CYTOTOXICITY OF OXIDIZED LDL TO PORCINE AORTIC SMOOTH-MUSCLE CELLS IS ASSOCIATED WITH THE OXYSTEROLS 7-KETOCHOLESTEROL AND 7-HYDROXYCHOLESTEROL

The cytotoxicity of oxidized low-density lipoprotein (Ox-LDL) to arterial smooth muscle cells (SMCs) may contribute to atherogenesis by causing cell death in core regions of plaques. The aim of the present study was to identify the components of copper-oxidized LDL responsible for its toxicity to porcine aortic SMCs. Toxicity to SMCs was assessed as the decrease in viable cell counts after 3-day cell incubation. Extracts of LDL were tested for toxicity at concentrations equivalent to that derived from 100 mu g LDL protein per milliliter. Lipid extracts of Ox-LDL but not native-LDL were toxic to SMCs. When separated into neutral and polar lipid classes, only the neutral lipids were toxic (89.7+/-0.7% cell loss). The neutral lipids were fractionated further by use of solid-phase extraction and high-performance liquid chromatography (HPLC). Two toxic fractions, causing 93.3% and 60.3% cell loss, were isolated from HPLC and analyzed by gas chromatography/mass spectrometry. The most toxic of these fractions contained 7-ketocholesterol, and the other contained 7 alpha- and 7 beta-hydroxycholesterol. Quantification of these unesterified oxysterols in LDL extracts and comparison with the toxicity of authentic standards indicate that 7-ketocholesterol and 7-hydroxycholesterol are present in Ox-LDL at levels sufficient to account for its toxicity to SMCs.