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

Abstract

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.

Genetic Networks Modulating Retinal Injury

“…We previously published CNS microarray analyses that examined the transcriptome response of injured rat retina and brain, the transcriptome of cultured human and rat astrocytes, and the regulatory loci controlling transcript expression in the mouse brain [Vazquez et al, 2004;Matzilevich et al, 2002;Dash et al, 2004;Chesler et al, 2004;Yang et al, 2004]. To complement our analysis of reactive CNS genes, we obtained publicly available data that examined the transcriptome response of injured rat spinal cord, the transcriptome of developing mouse retina, as well as the transcriptome of cultured mouse neurons, mouse astrocytes, and rat microglia ; Duker et al, We performed a meta-analysis of previous experimental data to select a group of acute phase genes that are part of a common response to injury in neural tissues.…”

“…To complement our analysis of reactive CNS genes, we obtained publicly available data that examined the transcriptome response of injured rat spinal cord, the transcriptome of developing mouse retina, as well as the transcriptome of cultured mouse neurons, mouse astrocytes, and rat microglia ; Duker et al, We performed a meta-analysis of previous experimental data to select a group of acute phase genes that are part of a common response to injury in neural tissues. Previously, we examined transcript levels in injured retina, injured brain, cultured astrocytes, and in forebrains from a panel of 35 BXD RI strains [Vazquez et al, 2004;Matzilevich et al, 2002;Dash et al, 2004;Chesler et al, 2004;Yang et al, 2004]. Microarray data sets describing transcript levels in injured spinal cord, cultured microglia, cultured neurons, and developing retina were obtained from publicly available published studies Duker et al, 2004;Moran et al, 2004;Kraft et al, 2004;Dorell et al, 2004].…”

“…Astrocytic, microglial, and neuronal expressions refer to moderate to high transcript levels in cultured astrocytes [Yang et al, 2004;Kraft et al, 2004], microglia [Duker et al, 2004;Moran et al, 2004], neurons [Kraft et al, 2004]. Chronic expression describes expression in rat retina from 3 to 30 days after injury [GEO data set GSE1001; Vazquez et al, 2004].…”

“…Our working group of wound-healing genes was defined by comparing injured retinal time points versus normal retina and using three criteria: fold changes > |±2|, Student's ttest (P < 0.05), and genes with moderate-to-high expression levels (please see Chapter 2 for discussion on expression thresholds). To complement our analysis of wound-healing genes, we obtained publicly available microarray analyses that examined the transcriptome response of developing mouse retina [Dorell et al, 2004]; the transcriptome of cultured mouse neurons [Kraft et al, 2004], mouse astrocytes [Kraft et al, 2004] rat Muller cells [Gerhardinger et al, 2005], human optic nerve astrocytes [Yang et al, 2004] ).…”

“…Gene expression patterns in the injured rat retina were obtained using our data available online at Gene Expression Omnibus (GEO, www.ncbi.hlm.nih.gov) as the group series GSE1001. Gene expression levels from cultured mouse neurons [Kraft et al, 2004], mouse astrocytes [Kraft et al, 2004], rat Muller cells [Gerhardinger et al, 2005], human optic nerve astrocytes [Yang et al, 2004], human U87 glioma cells [unpublished data, Eldon Geisert], and rat RPE cells [unpublished data, Eldon Geisert] were obtained from published and unpublished data. Asterisk represent genes with no probe set available.…”