M. Rosario Hernandez

The optic nerve head in glaucoma: role of astrocytes in tissue remodeling

Primary open angle glaucoma is a common eye disease characterized by loss of the axons of the retinal ganglion cells leading to progressive loss of vision. The site of damage to the axons is at the level of the lamina cribrosa in the optic nerve head. The mechanism of axonal loss is unknown but elevated intraocular pressure and age are the most common factors associated with the disease. Previous studies in human glaucoma and in experimental glaucoma in monkeys have established a relationship between chronic elevation of intraocular pressure and remodeling of the optic nerve head tissues known clinically as cupping of the optic disc. This review focuses on the astrocytes, the major cell type in the optic nerve head. Astrocytes participate actively in the remodeling of neural tissues during development and in disease. In glaucomatous optic neuropathy, astrocytes play a major role in the remodeling of the extracellular matrix of the optic nerve head, synthesize growth factors and other cellular mediators that may affect directly, or indirectly, the axons of the retinal ganglion cells. Due to the architecture of the lamina cribrosa, formed by the cells and the fibroelastic extracellular matrix, astrocytes may respond to changes in intraocular pressure in glaucoma, leading to some of the detrimental events that underlie axonal loss and retinal ganglion cell degeneration.

Differential gene expression in astrocytes from human normal and glaucomatous optic nerve head analyzed by cDNA microarray

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. GLIA 38:45–64, 2002.

Astrocyte responses in human optic nerve head with primary open-angle glaucoma.

PurposeTo identify and characterize astrocyte responses and reactivation in human optic nerve heads from patients with primary open-angle glaucoma. MethodsFifteen optic nerve heads with primary open-angle glaucoma and 13 normal controls were fixed in 4% paraformaldehyde, paraffin embedded, and stained for immunofluorescence and immunoperoxidase. The antibodies used were against glial fibrillary acidic protein (GFAP) and against neural cell adhesion molecule (N-CAM). ResultsTwo subpopulations of type 1 astrocytes exist in the normal optic nerve. Type IA astrocytes express only glial fibrillary acidic protein and type IB express both glial fibrillary acidic protein and neural cell adhesion molecule. These are the major cell subpopulations in the lamina cribrosa and prelaminar regions. In primary open angle glaucoma, type IB astrocytes in the prelaminar region showed increased immunoreactivity for glial fibrillary acidic protein and neural cell adhesion molecule, and cytoplasmic enlargement with thicker and longer cytoplasmic processes. At the level of the lamina cribrosa, type IB astrocytes appeared round and the cell bodies were no longer in the cribriform plates but located in the nerve bundles. Type IA astrocytes were not observed in the glaucomatous optic nerve head. ConclusionsAstrocyte responses in primary open angle glaucoma may underlie cellular changes that lead to axonal damage and optic nerve head remodeling. These responses may have pathogenic significance for glaucomatous optic neuropathy.

Astrocytes in glaucomatous optic neuropathy.

Glaucoma, the second most prevalent cause of blindness worldwide, is a degenerative disease characterized by loss of vision due to loss of retinal ganglion cells. There is no cure for glaucoma, but early intervention with drugs and/or surgery may slow or halt loss of vision. Increased intraocular pressure (IOP), age, and genetic background are the leading risk factors for glaucoma. Our laboratory and other investigators have provided evidence that astrocytes are the cells responsible for many pathological changes in the glaucomatous optic nerve head (ONH). Over several years, in vivo and in vitro techniques characterized the changes in quiescent astrocytes that lead to the reactive phenotype in glaucoma. Reactive astrocytes alter the homeostasis and integrity of the neural and connective tissues in the ONH of human and experimental glaucoma in monkeys. During the transition of quiescent astrocytes to the reactive phenotype altered astrocyte homeostatic functions such as cell-cell communication, migration, growth factor pathway activation, and responses to oxidative stress may impact pathological changes in POAG. Our data also suggests that the creation of a non-supportive environment for the survival of RGC axons through remodeling of the ONH by reactive astrocytes leads to progression of glaucomatous optic neuropathy.

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human optic nerve head astrocytes

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.

The Role of Glia, Mitochondria, and the Immune System in Glaucoma

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group

Expression of small heat shock proteins and intermediate filaments in the human optic nerve head astrocytes exposed to elevated hydrostatic pressure in vitro

The small heat shock proteins (sHSP), αB‐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 αB‐crystallin and Hsp27 with IF in cultured ONH astrocytes. Astrocyte monolayers were pressurized to 60 mm Hg (92% air 8% CO2) and incubated at 37°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, αB‐crystallin, vimentin or GFAP. Control astrocytes seemed flat, polygonal with short processes. αB‐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 αB‐crystallin in the perinuclear area. Western blot and metabolic labeling detected increased synthesis of Hsp27, GFAP and vimentin but no change in αB‐crystallin. These results indicated that GFAP and vimentin associate with Hsp27 and αB‐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. J. Neurosci. Res. 66:59–73, 2001.

Hydrostatic pressure stimulates synthesis of elastin in cultured optic nerve head astrocytes

Elastin is a major component of the extracellular matrix (ECM) of the lamina cribrosa in the optic nerve head in humans and nonhuman primates. The lamina cribrosa appears to be the site of damage to the retinal ganglion cell axons in glaucomatous optic neuropathy, characterized in many patients by elevated intraocular pressure (IOP). Type 1B astrocytes are the major cell type in the lamina, synthesize elastic fibers during development, express increased elastin mRNA, and synthesize abnormal elastin in glaucoma. In this study, we determined the effect of elevated hydrostatic pressure on the synthesis of elastin by type 1B astrocytes in culture. Type 1B astrocytes were exposed to gradients of hydrostatic pressure and tested for proliferation, morphology, synthesis, and deposition of elastin. Trichloroacetic acid (TCA) and immunoprecipitation of radiolabeled protein determined total new protein and elastin synthesis. Proteins from the conditioned media were analyzed by Western blot. Levels of elastin mRNA were determined by in situ hybridization. Cell proliferation increased ∼2‐fold after exposure to pressure for one day, ∼5‐fold after 3 and 5 days of exposure to pressure. Confocal and electron microscopic cytochemistry showed a marked increase in intracellular elastin in astrocytes exposed to pressure, as compared with controls. Intracellular elastin was associated with the RER‐Golgi region and with the cytoskeleton. Total protein and elastin synthesis increased significantly (P < 0.05) at 3‐ and 5‐day exposure to pressure, as well as the level of elastin mRNA. Elastin protein in the media increased with the level of pressure. These results indicate that hydrostatic pressure stimulates type 1B astrocytes to synthesize and secrete soluble elastin into the media. In glaucoma, type 1B astrocytes may respond to IOP‐related stress with increased expression of elastin and formation of elastotic fibers leading to loss of elasticity and tissue remodeling. GLIA 32:122–136, 2000.

In vitro evaluation of reactive astrocyte migration, a component of tissue remodeling in glaucomatous optic nerve head

In order to improve understanding of remodeling events in the glaucomatous optic nerve head, the migration of optic nerve head astrocytes was studied in vitro. Since elevated intraocular pressure is an important stress factor identified in glaucomatous eyes, optic nerve head astrocytes were incubated under physical stress created by elevated hydrostatic pressure. In addition, they were incubated in the presence of a chemical stimulus, lipolysaccharide (LPS). Migration of reactivated astrocytes in the presence of these stressors was examined using chambers in which cell migration through extracellular matrix‐coated pores is only possible following proteolytic digestion of the matrix. We observed that the migratory ability of optic nerve head astrocytes was approximately 4–6 times greater following exposure to elevated hydrostatic pressure or LPS for up to 48 h. Phosphoinositide 3‐kinase, protein kinase C, and tyrosine kinase were found to be involved in the signal transduction for activated migration of optic nerve head astrocytes in response to elevated hydrostatic pressure or LPS. In addition, we observed that the stress‐induced migration of optic nerve head astrocytes, which is accompanied by proteolytic degradation, resulted in the formation of culture cavities containing mucopolysaccharides. These in vitro findings provide a clearer understanding of the pathophysiologic mechanisms of characteristic tissue remodeling events that occur, in vivo, in the glaucomatous optic nerve head. GLIA 34:178–189, 2001.

Selective expression of neural cell adhesion molecule (NCAM)-180 in optic nerve head astrocytes exposed to elevated hydrostatic pressure in vitro.

Glaucomatous optic neuropathy is usually associated with elevated intraocular pressure. Optic nerve head astrocytes may respond to intraocular pressure by stimulation of pressure-sensitive mechanoreceptors on the cell surface. Neural cell adhesion molecule (NCAM) a transmembrane protein, mediates cell adhesion and migration. The NCAM 180 isoform increases in astrocytes of glaucomatous optic nerve head. We characterized the relative expression of NCAM isoforms in human optic nerve head astrocytes grown under elevated hydrostatic pressure. Astrocytes cultured from normal human optic nerve heads were exposed to either atmospheric or continuous hydrostatic pressure of 60 mm Hg, and analyzed at 6-48 h. Changes in cell shape, immunoreactivity, and distribution of GFAP, actin and NCAM were observed in pressure-treated cultures. Newly synthesized (35)S-labeled NCAM protein immunoprecipitated from cell lysates was increased 2-fold within 24 h after exposure to elevated pressure compared to control. The increase in NCAM synthesis was primarily due to the NCAM 180 isoform. A significant increase in NCAM 180 mRNA levels was detected by RT-PCR and Northern blots in cultured optic nerve head astrocytes within 6 h after exposure to elevated pressure. NCAM 180 mRNA and protein synthesis decreased after 24 h and returned to control levels by 48 h. Our data indicate that NCAM 180 transcription and synthesis in astrocytes is stimulated by elevated hydrostatic pressure. Because NCAM 180 interacts with the cytoskeleton through an extended cytoplasmic tail, a selective and transient increase in NCAM 180 in optic nerve head astrocytes exposed to elevated pressure may be relevant to the migration and interactions of reactive astrocytes in glaucoma.