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I.R. Rodriguez, S. Alam; Cytotoxic Effects of Oxidized LDL-cholesterol and Oxysterols on Cultured Human RPE Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1638.
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Purpose: The accumulation, oxidation and subsequent internalization of oxidized LDL-cholesterol by the RPE may be a potential mechanism for the pathogenesis of age-related macular degeneration. Thus, we are interested in determining which oxysterols present in oxidized LDL-cholesterol are most cytotoxic to human RPE cells. We are also interested in determining the mechanism by which RPE cells die when exposed to oxysterols. Methods: Two different human cultured RPE cells were tested, ARPE19 (ATCC, Rockville, MD) and hTERT (Clontech Laboratories, Inc., Palo Alto, CA). These cells were grown to confluency then exposed to different concentrations of oxysterols and oxidized LDL-cholesterol (OxLDL) in serum free media. The LDL was oxidized using 20 µM Cu+2 and analyzed for oxysterols content by HPLC. The oxysterol analysis was performed in a Waters 2790 HPLC system using an Xterra RP18 column heated to 60°C in an aqueous acetonitrile gradient. The oxysterols were detected using a Waters 996 PDA detector and an ESA Inc. ELSD detector. Cell viability was measured by MTT hydrolysis and/or by using a fluorescent cell cytometer (Guava Inc.). Results: Ten different oxysterols were analyzed with cholesterol and untreated cells used as controls. Most oxysterols demonstrated measurable cytotoxicity at concentrations of 20-50 µM within 48-72 hrs. Two common oxysterols, 7-ketocholesterol (7KCh) and 20α-hydroxycholesterol (20αHCh) were found to be highly cytotoxic. OxLDL was also found to be cytotoxic to RPE cells at concentrations of 1-2 µg protein/ml within 24 hrs. Analysis of the OxLDL revealed a mixture of oxysterols including 7KCh and 20αHCh but not all of the oxysterols were identified. Cells undergoing cytotoxicity were analyzed for activation of caspases, annexin V binding and DNA laddering. Staurosporin was used as a positive control for apoptosis. Conclusions: Two oxysterols 7KCh and 20αHCh were found to be cytotoxic to cultured human RPE cells. OxLDL was also found to be cytotoxic and to contain significant amounts of these oxysterols. No evidence of apoptosis was found using generally accepted assays. The cell death mechanism is under investigation using commercially available and custom made microarrays containing genes in known stress, cytotoxicity and signaling pathways. Microarrays containing all known oxysterol binding proteins as well as other related receptors are under construction.
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