Correction of apolipoprotein A-I-mediated lipid efflux and high density lipoprotein particle formation in human Niemann-Pick type C disease fibroblasts.

Impaired cell cholesterol trafficking in Niemann-Pick type C (NPC) disease results in the first known instance of impaired regulation of the ATP-binding cassette transporter A1 (ABCA1), a lipid transporter mediating the rate-limiting step in high density lipoprotein (HDL) formation, as a cause of low plasma HDL-cholesterol in humans. We show here that treatment of human NPC1(-/-) fibroblasts with the liver X receptor (LXR) agonist TO-901317 increases ABCA1 expression and activity in human NPC1(-/-) fibroblasts, as indicated by near normalization of efflux of radiolabeled phosphatidylcholine and a marked increase in efflux of cholesterol mass to apoA-I. LXR agonist treatment prior to and during apoA-I incubation resulted in reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes, as well as cholesterol mass, in NPC1(-/-) cells. HDL species in human NPC disease plasma showed the same pattern of diminished large, cholesterol-rich alpha-1 HDL particles as seen in isolated heterozygous ABCA1 deficiency. Incubating NPC1(-/-) fibroblasts with the LXR agonist normalized the pattern of HDL particle formation by these cells. ABCG1, another LXR target gene involved in cholesterol efflux to HDL, also showed diminished expression in NPC1(-/-) fibroblasts and increased expression upon LXR agonist treatment. These results suggest that NPC1 mutations can be largely bypassed and that NPC1 protein function is non-essential for the trafficking and removal of cellular cholesterol if the down-stream defects in ABCA1 and ABCG1 regulation in NPC disease cells are corrected using an LXR agonist.

Cholesterol and phospholipid efflux from cultured cells.

The removal of phospholipids and cholesterol from tissues is the major mechanism mediating the initial assembly of high density lipoproteins (HDL), as well as being the main reason HDL are thought to protect against atherosclerosis. Investigations of the mechanisms of HDL assembly and testing of novel HDL-raising agents typically involve assays to determine phospholipid and/or cholesterol removal or "efflux" from cultured cells. The purpose of this chapter is to describe experimental protocols that can be used in the determination of cholesterol and phospholipid efflux from cultured cells by HDL apolipoproteins for the formation of new HDL particles, and the testing of novel HDL-raising therapies in vitro. A protocol is also provided for determining the size and nature of HDL particles formed in cell-conditioned medium using two-dimensional gel electrophoresis.

Fatty acid oxidation products in human atherosclerotic plaque: an analysis of clinical and histopathological correlates.

Markers of lipid peroxidative damage have been shown to be elevated in individuals with risk factors for cardiovascular disease, and human atherosclerotic plaque contains products resulting from lipid peroxidation. In particular, the presence of fatty acid oxidation products such as hydroxyeicosatetraenoic acids (HETEs) has previously been suggested as a marker of plaque instability and symptomatic cerebrovascular disease. The aim of the present study was to quantitate the levels of various oxidation products of linoleic acid (HODEs) and arachidonic acid (HETEs), respectively, in human atherosclerotic plaque tissue and assess their level in relation to plaque histopathology, symptoms of cerebrovascular disease and preexisting atherosclerotic risk factors. We also assessed the correlation between the levels of the hydroxy fatty acid compounds and F(2)-isoprostanes, an established marker of in vivo free radical mediated oxidation. Hydroxy fatty acid oxidation products were identified in all histological subtypes of advanced plaque. However, there were no significant differences in levels between the histopathologically classified sub-groups or between patients symptomatic or asymptomatic for cerebrovascular disease. Arachidonic acid oxidation products were significantly higher in those subjects who also had symptomatic peripheral vascular disease. The level of linoleic acid oxidation products was significantly higher in individuals who consumed alcohol on a regular basis. While F(2)-isoprostanes and fatty acid oxidation products were highly correlated (P<0.01), levels of the hydroxy fatty acid compounds were 20-40-fold higher than F(2)-isoprostanes. Chiral analysis of the plaque extracts indicated that all HODEs and HETEs originated primarily from non-enzymatic lipid peroxidation. While our results do not support previous reports that fatty acid oxidation products such as the HETEs are associated with plaque instability and symptomatic cerebrovascular disease, further work is warranted to determine the potential of these compounds as circulating markers for underlying atherosclerotic disease and lipid peroxidative stress.