Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

BACKGROUND: Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogen-esis in retinal pigment epithelium (RPE). RESULTS: We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium-derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased. CONCLUSIONS: RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

Detection of specific proteins in the aqueous humor in primary open-angle glaucoma

To elucidate the mechanism of increased intraocular pressure in primary open-angle glaucoma (POAG), the protein profiles of aqueous humor obtained from POAG patients were compared with those of cataract patients as a control group. Aqueous humor proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by the ultrasensitive silver staining technique. In 79% of the samples taken from POAG patients, protein bands of 140,000 or 160,000 daltons were stained, but none were stained from cataract patients. The presence of these protein bands revealed statistically significant differences between the two groups. Protein bands of 140,000 or 160,000 daltons were evenly visible at all ages in POAG patients, and the positivity of bands had no correlation with sex or initial intraocular pressure level. It is possible that the ultrastructural changes of the aqueous outflow pathway in POAG may be related to the changes in the aqueous protein, presence of 140,000 or 160,000 daltons protein bands.

Efeito da radiaçäo gama nas proteínas de cristalino bovino in vitro / Effect of gama irradiation on in vitro bovine lens proteins

A radiossensibilidade das lentes oculares manifestada pela formaçäo de cataratas, tem sido de considerável interesse para pesquisadores no estudo dos efeitos biológicos das radiaçöes. A formaçäo da catarata se dá por diferentemente causas, e também pelo processo normal de envelhecimento. Neste trabalho o objetivo é desenvolver um sistema "on vitro", análogo à formaçäo da catarata. É usada uma soluçäo aquosa de cristalino bovino. Os cristalinos após remoçäo cirúrgica säo homogeneizados mecânicamente e por ultrasom. Está suspensäo é centrifugada e o sobrenadante é dializado e irradiado com diferentes doses de 60Co. A leitura do grau de opacificaçäo é feita por espectrofotometria.