Role of Free Apolipoprotein A-I in Cholesterol Efflux

Abstract

This article characterizes products formed by the interaction of purified apolipoprotein (apo) A-I and human fibroblasts. Fibroblasts were incubated with different concentrations of purified apoA-I (1 to 30 micrograms/mL) in tissue culture medium for different periods of time (0 to 24 hours). The medium was then characterized by one- (agarose) and two-dimensional (agarose: polyacrylamide nondenaturing gradient gel) electrophoresis. At any given concentration of apoA-I, the rate of cellular cholesterol efflux appeared linear over 24 hours. Incubating purified apoA-I with fibroblasts for 4 hours, we detected five pre-alpha lipoproteins with particle sizes between 114 and 684 kDa. Formation of pre-alpha lipoproteins was concentration-dependent. At low concentrations (below 5 micrograms/mL apoA-I), all purified apoA-I (with pre-beta mobility) was converted to pre-alpha lipoproteins. At higher concentrations (greater than 5 micrograms/mL apoA-I), more apoA-I remained with pre-beta mobility. The pre-alpha lipoproteins were characterized by colocalization of apoA-I particles with 14C-cholesterol and 32P-phospholipids. Results showed that the pre-alpha particle of lowest molecular weight contained phospholipid and apoA-I but no cholesterol. The remaining pre-alpha particles contained all three substances. When pre-alpha particles were subjected to ultracentrifugation, all particles floated at d < 1.21 g/mL with some of the smallest phospholipid apoA-I only particles being present in the d > 1.21 g/mL fraction. Based on these results, we postulated that in the first stages of reverse cholesterol transport, pre-alpha lipoproteins are formed by the interaction of lipid free apoA-I and peripheral cells.

Inactivation of Macrophage Scavenger Receptor Class B Type I Promotes Atherosclerotic Lesion Development in Apolipoprotein E–Deficient Mice

“…24,25 After transfer of lipoproteins to nitrocellulose membranes, apoAI-containing lipoproteins were immunolocalized with rabbit anti-mouse apoAI (Biodesign). Rabbit antibody was detected with 125 I-labeled anti-rabbit immunoglobulin (Zymed) prepared by the method of McFarlane.…”

“…28 Acetylated LDL (AcLDL) was prepared according to Basu et al 29 Human apoA-I was purified from human plasma HDL as described. 24,25 ApoA-I discs were prepared with apoA-I and 1-palmitoyl 2-oleoyl phosphatidylcholine at a ratio of 1:100 (wt/wt), according to Sparks et al, 30 and isolated by ultracentrifugation at a density of 1.063 to 1.21 g/mL.…”

Triacylglycerol-rich lipoproteins alter the secretion, and the cholesterol-effluxing function, of apolipoprotein E-containing lipoprotein particles from human (THP-1) macrophages

“…These particles were cholesterol-poor and enriched in sphingomyelin ; they promoted the efflux of cholesterol from fibroblasts [47]. The reasons for these differences are not clear but we hypothesize that, by analogy with lipid-poor ApoAI and pre-β " HDL [49][50][51], pre-β migrating LpE particles might reflect the progressive maturation of γ-LpE by the subsequent accumulation of cholesterol and phospholipid. Radiolabelled cellular cholesterol has been shown to accumulate in γ-, pre-β-and α-migrating LpE particles in plasma [29] and we have also noted the co-localization of cellular effluxed cholesterol with pre-β-migrating LpE particles without serum or exogenous lipoprotein acceptors (results not shown).…”

Apolipoprotein A-I, Phospholipid Vesicles, and Cyclodextrins as Potential Anti-Atherosclerotic Drugs: Delivery, Pharmacokinetics, and Efficacy

“…In rabbits where peripheral tissues are loaded with cholesterol through intravenous injection of acLDL or native human LDL (Miller et al 1985), changes in HDL (increase in cholesterol content), signifying cholesterol ef ux from cells, mirrors that observed in vitro. Complexes of apoA-I and PC or free apoA-I promote ef ux of UC from various cells in vitro, including the mouse monocyte/macrophage cell line J774A.1 (Westman et al 1993), resident MPMs (Gelissen et al 1996;Kritharides et al 1996), arterial smooth muscle cells (Stein and Stein 1973;Stein et al 1975), human skin broblasts (Asztalos et al 1997), and human monocyte-derived macrophages (HMDMs) (Westman et al 1998;Dass et al 1999a;Panzenböck et al, unpublished results).…”

“…Apolipoprotein-mediated cholesterol removal from microsomal membranes (Nunez and Swaney 1984), from CHO cells (Forte et al 1993), broblasts (Bielicki et al 1992;Asztalos et al 1997), endothelial cells (Savion and Kotev-Emeth 1993), Fu5AH rat hepatoma cells (Fournier et al 1996), and macrophages (Hara and Yokoyama 1991;Yancey et al 1995) requires PL association with the protein (Davidson et al 1995;Forte et al 1995;Sviridov et al 1996b). ApoA-I, in the absence of serum, facilitates cholesterol ef ux from certain types of cells including human monocyte-derived macrophages (HMDMs: Dass et al 1999a) and mouse peritoneal macrophages (MPMs: Gelissen et al 1996), probably by ef uxing and associating with PLs from the PM (Yancey et al 1995).…”