Satoru Kase

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.

AlphaB crystallin regulation of angiogenesis by modulation of VEGF

alphaB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in alphaB-crystallin knockout (alphaB-crystallin(-/-)) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1alpha protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in alphaB-crystallin(-/-) retina versus wild-type mice, whereas VEGF-R2 expression was not affected. Both alphaB-crystallin and its phosphorylated serine59 formwere expressed, and immunoprecipitation revealed alphaB-crystallin binding to VEGF-A but not transforming growth factor-beta in cultured retinal pigment epithelial (RPE) cells. alphaB-crystallin and VEGF-A are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. alphaB-crystallin(-/-) RPE showed low VEGF-A secretion under serum-starved conditions compared with wild-type cells. VEGF-A is polyubiquitinated in control and alphaB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF-A increases with alphaB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in alphaB-crystallin(-/-) than wild-type mice. Proteasomal inhibition in alphaB-crystallin(-/-) mice partially restores VEGF-A secretion and angiogenic phenotype in choroidal neovascularization. Our studies indicate an important role for alphaB-crystallin as a chaperone for VEGF-A in angiogenesis and its potential as a therapeutic target.

Expression of cyclo-oxygenase-2 is correlated with high intratumoral microvessel density and low apoptotic index in human esophageal squamous cell carcinomas

Abstract. Cyclo-oxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostanoids. COX-2 expression has been found in many malignancies. This study analyzed the correlation between COX-2 expression and angiogenesis or apoptosis in human esophageal carcinomas. The study examined the expression of COX-2 in six esophageal carcinoma cell lines and in 100 esophageal squamous cell carcinomas, comparing intratumoral microvessel density (IMVD) and apoptotic index (AI) by immunohistochemistry and TUNEL methods. COX-2 was variably expressed in all the cell lines examined. COX-2 immunoreactivity was observed mainly in the cytoplasm of carcinoma cells. Significantly higher mean IMVD and lower AI were noted in the 51 strong COX-2 expressing cases than in the 49 weak cases. IMVD and AI were negatively correlated. COX-2 expression was higher in the tumors with lymphatic invasion than in the others. These data indicate that COX-2 expression is associated with increased intratumoral microvessels and suppression of tumor cell apoptosis. Thus COX-2 might play an important role in the angiogenesis and regulation of apoptosis in esophageal squamous cell carcinomas.

Recombinant Human VEGF165b Inhibits Experimental Choroidal Neovascularization

PURPOSE Vascular endothelial growth factor (VEGF-A) is the principal stimulator of angiogenesis in wet age-related macular degeneration (AMD). However, VEGF-A is generated by alternate splicing into two families, the proangiogenic VEGF-A(xxx) family and the antiangiogenic VEGF-A(xxx)b family. It is the proangiogenic family that is responsible for the blood vessel growth seen in AMD. METHODS To determine the role of antiangiogenic isoforms of VEGF-A as inhibitors of choroidal neovascularization, the authors used a model of laser-induced choroidal neovascularization in the mouse eye and investigated VEGF-A(165)b effects on endothelial cells and VEGFRs in vitro. RESULTS VEGF-A(165)b inhibited VEGF-A(165)-mediated endothelial cell migration with a dose effect similar to that of ranibizumab and bevacizumab and 200-fold more potent than that of pegaptanib. VEGF-A(165)b bound both VEGFR1 and VEGFR2 with affinity similar to that of VEGF-A(165). After laser injury, mice were injected either intraocularly or subcutaneously with recombinant human VEGF-A(165)b. Intraocular injection of rhVEGF-A(165)b gave a pronounced dose-dependent inhibition of fluorescein leakage, with an IC(50) of 16 pg/eye, neovascularization (IC(50), 0.8 pg/eye), and lesion as assessed by histologic staining (IC(50), 8 pg/eye). Subcutaneous administration of 100 microg twice a week also inhibited fluorescein leakage and neovascularization and reduced lesion size. CONCLUSIONS These results show that VEGF-A(165)b is a potent antiangiogenic agent in a mouse model of age-related macular degeneration and suggest that increasing the ratio of antiangiogenic-to-proangiogenic isoforms may be therapeutically effective in this condition.

Expression of p27(KIP1) and cyclin D1, and cell proliferation in human pterygium

Background: The pterygium is a growth onto the cornea of fibrovascular tissue that is continuous with the conjunctiva, whereas the mechanisms of cell proliferation in pterygium epithelium are unknown. Aim: To analyse the histopathology and the expression of cell cycle-related molecules in pterygium tissues. Methods: Seven pterygia were surgically removed using the bare-sclera procedure, and three normal bulbar conjunctivas were also obtained. Formalin-fixed, paraffin-wax-embedded tissues were analysed by immunohistochemistry with anti-p27(KIP1), cyclin D1 and Ki-67 antibodies. Results: Conjunctival epithelium consisted of several layers of round cells with a few goblet cells. Nuclear immunoreactivity for p27(KIP1) was noted in many normal epithelial cells, where cyclin D1 and Ki-67-positive nuclei were intermingled. A variety of goblet cells were located in the superficial layer of the pterygium head as well as those of the body epithelia. Several pterygium epithelial cells were p27(KIP1) positive, whereas nuclear immunoreactivity for cyclin D1 and Ki-67 was detected in many epithelial cells. By contrast, immunoreactivity for p27(KIP1), cyclin D1 and Ki-67 was hardly detected in the pterygium stroma. Conclusion: It is suggested that pterygium growth and development are associated with the proliferation of epithelium, which is possibly involved in the expression of cell cycle-related molecules.

Expression of Fas and Fas ligand in human gastric adenomas and intestinal-type carcinomas: correlation with proliferation and apoptosis

Background. Fas (APO-1/CD95), a member of the tumor necrosis factor/nerve growth factor receptor superfamily, mediates apoptosis in response to agonistic antibodies or Fas ligand (FasL) binding. Previous reports indicated an upregulation of FasL in gastric carcinomas to evade host immune attack. Fas/FasL expression, however, has not been analyzed in terms of apoptosis and proliferation in gastric adenoma and carcinoma. Methods. This study was conducted on seven human gastric carcinoma cell lines, 47 gastric adenomas, and 75 intestinal-type adenocarcinomas (48 early and 27 advanced carcinomas). Fas/FasL expression was examined by immunohistochemistry, apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) method, and Fas gene mutation by a reverse transcriptase (RT) polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing method. Results. Fas and FasL expressions were noted in 18 (38.3%) and 17 (36.2%) adenomas, in 21 (43.8%) and 33 (68.8%) early carcinomas, and in 10 (37.0%) and 19 (70.4%) advanced carcinomas, respectively. The frequency of FasL expression was significantly higher in advanced carcinomas than in the early carcinomas and adenomas; in contrast, there was no significant difference in Fas expression among the three groups. The mean apoptotic index (AI) was 4.96 ± 0.51 in the adenomas, 2.96 ± 0.23 in the early carcinomas, and 1.67 ± 0.17 in the advanced carcinomas. A significantly higher AI was noted in the lesions with Fas expression than in those without Fas expression in all three groups. No missense mutations of the Fas gene were detected in any of the gastric carcinoma cell lines, or in the gastric adenomas or carcinomas. Conclusions. Upregulation of FasL may correlate with the progression of gastric carcinoma. Apoptosis in gastric adenoma and carcinoma cells may occur via Fas-dependent and -independent pathways, but further clarification is needed.

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.

Expression of Cyclooxygenase-1 and Cyclooxygenase-2 in Human Esophageal Mucosa, Dysplasia and Carcinoma

Objective: COX (cyclooxygenase), a prostaglandin H synthase, catalyzes the rate-limiting step in prostaglandin biosynthesis. Two isoforms of COX have been identified: COX-1 and COX-2. We examined the expression of COX-1 and COX-2 in esophageal normal mucosa, dysplasia and squamous cell carcinoma (SCC). Methods: The expression of COX-1 and COX-2 in 80 surgically removed esophagi due to SCC, as well as in 5 human esophageal SCC cell lines was analyzed, using immunohistochemistry and Western blot analyses. Results: COX-1 and COX-2 were variably expressed in the SCC cell lines. Higher COX-1 expression was noted in 31 (41.9%) of the 74 specimens of normal mucosa, in none of the 40 specimens of dysplastic mucosa and in 15 (18.8%) of the 80 specimens of SCC, the frequency being significantly higher in normal mucosa than in dysplasia or SCC (p < 0.0001, p = 0.0018, respectively). COX-1 expression was significantly higher in well-differentiated SCC than in moderately or poorly differentiated SCC (p < 0.01). Higher COX-2 expression was noted in none (0.0%) of the specimens of normal mucosa, in 12 (30%) of the specimens of dysplastic mucosa, and in 41 (51.3%) of the speciments of SCC, the frequency being significantly higher in SCC than in normal mucosa or dysplasia (p < 0.0001, p = 0.0278, respectively). Conclusions: COX-1 is expressed in normal esophageal mucosa and is occasionally induced in well-differentiated SCC, whereas COX-2 expression is more characteristic of dysplasia and carcinoma than of normal mucosa, implying a possible association with cell differentiation in the former, and esophageal tumorigenesis in the latter.

TNF-α Decreases VEGF Secretion in Highly Polarized RPE Cells but Increases It in Non-Polarized RPE Cells Related to Crosstalk between JNK and NF-κB Pathways

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.