Search

Purpose. To identify an effective method to prevent myopia progression by characterizing the regulation of matrix metalloproteinase- (MMP-) 2 expression and its secretion from scleral fibroblasts and retinal pigment epithelium (RPE) cells by miR-29a. Methods. The effects of miR-29a on the growth of scleral fibroblasts and RPE cells were assessed using the cell counting kit-8. The changes in MMP-2 mRNA levels in scleral fibroblasts and RPE cells after transfection with miR-29a mimics or inhibitor were measured by quantitative PCR. Enzyme-linked immunosorbent assays were used to determine the changes in MMP-2 secretion from scleral fibroblasts and RPE cells after transfection with miR-29a mimics or inhibitor. Results. The miR-29a mimics or inhibitor did not significantly alter the growth of scleral fibroblasts or RPE cells at 24, 48, or 72 hours after transfection. MMP-2 mRNA levels were significantly decreased in scleral fibroblasts and RPE cells transfected with the miR-29a mimics. The secretion of MMP-2 by scleral fibroblasts and RPE cells was significantly decreased in cells transfected with the miR-29a mimics. Conclusions. Suppression of scleral fibroblast and RPE cell expression and secretion of MMP-2 by miR-29a can be used as a therapeutic target for the prevention and treatment of myopia. [ABSTRACT FROM AUTHOR]

Butein is a polyphenol, one of the compounds of chalcones, which are flavonoids that are widely biosynthesized in plants, and exhibits different pharmacological activities. Plants containing butein have been used in Chinese traditional medicine. Recently, it has been reported that butein suppresses proliferation and triggers apoptosis in various human cancer cells in vitro and in vivo. The aim of this study was to investigate its pro-apoptotic effect and mechanisms in two cultured human ovarian cancer cells (ES-2 and TOV-21G). The effects of butein on cell viability were assessed by a MTT assay at 3, 10, 30, and 100 μ/M. The apoptotic pathway related factors, including the mitochondrial transmembrane potential (MTP), cytochrome c, caspase cascade, and Bcl-2 family proteins, were examined. MTT assay revealed that butein was cytotoxic to both ovarian cancer cells in a dose- and time-dependent manner. JC-1 flow cytometry, cytochrome c, and caspase activity assays revealed that butein damaged the MTP, increased the level of cytosol cytochrome c and the activities of caspase-3, -8, and -9 in the two ovarian cancer cells. Western blot analysis revealed that butein down-regulated the anti-apoptotic proteins Bcl-2 and Bcl-xL and increased the pro-apoptotic proteins Bax and Bad. These findings suggest that butein-induced apoptosis in ovarian cancer cells via the activation of both extrinsic and intrinsic pathways. In addition, butein also down-regulated the expressions of the inhibitor of apoptosis (IAP) proteins, XIAP, survivin, CIAP-1, and CIAP-2. This indicates that the inhibition of IAP proteins was also involved in butein-induced apoptosis. The results of our study suggest that butein may be a promising anticancer agent in treating ovarian cancer. [ABSTRACT FROM AUTHOR]

Hypoxia is the most important stimulus leading to upregulation of VEGF in the retina and this is caused by accumulation of hypoxia-inducible factors-1α (HIF-1α) protein. The effects of zeaxanthin, a natural phytochemical, on the VEGF and HIF-1α expression in the primary culture of human retinal pigment epithelial (RPE) cells were studied. An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2) to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without zeaxanthin under normoxic and hypoxic conditions. VEGF and HIF-1α protein and RNA levels were measured by ELISA kits and RT-PCR, respectively. Hypoxia caused a significant increase of VEGF expression and accumulation of HIF-1α in RPE cells. Zeaxanthin at 50–150 μM significantly inhibited the expression of VEGF and accumulation of HIF-1α protein caused by hypoxia but did not affect expression of VEGF and HIF-1α under normoxic conditions. This is the first report on the effect of zeaxanthin on VEGF and HIF-1α levels in cultured RPE cells and suggests that zeaxanthin may have potential value in the prevention and treatment of various retinal diseases associated with vascular leakage and neovascularization. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of the present study was to develop methods for isolation, purification and cultivation of human conjunctival melanocytes. Conjunctiva excised from donor eyes or corneal rims was subjected with various enzyme digestion methods or by the enzyme-microdissection method. Cells were cultured with F12 medium supplemented by fetal bovine serum, basic fibroblast growth factor, isobutylmethylxanthine and cholera toxin. Contaminant cells were eliminated by a selective cytotoxic agent, geneticin. Both trypsin digestion and dispase-microdissection methods provided pure conjunctival melanocyte cultures with high cell yields, good viability and rapid growth rate. Melanocytes isolated with dispase-microdissection method showed better viability and growth capacity. Cells grew well, could be passaged for 5–10 generations and divided 20 times in vitro. They maintained a constant melanin content per cell and produced measurable amounts of melanin in vitro. Melanogenesis correlated with the degree of pigmentation of the eyes (iris color). This method provides a valuable source of large numbers of human conjunctival melanocytes, which can be used to study their biological behavior, to compare with the epidermal and uveal melanocytes; and to compare them to their malignant counterparts in the exploration of the pathogenesis of conjunctival melanoma. [Copyright &y& Elsevier]

Uveal melanocytes and the ocular pigment epithelium are located in the middle and inner layers of the eye. Müller cells (a type of glial cell) are located in the neural retina. Melanocytes, retinal pigment epithelium (RPE), and Müller cells do not participate directly in the detection or transfer of visual information, but they have various functions that support the neural retina and are essential for the maintenance of vision. Methods for the isolation and cultivation of melanocytes, RPE, and Müller cells have been established by us and other investigators. These cultured cells can be used as in vitro model systems for studying the toxicology of visible light, ultraviolet (UV) radiation, drugs, and other potentially toxic agents. Toxic effects on these cells may give rise to altered retinal function and result in impaired vision. Both melanocytes and pigment epithelium contain melanin, which has the ability to bind organic amines and metal ions. This results in the accumulation of these substances in the eye. Melanin may protect cells from chemical stress by binding toxic chemicals; but in chronic exposure, increased and lengthy binding may cause damage to these cells. Two different types of melanin are found in the eye: eumelanin and pheomelanin, which may have photoprotective and phototoxic effects, respectively. Pigment epithelium contains mainly eumelanin, whereas melanocytes contain both eumelanin and pheomelanin. Melanin is an antioxidant and with age, the antioxidant properties may diminish to the point that it may even become a prooxidant. There are also other functions of pigment epithelium and uveal melanocytes not related to melanin and there are also several functions of Müller cells that play a role in the toxicological aspects of the eye. Cultured uveal melanocytes, pigment epithelial cells, and Müller cells can be used to study the toxicology of these cells in vitro. [ABSTRACT FROM AUTHOR]

IL-6 plays an important role in various inflammatory ocular diseases, including diabetic retinopathy. Müller cells are the major source of inflammatory mediators, including IL-6, in the retina. However, the mechanism of regulating IL-6 production in these cells remains unclear. Examination of signaling pathways in human retinal Müller cells (MIO-M1 cell line) cultured with IL-1β, TNF-α, IL-6, IL-8, VEGF, IFN-γ, glucose or mannitol showed that IL-1β was the most potent stimulator of IL-6 production. In addition, IL-1 β also increased NF-κB p50 protein level and phosphorylation of p38 MAPK, ERK1/2 and c-Jun. Induction of IL-6 production by IL-1β was significantly reduced by addition of p38 MAPK (SB203580), MEK1/2 (U0126) or NF-κB (BAY11-7082) inhibitors, with the highest effect being observed with SB203580. To explore the specific elements in IL-6 promoter responsible for IL-1β-induction of IL-6 expression, a series of plasmids bearing various IL-6 promoter mutations were transiently expressed in MIO-MI cells cultured in the presence or absence of IL-1β (10 ng/ml) and/or SB203580 (10 µM). Results showed that IL-6 promoter activity of the parent pIL-6-Luc651 was significantly enhanced by IL-1β, but the level was significantly attenuated by SB203580. Furthermore, the IL-6 promoter activity was also reduced upon deletion of NF-κB, AP-1 or C/EBP binding sites, with NF-κB deletion being the greatest. These results are the first demonstration that IL-1β induces IL-6 production in Müller cells by activation of IL-6 promoter activity predominantly through the p38 MAPK/NF-κB pathway. [ABSTRACT FROM AUTHOR]