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T.–H. Wu, J.T. Handa, J.D. Gottsch; LIGHT INDUCED–OXIDATIVE STRESS IN MOUSE CHORIOCAPILLARYENDOTHELIAL CELLS . Invest. Ophthalmol. Vis. Sci. 2004;45(13):767.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate light–induced activation of nuclear factor–ΚB (NF–ΚB) in mouse choriocapillary endothelial cells (CECs). Methods: CECs were isolated from the mouse RPE/choroid tissue using an anti–CD31 antibody and magnetic beads. CECs were characterized by the expression and localization of two endothelial cell markers, CD31 and P1H12, with double immunofluorescence staining. The quality and amount of RNA isolated from CECs were assessed on a bioanalyzer. The integrity of the CD31–transcript in the RNA was detected by RT–PCR. The contamination of the RNA was estimated by measuring the expression levels of a retinal pigment epithelial cell (RPE) maker RPE65 and two endothelial cell markers, CD31 and von Willebrand Factor (vWF). For mouse light exposure experiments, a green light was used at an illuminance of 3.1 to 3.5 klux for 3 h. The light–stressed CECs were then isolated for triple immunofluorescence staining for IΚBα, phosphorylated IΚBα (pIΚBα), and CD31. The expression levels of IΚBα in CECs were determined by real time quantitative PCR. Results: About 80–90% of isolated CECs expressed both CD31 and P1H12. Total RNA, 5ng, was obtained from CECs extracted from four mice. The ratio of 28S/18S in the total RNA was about 1.5. The CD31 transcript of the RNA could be amplified by RT–PCR primer sets for the 5' and the 3' ends of the CD31 transcript. The CD31 and vWF were predominantly expressed in CEC fraction whereas RPE65 predominantly in unfractionated RPE/choroid tissue. After a 3–h light exposure, IΚBa, the inhibitor of NF–ΚB, in CECs was phosphorylated. The changes in the expression levels of IΚBα were also observed. Conclusions: A method for isolation of CECs from the mouse choroid with preservation of RNA quality has been developed. Intense light exposure induces photo–oxidative stress related activation of the NF–ΚB signaling pathway in CECs. The methods developed may facilitate the identification of CEC specific genes and gene products that respond to oxidative stress, and to define a pathologic role of CEC in light–induced retinal degeneration.
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