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J.R. Smith, D. Choi, T.J. Chipps, Y. Pan, D. Zamora, S.R. Planck, M. Mori, J.T. Rosenbaum; Microarray Identifies Unique Gene Expression Profiles of Human Retinal and Choroidal Vascular Endothelium in Donor–Matched Cell Cultures . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2922.
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© ARVO (1962-2015); The Authors (2016-present)
Consistent with recent recognition that vascular beds throughout the body express different sets of cell adhesion molecules, we hypothesized that retinal vascular endothelium was a cell population of unique molecular phenotype. We compared gene expression profiles of human retinal and choroidal vascular endothelial cells (EC) both in unstimulated and stimulated (by infection with T. gondii tachyzoites) states using microarray.
Paired cultures of retinal and choroidal EC were prepared from 3 human cadaver donors by digestion with Type II collagenase 0.2% followed by purification using Dynal magnetic beads linked to anti–CD31 antibody. Confluent monolayers of EC, grown in modified MCDB–131 medium and used at passages 3–5, were incubated with or without tachyzoites (10:1, parasite:cell) for 4 hours. RNA was extracted, reverse transcribed into cDNAs, transcribed and amplified into biotin–labeled amplified RNAs and hybridized to oligonucleotide Affymetrix human genome focus arrays detecting 8500 genes. Intensities of perfect match probes were normalized by GCRMA–invariant set–medial polish. Differential expression was assessed by SAM with false discovery rate set at 5%.
Graphical descriptive analysis revealed that expression profiles varied between donors, and for retinal or choroidal EC following stimulation with tachyzoites. However, greatest differences in gene expression were observed when choroidal EC were compared with retinal EC. Comparison of unstimulated retinal and choroidal EC revealed differences in 478 genes (45% up–regulated and 55% down–regulated in retinal EC). Selecting for genes that showed a 2–fold change in the same direction in all 3 donors, 78 genes were up–regulated and 56 genes were down–regulated. Cluster analysis of genes (FatiGO, Level 6) revealed that 34% of the total up–regulated genes were associated with the immune response and 23% were associated with pathogen responses (eg. IL–8, GROα, GROß, GCP–2, IP–10). 59% of up–regulated genes and 35% of down–regulated genes were integral to the cell membrane.
Retinal vascular endothelium displays a unique pattern of gene expression in comparison to choroidal endothelium. Our findings are relevant to the understanding of mechanisms responsible for homing of leukocytes or microbes to the retina. This understanding may direct the development of new uveitis treatments that target tissue–specific interactions between retinal endothelium and an infiltrating cell or invading microbe.
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