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We previously identified cytosolic lipid-protein particles (CLPP) having size and density of HDL in rat astrocytes, to which apoA-I induces translocation of cholesterol, caveolin-1 and protein kinase Cα (PKCα) following its association with microtubules prior to cholesterol release/biogenesis of HDL (JBC 277: 7929, 2002; JLR 45: 2269, 2004). To further understand the physiological relevance of these findings, we investigated the CLPP/microtubule association and its role in intracellular cholesterol trafficking by using a technique of reconstituted microtubule-like filaments (rMT) in rat astrocyte cytosol. When the cells were pretreated with apoA-I, α-tubulin as a 52-kDa protein in rMT was found phosphorylated while α-tubulin in a soluble monomeric form was little phosphorylated. The phosphorylation took place coincidentally to apoA-I-induced association with rMT of CLPP, a complex containing PKCα, and was suppressed by a PKC inhibitor, Bis indolylmaleimide 1 (BIM). α-Tubulin dissociated from CLPP when phosphorylated, and it poorly bound to CLPP once dissociated. BIM did not influence association of PKCa with rMT but suppressed apoA-I-induced cholesterol translocation to the cytosol from the ER/Golgi apparatus and apoA-I-mediated cholesterol release. We thereby concluded that a-tubulin phosphorylation by PKCa on CLPP is involved in reversible CLPP association with the microtubules and intracellular cholesterol trafficking for apoA-I-dependent HDL biogenesis/cholesterol release in rat astrocytes. [ABSTRACT FROM AUTHOR]

Abstract: Liver X receptors (LXRα and LXRβ) are key transcription factors in cholesterol metabolism that regulate cholesterol biosynthesis/efflux and bile acid metabolism/excretion in the liver and numerous organs. In macrophages, LXR signaling modulates cholesterol handling and the inflammatory response, pathways involved in atherosclerosis. Since regulatory pathways of LXR transcription control are well understood, in the present study we aimed at identifying post-transcriptional regulators of LXR activity. MicroRNAs (miRs) are such post-transcriptional regulators of genes that in the canonical pathway mediate mRNA inactivation. In silico analysis identified miR-206 as a putative regulator of LXRα but not LXRβ. Indeed, as recently shown, we found that miR-206 represses LXRα activity and expression of LXRα and its target genes in hepatic cells. Interestingly, miR-206 regulates LXRα differently in macrophages. Stably overexpressing miR-206 in THP-1 human macrophages revealed an up-regulation and miR-206 knockdown led to a down-regulation of LXRα and its target genes. In support of these results, bone marrow-derived macrophages (BMDMs) from miR-206 KO mice also exhibited lower expression of LXRα target genes. The physiological relevance of these findings was proven by gain- and loss-of-function of miR-206; overexpression of miR-206 enhanced cholesterol efflux in human macrophages and knocking out miR-206 decreased cholesterol efflux from MPMs. Moreover, we show that miR-206 expression in macrophages is repressed by LXRα activation, while oxidized LDL and inflammatory stimuli profoundly induced miR-206 expression. We therefore propose a feed-back loop between miR-206 and LXRα that might be part of an LXR auto-regulatory mechanism to fine tune LXR activity. [Copyright &y& Elsevier]

Abstract: Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter A1 (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190–243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223–243) is essential for the HDL formation, the function of low lipid affinity region (residues 191–220) remains unclear. To evaluate the role of residues 191–220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191–220 apoA-I, in comparison to wild-type and Δ223–243 apoA-I. Although deletion of residues 191–220 has a slight effect on the tertiary structure of apoA-I, the Δ191–220 variant showed intermediate behavior between wild-type and Δ223–243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δ191–220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABCA1-expressing cells of Δ191–220 apoA-I was also intermediate between wild-type and Δ223–243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191–220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation. [Copyright &y& Elsevier]

Abstract: Hepatic lipase (HL) and endothelial lipase (EL) are negative regulators of plasma HDL cholesterol (HDLc) levels and presumably could affect two main HDL atheroprotective functions, macrophage-to-feces reverse cholesterol transport (RCT) and HDL antioxidant properties. In this study, we assessed the effects of both HL and EL deficiency on macrophage-specific RCT process and HDL ability to protect against LDL oxidation. HL- and EL-deficient and wild-type mice were injected intraperitoneally with [3H]cholesterol-labeled mouse macrophages, after which the appearance of [3H]cholesterol in plasma, liver, and feces was determined. The degree of HDL oxidation and the protection of oxidative modification of LDL co-incubated with HDL were evaluated by measuring conjugated diene kinetics. Plasma levels of HDLc, HDL phospholipids, apoA-I, and platelet-activated factor acetyl-hydrolase were increased in both HL- and EL-deficient mice. These genetically modified mice displayed increased levels of radiolabeled, HDL-bound [3H]cholesterol 48h after the label injection. The magnitude of macrophage-derived [3H]cholesterol in feces was also increased in both the HL- and EL-deficient mice. HDL from the HL- and EL-deficient mice was less prone to oxidation and had a higher ability to protect LDL from oxidation, compared with the HDL derived from the wild-type mice. These changes were correlated with plasma apoA-I and apoA-I/HDL total protein levels. In conclusion, targeted inactivation of both HL and EL in mice promoted macrophage-to-feces RCT and enhanced HDL antioxidant properties. [Copyright &y& Elsevier]

Abstract: To develop a detailed double belt model for discoidal HDL, we previously scored inter-helical salt bridges between all possible registries of two stacked antiparallel amphipathic helical rings of apolipoprotein (apo) A-I. The top score was the antiparallel apposition of helix 5 with 5 followed closely by appositions of helix 5 with 4 and helix 5 with 6. The rationale for the current study is that, for each of the optimal scores, a pair of identical residues can be identified in juxtaposition directly on the contact edge between the two antiparallel helical belts of apoA-I. Further, these residues are always in the ‘9th position’ in one of the eighteen 11-mer repeats that make up the lipid-associating domain of apoA-I. To illustrate our terminology, 129j (LL5/5) refers to the juxtaposition of the Cα atoms of G129 (in a ‘9th position’) in the pairwise helix 5 domains. We reasoned that if identical residues in the double belt juxtapositions were mutated to a cysteine and kept under reducing conditions during disc formation, we would have a precise method for determining registration in discoidal HDL by formation of a disulfide-linked apoA-I homodimer. Using this approach, we conclude that 129j (LL5/5) is the major rotamer orientation for double belt HDL and propose that the small ubiquitous gap between the pairwise helix 5 portions of the double belt in larger HDL discoidal particles is significantly dynamic to hinge off the disc edge under certain conditions, e.g., in smaller particles or perhaps following binding of the enzyme LCAT. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945–2010). [Copyright &y& Elsevier]

Abstract: In the previous paper, we reported that apolipoprotein (apo) A-I enhances generation of HDL-like lipoproteins in rat astrocytes to be accompanied with both increase in tyrosine phosphorylation of phospholipase Cγ (PL-Cγ) and PL-Cγ translocation to cytosolic lipid–protein particles (CLPP) fraction. In this paper, we studied the interaction between apoA-I and ATP-binding cassette transporter A1 (ABCA1) to relate with PL-Cγ function for generation of HDL-like lipoproteins in the apoA-I-stimulated astrocytes. ABCA1 co-migrated with exogenous apoA-I with apparent molecular weight over 260kDa on SDS-PAGE when rat astrocytes were treated with apoA-I and then with a cross-linker, BS3. The solubilized ABCA1 of rat astrocytes was associated with the apoA-I-immobilized Affi-Gel 15. An LXR agonist, To901317, increased the cellular level of ABCA1, association of apoA-I with ABCA1 and apoA-I-mediated lipid release in rat astrocytoma GA-1/Mock cells where ABCA1 expression at baseline is very low. PL-Cγ was co-isolated by apoA-I-immobilized Affi-Gel 15 and co-immunoprecipitated by anti-ABCA1 antibody along with ABCA1 from the solubilized membrane fraction of rat astrocytes. The SiRNA of ABCA1 suppressed not only the PL-Cγ binding to ABCA1 but also the tyrosine phosphorylation of PL-Cγ. A PL-C inhibitor, U73122, prevented generation of apoA-I-mediated HDL-like lipoproteins in rat astrocytes. To901317 increased the association of PL-Cγ with ABCA1 in GA-1/Mock cells dependently on the increase of cellular level of ABCA1 without changing that of PL-Cγ. These findings suggest that the exogenous apoA-I augments the interaction between PL-Cγ and ABCA1 to stimulate tyrosine phosphorylation and activation of PL-Cγ for generation of HDL-like lipoproteins in astrocytes. [Copyright &y& Elsevier]

Abstract: Gemfibrozil and fenofibrate, two of the fibrates most used in clinical practice, raise HDL cholesterol (HDLc) and are thought to reduce the risk of atherosclerotic cardiovascular disease. These drugs act as PPARα agonists and upregulate the expression of genes crucial in reverse cholesterol transport (RCT). In the present study, we determined the effects of these two fibrates on RCT from macrophages to feces in vivo in human apoA-I transgenic (hApoA-ITg) mice. [3H]cholesterol-labeled mouse macrophages were injected intraperitoneally into hApoA-ITg mice treated with intragastric doses of fenofibrate, gemfibrozil or a vehicle solution for 17days, and radioactivity was determined in plasma, liver and feces. Fenofibrate, but not gemfibrozil, enhanced [3H]cholesterol flux to plasma and feces of female hApoA-ITg mice. Fenofibrate significantly increased plasma HDLc, HDL phospholipids, hApoA-I levels and phospholipid transfer protein activity, whereas these parameters were not altered by gemfibrozil treatment. Unlike gemfibrozil, fenofibrate also induced the generation of larger HDL particles, which were more enriched in cholesteryl esters, together with higher potential to generate preβ-HDL formation and caused a significant increase in [3H]cholesterol efflux to plasma. Our findings demonstrate that fenofibrate promotes RCT from macrophages to feces in vivo and, thus, highlight a differential action of this fibrate on HDL. [ABSTRACT FROM AUTHOR]

Abstract: Transgenic over expression of apolipoprotein A-I (ApoA-I) the major structural apolipoprotein of HDL appears to convey the most consistent and strongest anti atherogenic effect observed in animal models so far. We tested the hypothesis that ApoA-I mediates its cardio protective effects additionally through ApoA-I induced differentiation of bone marrow-derived progenitor cells in vitro. This study demonstrates that lineage negative bone marrow cells (lin− BMCs) alter and differentiate in response to free ApoA-I. We find that lin− BMCs in culture treated with recombinant free ApoA-I at a concentration of 0.4 μM are twice as large in size and have altered cell morphology compared to untreated cells; untreated cells retain the original spheroid morphology. Further, the total number of CD31 positive cells in the ApoA-I treated population consistently increased by two fold. This phenotype was significantly reduced in untreated cells and points towards a novel ApoA-I dependent differentiation. A protein lacking its best lipid-binding region (ApoA-IΔ10) did not stimulate any changes in the lin−BMCs indicating that ApoA-I may mediate its effects by regulating cholesterol efflux. The increased CD31 correlates with an increased ability of the lin− BMCs to adhere to both fibronectin and mouse brain endothelial cells. Our results provide the first evidence that exogenous free ApoA-I has the capacity to change the characteristics of progenitor cell populations and suggests a novel mechanism by which HDL may mediate its cardiovascular benefits. [Copyright &y& Elsevier]

Abstract: Formation of discoidal high density lipoproteins (rHDL) by apolipoprotein A-I (apoA-I) mediated solubilization of dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles (MLV) was dramatically affected by bilayer cholesterol concentration. At a low ratio of DMPC/apoA-I (2 mg DMPC/mg apoA-I, 84/1 mol/mol), sterols (cholesterol, lathosterol, and β-sitosterol) that form ordered lipid phases increase the rate of solubilization similarly, yielding rHDL with similar structures. By changing the temperature and sterol concentration, the rates of solubilization varied almost 3 orders of magnitude; however, the sizes of the rHDL were independent of the rate of their formation and dependent upon the bilayer sterol concentration. At a high ratio of DMPC/apoA-I (10/1 mg DMPC/mg apoA-I, 420/1 mol/mol), changing the temperature and cholesterol concentration yielded rHDL that varied greatly in size, phospholipid/protein ratio, mol% cholesterol, and number of apoA-I molecules per particle. rHDL were isolated that had 2, 4, 6, and 8 molecules of apoA-I per particle, mean diameters of 117, 200, 303, and 396 Å, and a mol% cholesterol that was similar to the original MLV. Kinetic studies demonstrated that the different sized rHDL are formed independently and concurrently. The rate of formation, lipid composition, and three-dimensional structures of cholesterol-rich rHDL is dictated primarily by the original membrane phase properties and cholesterol content. The size speciation of rHDL and probably nascent HDL formed via the activity of the ABCA1 lipid transporter is mechanistically linked to the cholesterol content of the membranes from which they were formed. [Copyright &y& Elsevier]

Abstract: Plasma levels of high-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) are inversely correlated with the risk of cardiovascular disease. One major atheroprotective mechanism of HDL and apoA-I is their role in reverse cholesterol transport, i.e., the transport of excess cholesterol from foam cells to the liver for secretion. The ATP-binding cassette transporters ABCA1 and ABCG1 play a pivotal role in this process by effluxing lipids from foam cells to apoA-I and HDL, respectively. In the liver, ABCA1 activity is one rate-limiting step in the formation of HDL. In macrophages, ABCA1 and ABCG1 prevent the excessive accumulation of lipids and thereby protect the arteries from developing atherosclerotic lesions. However, the mechanisms by which ABCA1 and ABCG1 mediate lipid removal are still unclear. Particularly, three questions remain controversial and are discussed in this review: (1) Do apoA-I and HDL directly interact with ABCA1 and ABCG1, respectively? (2) Does cholesterol efflux involve retroendocytosis of apoA-I or HDL? (3) Which lipids are directly transported by ABCA1 and ABCG1? [Copyright &y& Elsevier]

Abstract: High density lipoproteins (HDL) and their main protein constituent, apolipoprotein A-I (apoA-I), exert potentially anti-atherogenic properties within the arterial wall. However, it is unknown how they are transported from the blood stream into the vascular wall. Here we investigated the interaction of apoA-I with endothelial cells. At 4 °C endothelial cells bound 125I-apoA-I with high affinity, K d =2.1 μg/ml and in a saturable manner (B max of 35 ng/mg cell protein). At 37 °C, the cell association of apoA-I revealed similar affinity as at 4 °C (K d =2.2 μg/ml) but the maximum specific cell association was much enhanced (B max =360 ng/mg cell protein). Binding and cell association was competed by excess unlabeled apoA-I and HDL but not by albumin. Biotinylation experiments and electron microscopy studies showed that endothelial cells internalize labeled apoA-I. Only minor amounts of the internalized apoA-I were degraded. Cultivated in a Transwell system, the cells transported a fraction of 125I-apoA-I from the apical to the basolateral compartment in a competable and temperature-sensitive manner. Furthermore, after specific transport the originally prebeta-mobile and lipid-free apoA-I was recovered as particles which have electrophoretic alpha-mobility. We conclude that endothelial cells transcytose and lipidate lipid-free apoA-I. [Copyright &y& Elsevier]

Background: Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis. Two isoforms of ACAT have been reported (ACAT-1 and ACAT-2). ACAT inhibitors cannot only prevent atherosclerosis formation, but may also induce its regression in animals. In humans, an ACAT inhibitor was shown to have a lipid-lowering effect. The present study was carried out to clarify the relationship between ACAT-1 gene variants and hyperlipidemia. Methods and results: To identify genetic variants, we screened 30 subjects with hyperlipidemia by direct sequencing. As a result, a missense variant (R526G) and a variant in the 5′ untranslated region (-77G→A) were identified. The genotype frequencies of each variant were determined in 178 unrelated normolipidemic and 441 unrelated hyperlipidemic subjects. The alleles frequencies of the R526G variant in normolipidemic and hyperlipidemic subjects were 0.676 and 0.633, respectively. The alleles frequencies of the -77G→A variant in normolipidemic and hyperlipidemic subjects were 0.503 and 0.515, respectively. Differences in allele frequencies between normolipidemic and hyperlipidemic subjects were not significant in both variants. R526G variant did not affect plasma concentrations of lipids or apolipoproteins in subjects studied. However, among hyperlipidemic subjects, plasma concentrations of HDL-C and apoA-I in subjects with -77G→A variant were significantly higher than those in subjects without variant. Conclusion: Two variants in ACAT-1 gene were identified in subjects with hyperlipidemia. -77G→A variant affects plasma HDL concentrations only in hyperlipidemic subjects. These data suggest that the intracellular FC concentration might modulate plasma HDL concentrations. [Copyright &y& Elsevier]

The effect of inhibition of acylCoA: cholesterol acyltransferase (ACAT) was studied on high density lipoprotein (HDL) metabolism. An inhibitor of ACAT, MCC-147, was given mouse peritoneal macrophages and expression of ATP-binding cassette transporter A1 (ABCA1) was examined. ABCA1 was increased both at the mRNA and protein levels, only when the cells are cholesterol-loaded and thereby the inhibitor decreased esterified cholesterol and increased unesterified cholesterol. In this condition, the ACAT inhibitor increased reversible binding of apoA-I to the cells and enhanced apoA-I-mediated release of cellular cholesterol and phospholipid, but did not influence nonspecific cellular cholesterol efflux to lipid microemulsion. It was therefore concluded that the ACAT inhibitor increased the release of cholesterol from the cholesterol-loaded macrophages by increasing the expression of ABCA1, putatively through shifting cholesterol distribution from the esterified to the free compartments. [Copyright &y& Elsevier]

We studied the mechanism of HDL denaturation with concomitant apoA-I dissociation with HDL preparations from 48 patients with a wide range of plasma HDL-C and evaluated the contribution of lipid-free apoA-I into cholesterol efflux from macrophage, in particular, mediated by cholesterol transporter ABCA1. We prepared HDL by precipitation of apoB-containing lipoproteins by polyethylene glycol and used the chaotropic agent urea to denature HDL preparations. Apo-I dissociation from urea-treated HDL was assessed by the increase of preβ-band fraction with agarose gel electrophoresis followed by electro transfer and immunodetection and by the increase of ABCA1-mediated efflux of fluorescent analogue BODIPY-Cholesterol from RAW 264.7 macrophages. The HDL denaturation is governed by a single transition to fully dissociated apoA-I and the transition cooperativity decreases with increasing HDL-C. The apoA-I release depends on phospholipid concentration of HDL preparation and HDL compositional and structural heterogeneity and is well described by apolipoprotein partition between aqueous and lipid phases. Dissociated apoA-I determines the increase of ABCA1-mediated efflux of BODIPY-Cholesterol from RAW 264.7 macrophages to patient HDL. The increase in apoA-I dissociation is associated with the increase of ABCA1 gene transcript in peripheral blood mononuclear cells from patients. The low level of plasma HDL particles may be compensated by their increased potency for apoA-I release, thus suggesting apoA-I dissociation as a new HDL functional property. Unlabelled Image • Denaturation of 48 HDL samples by urea proceeds with concomitant apoA-I dissociation • Dissociation of apoA-I results in the increase of ABCA1-mediated Chol efflux • ApoA-I partition between aqueous and lipid phases depends on PL and HDL heterogeneity • The increase in apoA-I release is associated with APOA1 transcript in patients' PBMC. • Low HDL-C level is equipoised by the increased potency of small HDL to release apoA-I. [ABSTRACT FROM AUTHOR]