linoleic-acid and cholestane-3-5-6-triol

Compared to cholesterol or linoleic acid (18:2), oxidized lipids such as cholestan-3 beta, 5 alpha, 6 beta-triol (triol) and hydroperoxy linoleic acid (HPODE) markedly impair endothelial barrier function in culture [Hennig and Boissonneault, 1987; Hennig et al. 1986]. Because proteoglycans contribute to vascular permeability properties, the effects of cholesterol and 18:2 and their oxidation products, triol and HPODE, on endothelial proteoglycan metabolism were determined. While cholesterol was without effect, a concentration-dependent decrease in cellular proteoglycans (measured by 35S incorporation) was observed after exposure to triol. Compared to control cultures, cholesterol reduced mRNA levels for the proteoglycans, perlecan and biglycan. Triol had a similar effect on biglycan but not an perlecan mRNA levels. Compared to 18:2, 1,3 and 5 microM HPODE depressed cellular proteoglycans. Perlecan mRNA levels were reduced more by HPODE when compared to 18:2. Biglycan mRNA levels were reduced by 3 microM, but not by 5 microM HPODE. These data demonstrate that oxidized lipids such as triol and HPODE can decrease cellular proteoglycan metabolism in endothelial monolayers and alter mRNA levels of major specific proteoglycans in a concentration-dependent manner. This may have implications in lipid-mediated disruption of endothelial barrier function and atherosclerosis.

Topics: Animals; Biglycan; Capillary Permeability; Cells, Cultured; Cholestanols; Cholesterol; Endothelium, Vascular; Extracellular Matrix Proteins; Gene Expression Regulation; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Oxidation-Reduction; Oxidative Stress; Proteoglycans; Pulmonary Artery; RNA, Messenger; Swine

The aim of this study was to investigate the effect of the oxysterol 5 alpha-cholestane-3 beta,5,6 beta-triol (triol) on the metabolism of linoleic acid (18:2n-6) to arachidonic acid (20:4n-6) and on the cell membrane fatty acid composition. Porcine kidney cells were incubated in medium with or without 10 microgram(s)/mL of triol for 24 h, then incubated for 1, 6, or 12 h in a medium which contained 50 muM of either [14C] linoleic acid or unlabeled linoleic acid. The cellular uptake of [14C] linoleic acid was significantly higher in the triol-treated cells than in control cells. After 1- and 6-h incubations despite the increase of [14C] linoleic acid pool size in the triol-treated cells, neither total n-6 polyunsaturated fatty acids (PUFA) metabolites nor arachidonic acid were increased in the triol-treated cells as compared to the control cells, but trienoic acids accumulated to a greater extent in the triol-treated cells. Therefore, the ratios of n-6 PUFA metabolites vs. pool size of linoleic acid and of tetraenoic acids vs. dienoic acids were significantly decreased in triol-treated cells as compared to the control cells. The cellular fatty acid composition also showed that linoleic acid percentage was significantly increased while arachidonic acid percentage was significantly decreased in the triol-treated cells, and that the accumulation of trienoic acids (18:3n-6 + 20:3n-6) observed from the [14C] linoleic acid experiment was due solely to increased 20:3n-6 content. This latter finding indicates that a decrease of elongase activity by triol is unlikely. Our results also showed that the triol-treated cells had a lower level of free cholesterol but higher levels of phospholipid and triol in their membranes, suggesting that triol displaced free cholesterol from the cell membrane.

Topics: Animals; Arachidonic Acid; Carbon Radioisotopes; Cell Line; Cell Membrane; Cholestanols; Fatty Acids; Fatty Acids, Omega-6; Fatty Acids, Unsaturated; Kidney; Linoleic Acid; Linoleic Acids; Swine

Disturbances in arterial wall elastin metabolism appear to be important factors in atherosclerosis development. To evaluate this hypothesis, elastase-like activity was determined in cultured endothelial cells and their surrounding media after exposure to tumor necrosis factor-alpha (TNF), cholestan-3 beta,5 alpha,6 beta-triol (Triol) and linoleic acid (18:2). Significant increases in elastase-like activity both in the cells and in the media were observed when subconfluent endothelial cells were treated with 12 microM Triol, 500 U TNF/ml, or 90 microM 18:2, for 72 h in the presence of 5% calf serum. Even higher activities were measured when endothelial cells were seeded directly into media enriched with 18:2, TNF or Triol and treated for 72 h. Vitamin E supplementation (25 microM) attenuated elastase-like activity in cells and media, independent of treatment. These results suggest that elastase-like enzyme induction in endothelial cells may be involved in cellular perturbations induced by certain lipids and cytokines. Vitamin E may provide a protective function by preventing the induction of elastolytic enzymes. This may have implications in elastin metabolism and atherosclerosis.

Topics: Animals; Cells, Cultured; Cholestanols; Culture Media; Endothelium, Vascular; Enzyme Induction; Hypolipidemic Agents; Linoleic Acid; Linoleic Acids; Pancreatic Elastase; Swine; Tumor Necrosis Factor-alpha; Vitamin E