Ignacio R Rodriguez1, Shahabuddin Alam, Jung Wha Lee. 1. Laboratory of Retinal Cell and Molecular Biology, Section on Mechanisms of Retinal Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. rodriguezi@nei.nih.gov
Abstract
PURPOSE: To determine which components present in oxidized LDL are responsible for the cytotoxicity associated with its internalization by culture ARPE19 cells. METHODS: ARPE19 cells were grown in 24-well and 96-well plates. Cell viability was measured by MTT and/or adenosine triphosphate (ATP) content. LDL was oxidized with Cu(+2) and oxysterol content analyzed by a novel HPLC method. RESULTS: OxLDL showed increased cytotoxicity with prolonged oxidation. Analysis of the oxLDL showed a predominance of the 7-oxygenated products, 7 alpha-hydroxycholesterol (7 alpha HCh), 7 beta-hydroxycholesterol (7 beta HCh), and 7-ketocholesterol (7kCh). Addition of these oxysterols to the ARPE19 cell in free form indicated that 7kCh is the most cytotoxic of the oxysterols but at physiologically unrealistic concentrations. Partitioning of individual oxysterols into nonoxidized LDL at concentrations similar to those found in the oxLDL also indicated that 7kCh is the most cytotoxic of the oxysterols. Transition metals are tightly bound by LDL and play an important role in the oxidation of LDL, but do not seem to enhance its cytotoxicity directly. CONCLUSIONS: Prolonged oxidation of LDL increases the levels of 7kCh due to further oxidation of 7 alpha HCh and 7 beta HCh. The formation of 7KCh seems to be responsible for most of the cytotoxicity associated with oxLDL internalization in ARPE19 cells.
PURPOSE: To determine which components present in oxidized LDL are responsible for the cytotoxicity associated with its internalization by culture ARPE19 cells. METHODS: ARPE19 cells were grown in 24-well and 96-well plates. Cell viability was measured by MTT and/or adenosine triphosphate (ATP) content. LDL was oxidized with Cu(+2) and oxysterol content analyzed by a novel HPLC method. RESULTS: OxLDL showed increased cytotoxicity with prolonged oxidation. Analysis of the oxLDL showed a predominance of the 7-oxygenated products, 7 alpha-hydroxycholesterol (7 alpha HCh), 7 beta-hydroxycholesterol (7 beta HCh), and 7-ketocholesterol (7kCh). Addition of these oxysterols to the ARPE19 cell in free form indicated that 7kCh is the most cytotoxic of the oxysterols but at physiologically unrealistic concentrations. Partitioning of individual oxysterols into nonoxidized LDL at concentrations similar to those found in the oxLDL also indicated that 7kCh is the most cytotoxic of the oxysterols. Transition metals are tightly bound by LDL and play an important role in the oxidation of LDL, but do not seem to enhance its cytotoxicity directly. CONCLUSIONS: Prolonged oxidation of LDL increases the levels of 7kCh due to further oxidation of 7 alpha HCh and 7 beta HCh. The formation of 7KCh seems to be responsible for most of the cytotoxicity associated with oxLDL internalization in ARPE19 cells.
Authors: Bruce A Pfeffer; Libin Xu; Ned A Porter; Sriganesh Ramachandra Rao; Steven J Fliesler Journal: Exp Eye Res Date: 2016-02-13 Impact factor: 3.467
Authors: B Dugas; S Charbonnier; M Baarine; K Ragot; D Delmas; F Ménétrier; J Lherminier; L Malvitte; T Khalfaoui; A Bron; C Creuzot-Garcher; N Latruffe; Gérard Lizard Journal: Eur J Nutr Date: 2010-03-27 Impact factor: 5.614
Authors: Jung Wha Lee; Hirotoshi Fuda; Norman B Javitt; Charles A Strott; Ignacio R Rodriguez Journal: Exp Eye Res Date: 2006-03-20 Impact factor: 3.467