Genome–Wide Microarray Analysis of the Gene Expression Profile of Normal and Primary Open Angle Glaucoma Human Trabecular Meshwork (HTM) Tissue and Cultured HTM Cells

Abstract

Abstract: : Purpose: To generate comprehensive profiles of gene expression from normal and primary open angle glaucoma HTM tissue, as well as cultured HTM cells. Methods: The anterior segments of two donor eyes without a history of eye disease (ages 78 and 85) and two donors with a confirmed history of POAG (ages 59 and 77) were fixed in RNAlater less than 5 hours postmortem. TMs were dissected, and total RNA was extracted. Two primary HTM cell lines at passage 3 from donors without glaucoma were grown to confluency, and total RNA was extracted. RNA samples were labeled with biotin and hybridized to Affymetrix U133 plus 2.0 arrays containing over 54,675 probe sets representing 47,000 transcripts. Array data was analyzed using the Genespring software from Silicon Genetics, and differential expression of selected genes was confirmed by quantitative PCR. Results: Array analysis of HTM normal tissue RNA identified 19,377 gene transcripts present in all four HTM samples analyzed. POAG samples showed relatively small changes compared to normal controls. These changes included upregulation of several genes associated with inflammation, including interleukin–8, immune–associated nucleotide, and selectin–E, as well as down–regulation of several genes potentially important in outflow modulation, including MMP–10 (stromelysin 2), WNT inhibitory factor–1, contactin binding protein–2, and latrophilin–3. Analysis of cultured HTM cells demonstrated that although the expression of some genes characteristic of the HTM tissue, like CHI3L1 was retained, a number of genes potentially important in HTM physiology including MYOC, CDT6, SOD–1, and matrix GLA were down–regulated. Cell culture conditions were also associated with increased expression of certain genes involved in extracellular matrix formation including fibronectin. Conclusions: Comparative analysis of the gene expression profiles of POAG donors and age–matched controls suggests that, similar to other age–related diseases like atherosclerosis, POAG might be associated with chronic activation of a proinflammatory–like response. Certain genes down–regulated in cell culture conditions may be associated with specialized functions of the TM cells in vivo. Down–regulation of these genes might result from the fact that standard cell culture conditions do not include the same biomechanical forces, tri–dimensional tissue structure, and specific aqueous humor composition influencing the cells of the TM in vivo.

Keywords: gene microarray • trabecular meshwork • gene/expression