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T. J. Chipps, M. H. Davies, Y. Pan, D. O. Zamora, S. R. Planck, M. R. Powers, J. T. Rosenbaum, J. R. Smith; High Expression of Gene Transcripts Related to the Immune Response in Human Retinal Vascular Endothelial Cells: A Quantitative Real-Time RT-PCR Study. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5179.
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Posterior uveitis is a diverse group of inflammatory or infectious diseases involving the posterior segment of the eye with potential to cause substantial ocular morbidity. Retinal vasculitis commonly accompanies the uveitis, and work by other groups indicates that leukocytes migrate into the posterior eye via the retinal vasculature. Previous work from our group using microarray suggests that human retinal vascular endothelial cells (ECs) have a unique pattern of gene expression, and, in particular, demonstrate relatively high expression of genes related to the immune response. In this study we sought to confirm this observation using cultured retinal and choroidal ECs and quantitative real-time RT-PCR (qRT-PCR).
Retina and choroid were dissected from both eyes of a 36-year-old male cadaver, and ECs were isolated from each tissue by enzymatic digestion with 0.5 - l mg/mL Type II collagenase and subsequent purification using Dynal magnetic beads conjugated to anti-human CD31 antibody. Following expansion in modified MCDB-131 medium, ECs in 3rd passage were grown to confluence. After extraction of total RNA, relative expression of the following gene transcripts in retinal and choroidal ECs was determined by qRT-PCR using the Chromo4 Thermocycler (Bio-Rad): intercellular adhesion molecule 1 (ICAM-1); vascular cell adhesion molecule 1 (VCAM-1); ephrin B2; E-selectin; interleukin (IL)-8; interferon gamma receptor 1 (IFNγR1); CCL2; CXCL6; CX3CL1; toll-like receptor 1 (TLR-1); stromelysin 2 (MMP-10); and, as controls, von Willebrand factor (vWF) and CD31.
There was significantly greater expression of all assayed transcripts in retinal ECs than in choroidal ECs. The fold differences for each transcript were as follows: ICAM-1, 4.6-fold; VCAM-1, 10.6-fold; ephrin B2, 2.6-fold; E-selectin, 5.6-fold; IL-8, 24.9-fold; IFNγR1, 2.1-fold; CCL2, 109-fold; CXCL6, 92.5-fold; CX3CL1, 9.4-fold; TLR-1, 3.8-fold; and MMP-10, 3.0-fold. Control transcripts demonstrated < 2-fold difference in abundance between retinal and choroidal ECs.
Human retinal ECs express higher basal levels of selected gene transcripts encoding proteins involved in the immune response in comparison to choroidal ECs. When immune privilege is breached and posterior uveitis is initiated, the retinal vascular endothelium is appropriately constituted to facilitate leukocyte migration. Treatments that target the retinal EC may have utility in the management of posterior uveitis.
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