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T.J. Chipps, B. Appukuttan, J.T. Stout, J.T. Rosenbaum, J.R. Smith; Regulation of Intercellular Adhesion Molecule 1 Expression in Human Retinal Vascular Endothelium . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5159.
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© ARVO (1962-2015); The Authors (2016-present)
Intercellular adhesion molecule 1 (ICAM–1) is a cell adhesion molecule expressed constitutively on vascular endothelium, but up–regulated by many stimuli including microbes, inflammatory cytokines, advanced glycation end products, and oxidative stress. It is implicated in a range of retinal vascular diseases and our unpublished data from gene expression microarray analysis suggests that retinal endothelium expresses approximately 5–fold more ICAM–1 than choroidal endothelium. Transcription of the ICAM–1 gene is regulated by multiple transcription factors. Our goal was to develop a method for determination of specific cis–regulatory elements controlling gene expression within retinal vascular endothelial cells (HREC) in response to different stimuli.
Efficiency of DNA transfer into primary HREC was determined using a plasmid encoding GFP (pGFP) and various protocols on the NucleofectorTM electroporation device (Amaxa). The promoter region of the human ICAM–1 gene was amplified from gDNA and cloned into a plasmid with a secretable alkaline phosphatase reporter gene (pSEAP). HREC were grown to confluence in modified MCDB–131 medium, nucleofected with pSEAP–ICAM–1 promoter or pSEAP without insert, recovered overnight, and stimulated by infection with T. gondii tachyzoites. Cell supernatant, collected 4 hours after infection, was assayed in triplicate for the presence and amount of SEAP, using a chemiluminescent assay.
Of 5 protocols tested, Program M–03 on NucleofectorTM gave the highest efficiency for transfection of HREC with pGFP, with 75 % efficiency and 90 % cell viability. Sequencing analysis confirmed successful cloning of a 1327 bp fragment of the 5’ flanking promoter of human ICAM–1, starting 20 bp upstream of the translation start site. This promoter fragment contained 2 alternative transcription initiation sites and at least 17 cis–regulatory elements including NF–ΚB, GAS, TRE and C/EBP. A preliminary experiment indicated that, after infection with tachyzoites, significantly more SEAP was present in supernatant from HREC transfected with pSEAP–ICAM–1 promoter than in supernatant from control cells transfected with pSEAP without insert (p=0.019).
We have developed an assay that, with application of deletion construction technology, promises to allow determination of stimulus–specific transcription factor binding sites within the ICAM–1 promoter. Transcription factors that control expression of the ICAM–1 gene in retinal endothelium may present specific targets for the treatment of retinal vascular diseases.
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