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A. C. Browning, E. Halligan, R. Dove, E. Elton, W. M. K. Amoaku; Gene Expression Profiling of Human Ocular Microvascular Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2933.
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© ARVO (1962-2015); The Authors (2016-present)
To compare the gene expression profiles of HUVEC and unpassaged, proliferating, human iris, retinal and choroidal microvascular endothelial cells.
Human microvascular endothelial cells were isolated from fresh human eyes using anti-CD31 coated dynabeads. HUVECs (unpassaged) were obtained from Promocell. All cells were grown to approximately 80% confluence and the RNA isolated using the Qiagen RNeasy minikit. Biotinylated cRNA probes were prepared from the total RNA samples. RNA was reverse transcribed to cDNA which was then amplified by PCR and converted to cRNA The cRNA samples were then fragmented and hybridised onto Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. The chips were scanned using Affymetrix GeneChip protocols. Comparative analysis of gene expression profiles was performed using the dCHIP software and Significance Analysis of Microarrays (SAM) The sorting and classification of significant genes into different biological processes was conducted using PANTHER.
Examination of genes showing a two-fold or higher expression demonstrated a dramatic difference in expression patterns reflecting sites of EC origin. This revealed that there were 149 genes upregulated in HUVEC cells and 317 upregulated in ocular microvascular ECs. These differences between macro and microvascular ECs were reflected in differences in expression of genes involved in a range of biological processes. Using SAM analysis, 1804 genes were differentially expressed between proliferating human retinal and choroidal endothelial cells while 76 genes were differentially expressed between unpassaged proliferating human iris and choroidal endothelial cells.
Gene expression profiling revealed marked differences between macrovascular and ocular microvascular endothelial cells. This suggests that HUVEC cells may not be a suitable surrogate for ocular microvascular ECs when studying ocular disorders. The results also reveal significant differences between the gene expression of human retinal and choroidal ECs. These differences may be important in the mechanisms of various site specific ocular diseases. Interestingly, there were very few differences in the gene expression of iris and choroidal ECs.
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