Hydrostatic Pressure Responsive Genes in Optic Nerve Head Astrocytes

Abstract

Abstract: : Purpose: To identify genes differentially expressed under elevated hydrostatic pressure (HP) in optic nerve head (ONH) astrocytes. Methods: Cultured ONH astrocytes from two different donors were exposed to 60 mm Hg HP for 6, 24 and 48h. Total RNA was isolated from astrocytes exposed to pressure and matched controls. Labeled cRNA was hybridized to 47 human Genome U95Av2 chips using GeneChip Instrument System. Affymetrix spiked probes and probes that were absent across all chips (N=5,466) were deleted leaving 7092 probes for subsequent analyses. Data were normalized across chips within each gene. A linear regression model was applied to each gene to test the effect of time and pressure on changes in expression level. Significance was set at p<0.05 and no adjustment made for multiple testing. Expression of selected genes was confirmed by real time RT-PCR, corresponding protein level was detected by western blot. Results: The linear regression model identified 744 genes differentially expressed under HP over time, of which 371 genes increased and 373 genes decreased. Using GeneSpring analysis we selected genes whose average expression level increased or decreased more than 1.5 fold at 6, 24 or 48 h. Under HP expression of 12 genes increased at 6 h, 8 genes at 24 h and 26 genes at 48 h. Amongst genes highly responsive to HP were MCF2 encoding a guanine nucleotide exchange factor and RASA2 encoding a Ras GTPase activating protein. Genes that increased significantly only at 48h included IL13 receptor, solute carriers, genes associated with ocular and brain development, and two genes with unidentified protein products. 17 genes decreased at 6 h, 22 genes at 24 h and 101 genes at 48 hr. Decreased genes encoded for ion channels, protein phosphatases and cell adhesion. Conclusions: In this study we selected a subset of novel and specific hydrostatic pressure responsive genes expressed by ONH astrocytes. Activation of gene products involved in intracellular signaling may regulate astrocyte responses to pressure.

Keywords: gene/expression • gene microarray