF KO mice (32.1+/-12.7) 24h after UV exposure. The number of c-Jun positive nuclei was significantly higher in MIF Tg (p<0.01) than in WT and MIF KO mice. Serial observation revealed that BrdU incorporation was significantly upregulated in MIF Tg (p<0.01), but downregulated in MIF KO (p<0.01) than WT mice. MIF expression may thus be related to the amelioration of UVB-caused corneal injury, and this association was attributable to the upregulation of cell proliferation after acute UV-induced corneal damage, which involves the c-Jun dependent pathway. In conclusion, UV-damaged cornea is recoverable without MIF, however it takes longer time than normal condition. Cornea is less damaged and can make a quick recovery when ocular tissue is enough supplied with MIF.