Hydrostatic pressure-dependent changes in cyclic AMP signaling in optic nerve head astrocytes from Caucasian and African American donors.

PURPOSE: Investigate the effect of hydrostatic pressure (HP) on 3', 5'-cyclic adenosine monophosphate (cAMP) levels and downstream signaling in cultures of normal optic nerve head (ONH) astrocytes from Caucasian American (CA) and African American (AA) donors. METHODS: Intracellular cAMP levels were assayed after exposing ONH astrocytes to HP for varying times. Quantitative RT-PCR was used to determine the expression levels of selected cAMP pathway genes in human ONH astrocytes after HP treatment. Western blots were used to measure changes in the phosphorylation state of cAMP response element binding protein (CREB) in astrocytes subjected to HP, ATP, and phosphodiesterase or kinase inhibitors. RESULTS: The basal intracellular cAMP level is similar among AA and CA astrocytes. After exposure to HP for 15 min and 30 min in the presence of a phosphodiesterase inhibitor a further increase of intracellular cAMP was observed in AA astrocytes, but not in CA astrocytes. Consistent with activation of the cAMP-dependent protein kinase pathway, CREB phosphorylation (Ser-133) was increased to a greater extent in AA than in CA astrocytes after 3 h of HP. Exposure to elevated HP for 3-6 h differentially altered the expression levels of selected cAMP pathway genes (ADCY3, ADCY9, PTHLH, PDE7B) in AA compared to CA astrocytes. Treatment with ATP increased more CREB phosphorylation in CA than in AA astrocytes, suggesting differential Ca(2+) signaling in these populations. CONCLUSIONS: Activation of the cAMP-dependent signaling pathway by pressure may be an important contributor to increased susceptibility to elevated intraocular pressure and glaucoma in AA, a population at higher risk for the disease.

Quantification of retinal transneuronal degeneration in human glaucoma: a novel multiphoton-DAPI approach.

PURPOSE: Glaucoma is presumed to result in the selective loss of retinal ganglion cells. In many neural systems, this loss would initiate a cascade of transneuronal degeneration. The quantification of changes in neuronal populations in the middle layers of the retina can be difficult with conventional histologic techniques. A method was developed based on multiphoton imaging of 4',6'-diamino-2-phenylindole (DAPI)-stained tissue to quantify neuron loss in postmortem human glaucomatous retinas. METHODS: Retinas from normal and glaucomatous eyes fixed in 4% paraformaldehyde were incubated at 4 degrees C overnight in DAPI solution. DAPI-labeled neurons at different levels of the retina were imaged by multiphoton confocal microscopy. Algorithms were developed for the automated identification of neurons in the retinal ganglion cell layer (RGCL), inner nucleus layer (INL), and outer nuclear layer (ONL). RESULTS: In glaucomatous retinas, the mean density of RGCs within 4 mm eccentricity was reduced by approximately 45%, with the greatest RGC loss occurring in a region that corresponds to the central 6 degrees to 14 degrees of vision. Significant neuron loss in the INL and ONL was also seen at 2 to 4 mm and 2 to 3 mm eccentricities, respectively. The ratios of neuron densities in the INL and ONL relative to the RGCL (INL/RGC and ONL/RGC, respectively) were found to increase significantly at 3 to 4 mm eccentricity. CONCLUSIONS: The data confirm that the greatest neuronal loss occurs in the RGCL in human glaucoma. Neuronal loss was also observed in the outer retinal layers (INL and ONL) that correlated spatially with changes in the RGCL. Further work is necessary to confirm whether these changes arise from transneuronal degeneration.

Bioinformatic and statistical analysis of the optic nerve head in a primate model of ocular hypertension.

BACKGROUND: The nonhuman primate model of glaucomatous optic neuropathy most faithfully reproduces the human disease. We used high-density oligonucleotide arrays to investigate whole genome transcriptional changes occurring at the optic nerve head during primate experimental glaucoma. RESULTS: Laser scarification of the trabecular meshwork of cynomolgus macaques produced elevated intraocular pressure that was monitored over time and led to varying degrees of damage in different samples. The macaques were examined clinically before enucleation and the myelinated optic nerves were processed post-mortem to determine the degree of neuronal loss. Global gene expression was examined in dissected optic nerve heads with Affymetrix GeneChip microarrays. We validated a subset of differentially expressed genes using qRT-PCR, immunohistochemistry, and immuno-enriched astrocytes from healthy and glaucomatous human donors. These genes have previously defined roles in axonal outgrowth, immune response, cell motility, neuroprotection, and extracellular matrix remodeling. CONCLUSION: Our findings show that glaucoma is associated with increased expression of genes that mediate axonal outgrowth, immune response, cell motility, neuroprotection, and ECM remodeling. These studies also reveal that, as glaucoma progresses, retinal ganglion cell axons may make a regenerative attempt to restore lost nerve cell contact.

Population differences in elastin maturation in optic nerve head tissue and astrocytes.

PURPOSE: Glaucomatous optic neuropathy is characterized by remodeling of the extracellular matrix with disorganization of elastic fibers in the optic nerve head (ONH). There are significant differences in prevalence of glaucomatous optic neuropathy between African Americans (AAs) and Caucasian Americans (CAs). The goal of this study was to evaluate differences in elastin synthesis and maturation in ONH tissue and cells of AA and CA donors with no eye disease, to provide a basis for underlying racial differences in susceptibility to elevated intraocular pressure. METHODS: The amount of mature elastin in ONHs from each group of donors was evaluated by desmosine radioimmunoassay. The distribution of elastic fibers in ONH tissue was investigated by immunofluorescent staining. Elastin and lysyl oxidase mRNA levels and alternative splicing of elastin in ONH astrocytes were investigated by quantitative PCR. Tropoelastin protein expression was assessed by immunoblot analysis. RESULTS: ONHs from AA donors had significantly reduced levels of desmosine compared with those of CAs. In contrast, elastin mRNA and tropoelastin synthesis were elevated in ONH astrocytes from AA individuals. The inclusion of exon 23 in elastin mRNA and lysyl oxidase-like 2 mRNA levels was significantly reduced in astrocytes from AA compared with CA donors. CONCLUSIONS: A reduced number of cross-linking domains in elastin and decreased lysyl oxidase-like 2 expression leads to decreased amount of mature elastin in ONHs from healthy AA individuals compared with CA donors. These results suggest ELN and LOXL2 as candidate susceptibility genes for population-specific genetic risk of primary open-angle glaucoma (POAG).

Expression of ephrinB1 and its receptor in glaucomatous optic neuropathy.

OBJECTIVE: To determine ephrinB1, ephrinB2 and EphB1 expression in the optic nerve head (ONH) and retina of monkeys with glaucoma and in human ONH astrocytes. METHODS: Using immunohistochemistry, the localisation of ephrinB1, ephrinB2 and EphB1 was determined in the ONH and retina bilaterally in monkeys with monocular laser-induced glaucoma. RT-PCR, western blot and immunocytochemistry were used to study ephrinB1, ephrinB2 and EphB1 expression in cultured human ONH astrocytes from donors with and without glaucoma. RESULTS: There was an increase in ephrinB1 and EphB1 expression in mild to moderate glaucoma. In the ONH, both ephrinB1 and EphB1 were localised to astrocytes and EphB1 was also localised to lamina cribrosa cells and perivascular cells. In the retina, ephrinB1 localised to Muller cells and astrocytes, and EphB1 was found in retinal ganglion cells. In ONH astrocytes in humans with glaucoma, ephrinB1 and EphB1 were up-regulated but barely present in donors without glaucoma. CONCLUSIONS: Ephrins are activated in early and moderate glaucoma in the ONH and retina. We postulate that the up-regulation of Eph/ephrin pathway may play a protective role by limiting axonal damage and inflammatory cell invasion. Loss of ephrin signalling in advanced glaucoma may explain macrophage activation.

Optic nerve glioma in mice requires astrocyte Nf1 gene inactivation and Nf1 brain heterozygosity.

Whereas biallelic neurofibromatosis 1 (NF1) inactivation is observed in NF1-associated gliomas, astrocyte-restricted Nf1 conditional knockout mice do not develop gliomas. These observations suggest that NF1 glioma formation requires additional cellular or genetic conditions. To determine the effect of an Nf1 heterozygous brain environment on NF1 glioma formation, we generated Nf1+/- mice lacking Nf1 expression in astrocytes. In contrast to astrocyte-restricted Nf1 conditional knockout mice, Nf1+/- mice lacking Nf1 in astrocytes develop optic nerve gliomas. This mouse model demonstrates that Nf1+/- cells contribute to the pathogenesis of gliomas in NF1 and provides a tool for the preclinical evaluation of potential therapeutic interventions for these tumors.

Genetic networks controlling retinal injury.

PURPOSE: The present study defines genomic loci underlying coordinate changes in gene expression following retinal injury. METHODS: A group of acute phase genes expressed in diverse nervous system tissues was defined by combining microarray results from injury studies from rat retina, brain, and spinal cord. Genomic loci regulating the brain expression of acute phase genes were identified using a panel of BXD recombinant inbred (RI) mouse strains. Candidate upstream regulators within a locus were defined using single nucleotide polymorphism databases and promoter motif databases. RESULTS: The acute phase response of rat retina, brain, and spinal cord was dominated by transcription factors. Three genomic loci control transcript expression of acute phase genes in brains of BXD RI mouse strains. One locus was identified on chromosome 12 and was highly correlated with the expression of classic acute phase genes. Within the locus we identified the inhibitor of DNA binding 2 (Id2) as a candidate upstream regulator. Id2 was upregulated as an acute phase transcript in injury models of rat retina, brain, and spinal cord. CONCLUSIONS: We defined a group of transcriptional changes associated with the retinal acute injury response. Using genetic linkage analysis of natural transcript variation, we identified regulatory loci and candidate regulators that control transcript levels of acute phase genes.

Vitreous glutamate concentration and axon loss in monkeys with experimental glaucoma.

OBJECTIVE: To evaluate vitreous glutamate concentration and axon loss in monkeys with experimental glaucoma. METHODS: We induced unilateral chronic glaucoma by means of laser trabecular destruction in 14 rhesus and 6 cynomolgus monkeys. Intraocular pressure (IOP) was monitored weekly. We assessed optic nerve damage clinically and photographically. Vitreous, sampled immediately before enucleation, was analyzed for glutamate concentration by means of high-performance liquid chromatography. We quantified percentage of axon loss after histopathologic sectioning of the optic nerve, compared median glutamate concentration ratios, and assessed correlation of glutamate concentration, axon count, IOP, cup-disc ratio, duration of IOP elevation, and age. RESULTS: Median vitreous glutamate concentration in glaucomatous eyes was 7.0 micromol/L (range, 3.0-88.6 micromol/L) vs 6.7 micromol/L (range, 2.8-87.4 micromol/L) in control eyes. The ratio (glaucomatous to control eyes) was 1.08. We found no significant correlation between vitreous glutamate concentration ratio and any of the other variables. The IOP, disc cupping, and axon loss were correlated. CONCLUSIONS: We found no difference between vitreous glutamate concentration in glaucomatous and contralateral control monkey eyes when the entire data set was examined and no evidence of correlation between vitreous glutamate concentration and axon loss. CLINICAL RELEVANCE: Vitreous concentration of the excitotoxic amino acid glutamate, thought to be associated with retinal ganglion cell death in glaucoma, was not altered in this study.

DNA microarray analysis of gene expression in human optic nerve head astrocytes in response to hydrostatic pressure.

There is clinical and experimental evidence that elevated intraocular pressure (IOP), a mechanical stress, is involved in the pathogenesis of glaucomatous optic neuropathy. The mechanism by which astrocytes in the optic nerve head (ONH) respond to changes in IOP is under study. Gene transcription by ONH astrocytes exposed either to 60 mmHg hydrostatic pressure (HP) or control ambient pressure (CP) for 6, 24, and 48 h was compared using Affymetrix GeneChip microarrays to identify HP-responsive genes. Data were normalized across arrays within each gene. A linear regression model applied to test effect of time and HP on changes in expression level identified 596 genes affected by HP over time. Using GeneSpring analysis we selected genes whose average expression level increased or decreased more than 1.5-fold at 6, 24, or 48 h. Expression of selected genes was confirmed by real-time RT-PCR; protein levels were detected by Western blot. Among the genes highly responsive to HP were those involved in signal transduction, such as Rho nucleotide exchange factors, Ras p21 protein activator, tyrosine kinases and serine threonine kinases, and genes involved in transcriptional regulation, such as c-Fos, Egr2, and Smad3. Other genes that increased expression included ATP-binding cassettes, solute carriers, and genes associated with lipid metabolism. Among the genes that decreased expression under HP were genes encoding for dual activity phosphatases, transcription factors, and enzymes involved in protein degradation. These HP-responsive genes may be important in the establishment and maintenance of the ONH astrocyte phenotype under conditions of elevated IOP in glaucoma.

Tissue differential microarray analysis of dexamethasone induction reveals potential mechanisms of steroid glaucoma.

PURPOSE: To identify myocilin (TIGR/MYOC) properties that are specific to the human trabecular meshwork (HTM). To search for genes highly expressed in dexamethasone (DEX)-induced HTM cells that are barely expressed or absent in DEX-induced cells from other tissues. METHODS: TIGR/MYOC induction by DEX (10(-7) M for 8-10 days) was analyzed by Northern and Western blot analyses in HTM, human umbilical vein endothelial cells, HeLa cells, and human embryonic skeletal muscle cells and optic nerve head (ONH) astrocytes at confluence. Processing and secretion were analyzed after the cells were infected with adenoviruses overexpressing wild-type and mutant forms of TIGR/MYOC. Affymetrix U95Av2 GeneChips (n = 6) and software were used to compare paired expression profiles of HTM, HTM-DEX, ONH astrocytes, and ONH astrocytes-DEX. Identification of HTM-DEX-specific genes (compared with ONH astrocytes-DEX) was performed by selecting genes with the highest fold change values (>/=20). Genes with fold change values of four or more were matched with loci linked to glaucoma, by using gene databases. RESULTS: TIGR/MYOC induction by DEX occurred only in HTM cells. Secretory and glycosylation characteristics remained the same across cell types. Expression profile analysis revealed multiple genes differentially upregulated in HTM-DEX including, in addition to TIGR/MYOC, a serine protease inhibitor (alpha1-antichymotrypsin), a neuroprotective factor (pigment epithelium-derived factor), an antiangiogenesis factor (cornea-derived transcript 6), and a prostaglandin synthase (prostaglandin D(2) synthase). Fifteen of the 249 genes with fold change values of four or more mapped to glaucoma-linked loci. CONCLUSIONS: The induction of TIGR/MYOC by DEX is HTM-specific, whereas its secretory and glycosylation characteristics are ubiquitous. The known functions of HTM-DEX-specific genes reveal the presence of protective and damaging mechanisms for regulation of IOP during DEX treatment. Besides TIGR/MYOC, other HTM-DEX-specific genes may be good candidates for linkage to glaucoma.