Recent advances in cDNA microarray technology have made it possible to analyze expression of several thousand genes at the same time. Using this technique, gene expression in human astrocytes cultured from glaucomatous and normal optic nerve heads (ONH) was compared. One hundred-fifty genes were differentially expressed more than 5-fold in glaucomatous cell cultures compared with normal. These genes are involved in a number of biological processes, including signal transduction, cell adhesion and proliferation, ECM synthesis, and degradation. Confirmation of differential gene expression was performed by quantitative RT-PCR. Western blots and immunohistochemistry demonstrated gene products in cell cultures or in human ONH tissues. Proliferation, adhesion and migration assays tested physiological responses suggested by differential gene expression. Our study suggests that cultured glaucomatous ONH astrocytes retain in culture many phenotypic characteristics that may be relevant to their role in the pathogenesis of glaucoma and, in general to reactive astrocytes in the CNS. Potential applications of these data include the identification and characterization of signaling pathways involved in astrocyte function, studies of the role of steroid-metabolizing enzymes in the glaucomatous ONH, and further exploration of the role of selected identified genes in experimental animal and in vitro models of glaucoma.

Glaucomatous optic neuropathy is a common blinding disease characterized by remodeling of the extracellular matrix (ECM) and loss of retinal ganglion cell (RGC) axons at the level of the optic nerve head (ONH). Astrocytes, the major cell type in ONH, may participate in this process by production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). In normal and glaucomatous ONH, we detected MMP and TIMP expression by immunohistochemistry. Cultured astrocytes were used to characterize expression of MMPs and TIMPs by zymography, Western blot, and RNase protection assay. MMP production was stimulated with phorbol 12‐myristate 13‐acetate (PMA). Astrocytes expressed MMP1, MT1‐MMP, MMP2, TIMP1, and TIMP2 in normal and glaucomatous ONH. MMP2, TIMP1, and TIMP2 localized to RGCs and their axons. Increased MMP1 and MT1‐MMP expression was demonstrated in glaucoma. Cultured astrocytes constitutively expressed MMP2, MT1‐MMP, TIMP1, and TIMP2, whereas MMP3, MMP7, MMP9, and MMP12 were not detectable in tissues or in cultured astrocytes. Our findings demonstrate the presence of specific MMPs and TIMPs in the ONH that may participate in the homeostasis and remodeling of the ECM in glaucoma. Expression of the same MMPs and TIMPs in cultured ONH astrocytes will allow further studies on the mechanisms regulating these enzymes. GLIA 33:205–216, 2001. © 2001 Wiley‐Liss, Inc.

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The small heat shock proteins (sHSP), alpha B-crystallin and Hsp27 are chaperone molecules that maintain the integrity of intermediate filament (IF) network and prevent unfolding of cellular proteins induced by stress. In the optic nerve head (ONH) of eyes with glaucoma, reactive astrocytes expressed Hsp27, perhaps in response to stress related to elevated intraocular pressure. In this study, we determined the effect of elevated hydrostatic pressure (HP) in the synthesis, distribution and co-localization of alpha B-crystallin and Hsp27 with IF in cultured ONH astrocytes. Astrocyte monolayers were pressurized to 60 mm Hg (92% air 8% CO(2)) and incubated at 37 degrees C for 6, 24 or 48 hr. Controls were exposed to ambient pressure. Cells were analyzed by immunocytochemistry, Western blot and immunoprecipitation using antibodies to Hsp27, alpha B-crystallin, vimentin or GFAP. Control astrocytes seemed flat, polygonal with short processes. alpha B-crystallin appeared granular in the perinuclear area and filamentous in the cell periphery. Fine granular Hsp27 was distributed throughout the cytoplasm. GFAP and vimentin co-localized with Hsp27 in the cytoplasm. Astrocytes exposed to HP were star-shaped with long processes. Hsp27 was condensed in large granules around the nucleus. GFAP and vimentin co-localized with Hsp27 and alpha B-crystallin in the perinuclear area. Western blot and metabolic labeling detected increased synthesis of Hsp27, GFAP and vimentin but no change in alpha B-crystallin. These results indicated that GFAP and vimentin associate with Hsp27 and alpha B-crystallin in ONH astrocytes. HP affected the integrity of the cytoskeleton consistent with morphological changes. Small HSP may reinforce and maintain IF integrity in response to HP.

In this study, we examined the effects of mechanical stress induced by elevated hydrostatic pressure (HP) on the migration of human optic nerve head (ONH) astrocytes, using an in vitro model that follows repopulation of a cell-free area (CFA) created on a monolayer of cultured astrocytes. alpha-Tubulin staining detected phenotypic changes in astrocytes exposed to HP. The influence of proliferation in closure of the CFA was determined by incorporation of BrdU under 1.5-cm H2O, control pressure (CP), and 10-cm H2O HP with or without 5-fluorouracil. Under control and experimental conditions, closure of the CFA occurred mostly by migration and less by proliferation. Exposure to 10-cm H2O HP induced faster closure of the CFA at 1, 3, and 5 days. The signaling pathways involved in responses to HP were determined using genistein, tyrphostin A25, AG1478, and AG1295, inhibitors of receptor tyrosine kinases; wortmannin and LY294002, inhibitors of phosphatidyl inositol 3-kinase (PI-3K); and SC58236, an inhibitor of inducible cyclooxygenase-2 (COX2). Genistein and tyrphostin A25 blocked HP-induced migration at 1, 3, and 5 days, but did not affect closure of the CFA under CP. AG1478 and AG1295 blocked HP-induced migration and partially inhibit closure of the CFA under CP. LY294002 blocked HP-induced migration. SC58236 markedly inhibited closure of the CFA under CP by inhibiting COX2 activity. Exposure to HP, a physical stress, induced faster closure of the CFA via activation of members of the epidermal growth factor receptor (EGFR) family and PI-3K pathways. Under CP, closure of the CFA in response to denudation, a form of injury, is due to activation of COX2 in ONH astrocytes.