Expression of ephrin B1 and ephrin B2 in normal and glaucomatous astrocytes in the optic nerve head and retina of monkeys and human.

Abstract

Abstract: : Purpose: Ephrins are the ligands of the Eph receptors, which are the largest family of tyrosine kinase receptors. In this study we determined the expression of ephrin B1 and ephrin B2 in the adult monkey normal and glaucomatous retina and optic nerve head (ONH). We also determined the expression of ephrin B1 and ephrin B2 in cultured human glaucomatous and normal human ONH astrocytes. Methods: Eyes from six adult male and female rhesus monkeys (Macaca mulatta) with unilateral laser induced ocular hypertension were used in this study. The contralateral eyes served as control. Double immunostaining was used to identify and localize the expression of ephrin B1 and ephrin B2 to specific cell types in retina and ONH. Western blots and quantitative real time RT–PCR were used to determine the level of expression of ephrins B1 and B2 mRNA and protein in cultured human glaucomatous and normal astrocytes. Results: In vivo, the expression of ephrin B1 in the normal adult monkey retina and ONH was low and localized to astrocytes in the nerve fiber layer. Ephrin B2 expression was limited to the photoreceptors and the blood vessels. In laser–induced glaucoma, the expression of Ephrin B1 was markedly increased in the ONH and retina, but ephrin B2 expression remained very low. Ephrin B1 was founded specifically in astrocytes and Muller cells of the retinal ganglion layer and nerve fiber layer, and its immunoreactivity was stronger in eyes with moderate and severe glaucomatous damage. Western blot and real time RT–PCR showed that in human normal astrocytes there was low expression level of ephrin B1 and that level of ephrin B2 was barely detectable. In human glaucomatous astrocytes, the expression of ephrin B1 increased three fold compared to normals, but ephrin B2 remained low. Conclusions: Ephrins are involved in cell migration and in cell contact–mediated interactions; in particular, they have been shown to establish repulsive boundaries during development and as a response to injuries in the adult CNS. The over–expression of ephrin B1 in the glaucomatous astrocytes in the ONH and retina in vivo and in vitro compared to normal controls, suggests that ephrin B1 is important in the maintenance of reactive phenotype, and presumably in preserving the blood–nerve barrier and preventing invasion of non– neural cells into areas of axonal loss. Further studies are needed to understand the signaling pathways activated in response to ephrin B1, and to elucidate its role in the maintenance of a reactive phenotype triggered by ocular hypertension.

Keywords: astrocytes: optic nerve head • intraocular pressure • retinal glia