Interconversion between apolipoprotein A-I-containing lipoproteins of pre-beta and alpha electrophoretic mobilities.

Apolipoprotein (apo) A-I-containing lipoproteins can be separated into two subfractions, pre-beta HDL and alpha HDL (high density lipoproteins), based on differences in their electrophoretic mobility. In this report we present results indicating that these two subfractions are metabolically linked. When plasma was incubated for 2 h at 37 degrees C, apoA-I mass with pre-beta electrophoretic mobility disappeared. This shift in apoA-I mass to alpha electrophoretic mobility was blocked by the addition of either 1.4 mM DTNB or 10 mM menthol to the plasma prior to incubation, suggesting that lecithin:cholesterol acyltransferase (LCAT) activity was involved. There was no change in the electrophoretic mobility of either pre-beta HDL or alpha HDL when they were incubated with cholesterol-loaded fibroblasts. However, after exposure to the fibroblasts, the cholesterol content of the pre-beta HDL did increase approximately sixfold, suggesting that pre-beta HDL can associate with appreciable amounts of cellular cholesterol. Pre-beta HDL-like particles appear to be generated by the incubation of alpha HDL with cholesteryl ester transfer protein (CETP) and either very low density lipoproteins (VLDL) or low density lipoproteins (LDL). This generation of pre-beta HDL-like particles was documented both by immunoelectrophoresis and by molecular sieve chromatography. Based on these findings, we propose a cyclical model in which 1) apoA-I mass moves from pre-beta HDL to alpha HDL in connection with the action of LCAT and the generation of cholesteryl esters within the HDL, and 2) apoA-I moves from alpha HDL to pre-beta HDL in connection with the action of CETP and the movement of cholesteryl esters out of the HDL. Additionally, we propose that the relative plasma concentrations of pre-beta HDL and alpha HDL reflect the movement of cholesteryl esters through the HDL. Conditions that result in the accumulation of HDL cholesteryl esters will be associated with low concentrations of pre-beta HDL, whereas conditions that result in the depletion of HDL cholesteryl esters will be associated with elevated concentrations of pre-beta HDL. This postulate is consistent with published findings in patients with hypertriglyceridemia and LCAT deficiency.     

Effect of apolipoprotein A-I lipidation on the formation and function of pre-beta and alpha-migrating LpA-I particles

A unique class of lipid-poor high-density lipoprotein, pre-beta1 HDL, has been identified and shown to have distinct functional characteristics associated with intravascular cholesterol transport. In this study we have characterized the structure/function properties of poorly lipidated HDL particles and the factors that mediate their conversion into multimolecular lipoprotein particles. Studies were undertaken with homogeneous recombinant HDL particles (LpA-I) containing apolipoprotein (apo) A-I and various amounts of palmitoyloleoylphosphatidylcholine (PC) and cholesterol. Complexation of apoA-I with small amounts of PC and cholesterol results in the formation of discrete lipoprotein structures that have a hydrated diameter of about 6 nm but contain only one molecule of apoA-I (Lp1A-I). While the molecular charge and alpha-helix content of apoA-I are unaffected by lipidation, the thermodynamic stability of the protein is reduced significantly (from 2.4 to 0.9 kcal/mol of apoA-I). Evaluation of apoA-I conformation by competitive radioimmunoassay with monoclonal antibodies shows that addition of small amounts of PC and cholesterol to apoA-I significantly increases the immunoreactivity of a number of domains over the entire molecule. Increasing the ratio of PC:apoA-I to 10:1 in the Lp1A-I complex is associated with increases in the alpha-helix content and stability of apoA-I. However, incorporation of 10-15 mol of PC destabilizes the Lp1A-I complex and promotes the formation of more thermodynamically stable (1.8 kcal/mol of apoA-I) bimolecular structures (Lp2A-I) that are approximately 8 nm in diameter. The formation of an Lp2A-I particle is associated with an increased immunoreactivity of most of the epitopes studied, with the exception of one central domain (residues 98-121), which becomes significantly less exposed. This structural change parallels a significant increase in the net negative charge on the complex. Characterization of the ability of these lipoproteins to act as substrates for lecithin:cholesterol acyltransferase (LCAT) shows that unstable Lp1A-I complexes stimulate a higher rate of cholesterol esterification by LCAT than the small but more stable Lp2A-I particles (Vmax values are 5.8 and 0.3 nmol of free cholesterol esterified/h, respectively). The ability of LCAT to interact with lipid-poor apoA-I suggests that LCAT does not need to bind to the lipid interface on an HDL particle but that LCAT may directly interact with apoA-I. The data suggests that lipid-poor HDL particles may be metabolically reactive particles because they are thermodynamically unstable.     

Dissociation of high density lipoprotein precursors from apolipoprotein B-containing lipoproteins in the presence of unesterified fatty acids and a source of apolipoprotein A-I

Incubation of low (LDL), intermediate (IDL), or very low density lipoproteins (VLDL) with palmitic acid and either high density lipoproteins (HDL), delipidated HDL, or purified apolipoprotein (apo) A-I resulted in the formation of lipoprotein particles with discoidal structure and mean particle diameters ranging from 146 to 254 A by electron microscopy. Discs produced from IDL or LDL averaged 26% protein, 42% phospholipid, 5% cholesteryl esters, 24% free cholesterol, and 3% triglycerides; preparations derived from VLDL contained up to 21% triglycerides. ApoA-I was the predominant protein present, with smaller amounts of apoA-II. Crosslinking studies of discs derived from LDL or IDL indicated the presence of four apoA-I molecules per particle, while those derived from large VLDL varied more in size and contained as many as six apoA-I molecules per particle. Incubation of discs derived from IDL or LDL with purified lecithin:cholesterol acyltransferase (LCAT), albumin, and a source of free cholesterol produced core-containing particles with size and composition similar to HDL2b. VLDL-derived discs behaved similarly, although the HDL products were somewhat larger and more variable in size. When discs were incubated with plasma d greater than 1.21 g/ml fraction rather than LCAT, core-containing particles in the size range of normal HDL2a and HDL3a were also produced. A variety of other purified free fatty acids were shown to promote disc formation. In addition, some mono and polyunsaturated fatty acids facilitated the formation of smaller, spherical particles in the size range of HDL3c. Both discoidal and small spherical apoA-I-containing lipoproteins were generated when native VLDL was incubated with lipoprotein lipase in the presence of delipidated HDL. We conclude that lipolysis product-mediated dissociation of lipid-apoA-I complexes from VLDL, IDL, or LDL may be a mechanism for formation of HDL subclasses during lipolysis, and that the availability of different lipids may influence the type of HDL-precursors formed by this mechanism.     

Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma

The effect of cholesterol esterification on the distribution of apoA-IV in human plasma was investigated. Human plasma was incubated in the presence or absence of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and immediately fractionated by 6% agarose column chromatography. Fractions were monitored for apoA-IV, apoE, and apoA-I by radioimmunoassay (RIA). Incubation resulted in an elevated plasma concentration of cholesteryl ester and in an altered distribution of apoA-IV. After incubation apoA-IV eluted in the ordinarily apoA-IV-poor fractions of plasma that contain small VLDL particles, LDL, and HDL2. Inclusion of DTNB during the incubation resulted in some enlargement of HDL; however, both cholesterol esterification and lipoprotein binding of apoA-IV were inhibited. Addition of DTNB to plasma after incubation and prior to gel filtration had no effect on the apoA-IV distribution when the lipoproteins were immediately fractionated. Fasting plasma apoE was distributed in two or three peaks; in some plasmas there was a small peak that eluted with the column void volume, and, in all plasmas, there were larger peaks that eluted with the VLDL-LDL region and HDL2. Incubation resulted in displacement of HDL apoE to larger lipoproteins and this effect was observed in the presence or absence of DTNB. ApoA-I was distributed in a single broad peak that eluted in the region of HDL and the gel-filtered distribution was unaffected by incubation either in the presence or absence of DTNB. Incubation of plasma that was previously heated to 56 degrees C to inactivate LCAT resulted in no additional movement of apoA-IV onto lipoproteins, unless purified LCAT was present during incubation. The addition of heat-inactivated LCAT to the incubation, had no effect on movement of apoA-IV. These data suggest that human apoA-IV redistribution from the lipoprotein-free fraction to lipoprotein particles appears to be dependent on LCAT action. The mechanism responsible for the increased binding of apoA-IV to the surface of lipoproteins when LCAT acts may involve the generation of "gaps" in the lipoprotein surface due to the consumption of substrate from the surface and additional enlargement of the core. ApoA-IV may bind to these "gaps," where the packing density of the phospholipid head groups is reduced.     

Role of LCAT in HDL remodeling: investigation of LCAT deficiency states

To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (-77% and -13%), and LCAT activities (-99% and -39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated prebeta-1 and alpha-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with alpha-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, prebeta-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with prebeta-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of prebeta-1 into alpha-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles.     

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.     

Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids

Peripheral lymph lipoproteins have been characterized in animals, but there is little information about their composition, and none about their ultrastructure, in normal humans. Therefore, we collected afferent leg lymph from 16 healthy males and quantified lipids and apolipoproteins in fractions separated by high performance-size exclusion chromatography. Apolipoprotein B (apoB) was found almost exclusively in low density lipoproteins. The distribution of apoA-I, particularly in lipoprotein A-I (LpA-I) without A-II particles, was shifted toward larger particles relative to plasma. The fractions containing these particles were also enriched in apoA-II, apoE, total cholesterol, and phospholipids and had greater unesterified cholesterol-to-cholesteryl ester ratios than their counterparts in plasma. Fractions containing smaller apoA-I particles were enriched in phospholipid. Most apoA-IV was lipid poor or lipid free. Most apoC-III coeluted with large apoA-I-containing particles. Electron microscopy showed that lymph contained discoidal particles not seen in plasma. These findings support other evidence that high density lipoproteins (HDL) undergo extensive remodeling in human tissue fluid. Total cholesterol concentration in lymph HDL was 30% greater (P < 0.05) than could be explained by the transendothelial transfer of HDL from plasma, providing direct confirmation that HDL acquire cholesterol in the extravascular compartment. Net transport rates of new HDL cholesterol in the cannulated vessels corresponded to a mean whole body reverse cholesterol transport rate via lymph of 0.89 mmol (344 mg)/day.     

GPI-specific phospholipase D associates with an apoA-I- and apoA-IV-containing complex

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and associates with high density lipoproteins (HDL). We have characterized the distribution of GPI-PLD among lipoproteins in human plasma. Apolipoprotein (apo)-specific lipoproteins containing apoB (Lp[B]), apoA-I and A-II (Lp[A-I, A-II]), or apoA-I only (Lp[A-I]) were isolated using dextran sulfate and immunoaffinity chromatography. In six human plasma samples with HDL cholesterol ranging from 39 to 129 mg/dl, 79 +/- 14% (mean +/- SD) of the total plasma GPI-PLD activity was associated with Lp[A-I], 9 +/- 12% with Lp[A-I, A-II], and 1 +/- 1% with Lp[B]; and 11 +/- 10% was present in plasma devoid of these lipoproteins. Further characterization of the GPI-PLD-containing lipoproteins by gel-filtration chromatography and nondenaturing polyacrylamide and agarose gel electrophoresis revealed that these apoA-I-containing particles/complexes were small (8 nm) and migrated with pre-beta particles on agarose electrophoresis. Immunoprecipitation of GPI-PLD with a monoclonal antibody to GPI-PLD co-precipitated apoA-I and apoA-IV but little or no apoA-II, apoC-II, apoC-III, apoD, or apoE. In vitro, apoA-I but not apoA-IV or bovine serum albumin interacted directly with GPI-PLD, but did not stimulate GPI-PLD-mediated cleavage of a cell surface GPI-anchored protein. Thus, the majority of plasma GPI-PLD appears to be specifically associated with a small, discrete, and minor fraction of lipoproteins containing apoA-I and apoA-IV. -- Deeg, M. A., E. L. Bierman, and M. C. Cheung. GPI-specific phospholipase D associates with an apoA-I- and apoA-IV-containing complex. J. Lipid Res. 2001. 42: 442--451.     

The interplay between size, morphology, stability, and functionality of high-density lipoprotein subclasses

High-density lipoprotein (HDL) mediates reverse cholesterol transport (RCT), wherein excess cholesterol is conveyed from peripheral tissues to the liver and steroidogenic organs. During this process HDL continually transitions between subclass sizes, each with unique biological activities. For instance, RCT is initiated by the interaction of lipid-free/lipid-poor apolipoprotein A-I (apoA-I) with ABCA1, a membrane-associated lipid transporter, to form nascent HDL. Because nearly all circulating apoA-I is lipid-bound, the source of lipid-free/lipid-poor apoA-I is unclear. Lecithin:cholesterol acyltransferase (LCAT) then drives the conversion of nascent HDL to spherical HDL by catalyzing cholesterol esterification, an essential step in RCT. To investigate the relationship between HDL particle size and events critical to RCT such as LCAT activation and lipid-free apoA-I production for ABCA1 interaction, we reconstituted five subclasses of HDL particles (rHDL of 7.8, 8.4, 9.6, 12.2, and 17.0 nm in diameter, respectively) using various molar ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, free cholesterol, and apoA-I. Kinetic analyses of this comprehensive array of rHDL particles suggest that apoA-I stoichiometry in rHDL is a critical factor governing LCAT activation. Electron microscopy revealed specific morphological differences in the HDL subclasses that may affect functionality. Furthermore, stability measurements demonstrated that the previously uncharacterized 8.4 nm rHDL particles rapidly convert to 7.8 nm particles, concomitant with the dissociation of lipid-free/lipid-poor apoA-I. Thus, lipid-free/lipid-poor apoA-I generated by the remodeling of HDL may be an essential intermediate in RCT and HDL's in vivo maturation.     

Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase

Previous studies have provided detailed information on the formation of spherical high density lipoproteins (HDL) containing apolipoprotein (apo) A-I but no apoA-II (A-I HDL) by an lecithin:cholesterol acyltransferase (LCAT)-mediated process. In this study we have investigated the formation of spherical HDL containing both apoA-I and apoA-II (A-I/A-II HDL). Incubations were carried out containing discoidal A-I reconstituted HDL (rHDL), discoidal A-II rHDL, and low density lipoproteins in the absence or presence of LCAT. After the incubation, the rHDL were reisolated and subjected to immunoaffinity chromatography to determine whether A-I/A-II rHDL were formed. In the absence of LCAT, the majority of the rHDL remained as either A-I rHDL or A-II rHDL, with only a small amount of A-I/A-II rHDL present. By contrast, when LCAT was present, a substantial proportion of the reisolated rHDL were A-I/A-II rHDL. The identity of the particles was confirmed using apoA-I rocket electrophoresis. The formation of the A-I/A-II rHDL was influenced by the relative concentrations of the precursor discoidal A-I and A-II rHDL. The A-I/A-II rHDL included several populations of HDL-sized particles; the predominant population having a Stokes' diameter of 9.9 nm. The particles were spherical in shape and had an electrophoretic mobility slightly slower than that of the alpha-migrating HDL in human plasma. The apoA-I:apoA-II molar ratio of the A-I/A-II rHDL was 0.7:1. Their major lipid constituents were phospholipids, unesterified cholesterol, and cholesteryl esters. The results presented are consistent with LCAT promoting fusion of the A-I rHDL and A-II rHDL to form spherical A-I/A-II rHDL. We suggest that this process may be an important source of A-I/A-II HDL in human plasma.     

Hyperlipoproteinemia in Nagase analbuminemic rats: effects of pravastatin on plasma (apo)lipoproteins and lecithin:cholesterol acyltransferase activity.

The present study demonstrates very high levels of plasma lipids and high density lipoprotein (HDL) apolipoproteins (apoA-I and apoE) in female Nagase analbuminemic rats (NAR) fed a semi-synthetic diet in order to further increase the hyperlipidemia present in this strain. Plasma apoB-containing lipoproteins (very low, intermediate, and low density lipoproteins) were also elevated in NAR. Plasma cholesterol was mainly present in lipoprotein particles with a density between 1.02 and 1.12 g/ml. Separation of lipoprotein classes by gel filtration showed that the major cholesterol-carrying lipoprotein fractions in NAR plasma are apoE-rich HDL and apoA-I-rich HDL. The high HDL levels in NAR are explained, at least partly, by the two- to threefold elevated activity of plasma lecithin:cholesterol acyltransferase (LCAT). The lysophosphatidylcholine generated in the LCAT reaction, as well as plasma free fatty acids, are bound to lipoproteins in NAR plasma. A study was carried out to determine whether the elevated LDL and aopoE-rich HDL levels could be corrected by administration of the HMG-CoA reductase inhibitor pravastatin (at a dose of 1 mg/kg per day). Pravastatin treatment results in a 43% decrease in plasma triglycerides in NAR, but not in Sprague-Dawley (SDR) rats, and had no significant effect on plasma total cholesterol, phospholipids apolipoproteins A-I, A-IV, B, or E, as well as on plasma LCAT activity levels in NAR or SDR.     

LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer’s disease, including facilitating β-amyloid (Aβ) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aβ clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aβ or amyloid in APP/PS1 LCAT^−/− mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer’s-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aβ clearance.     

LCAT can rescue the abnormal phenotype produced by the natural ApoA-I mutations (Leu141Arg)Pisa and (Leu159Arg)FIN

To explain the etiology and find a mode of therapy of genetically determined low levels of high-density lipoprotein (HDL), we have generated recombinant adenoviruses expressing apolipoprotein A-I (apoA-I)(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN and studied their properties in vitro and in vivo. Both mutants were secreted efficiently from cells but had diminished capacity to activate lecithin/cholesterol acyltransferase (LCAT) in vitro. Adenovirus-mediated gene transfer of either of the two mutants in apoA-I-deficient (apoA-I-/-) mice resulted in greatly decreased total plasma cholesterol, apoA-I, and HDL cholesterol levels. The treatment also decreased the cholesteryl ester to total cholesterol ratio (CE/TC), caused accumulation of prebeta1-HDL and small size alpha4-HDL particles, and generated only few spherical HDL particles, as compared to mice expressing wild-type (WT) apoA-I. Simultaneous treatment of the mice with adenoviruses expressing either of the two mutants and human LCAT normalized the plasma apoA-I, HDL cholesterol levels, and the CE/TC ratio, restored normal prebeta- and alpha-HDL subpopulations, and generated spherical HDL. The study establishes that apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN inhibit an early step in the biogenesis of HDL due to inefficient esterification of the cholesterol of the prebeta1-HDL particles by the endogenous LCAT. Both defects can be corrected by treatment with LCAT.     

Lipoprotein abnormalities associated with lipopolysaccharide-induced lecithin: cholesterol acyltransferase and lipase deficiency

Density gradient ultracentrifugation was used to isolate and characterize the plasma lipoproteins from African green monkeys before and 24 and 48 h after subcutaneous injection of 300 micrograms/kg lipopolysaccharide (LPS) to induce an acute phase response. Compared with 0 h values, reductions occurred in plasma cholesterol (39%), high density lipoprotein (HDL) cholesterol (54%), lecithin:cholesterol acyltransferase (LCAT) activity (55%), and post-heparin plasma lipase activity (68%) 48 h after LPS injection while plasma triglyceride concentrations increased 700%. Cholesterol distribution among lipoproteins shifted from 7 to 41% in very low density lipoproteins (VLDL), 65 to 38% in low density lipoproteins (LDL), and 28 to 21% in HDL after LPS injection. At 48 h after LPS injection, all lipoprotein classes were relatively enriched in phospholipid and triglyceride and depleted of cholesteryl ester. The plasma concentration of all chemical constituents in VLDL was increased 3-9-fold within 48 h after LPS injection. By negative stain electron microscopy, HDL were discoidal in shape while VLDL and LDL appeared to have excess surface material present. Even though total HDL protein concentration in plasma was unaffected, the plasma mass of the smallest HDL subfractions (HDL3b,c) doubled while the mass of intermediate-sized subfractions (HDL3a) was dramatically decreased within 24 h after treatment. HDL became enriched in apoE, acquired apoSAA, and became depleted of apoA-I, A-II, and Cs by 48 h after LPS injection while apoB-100 remained the major apoprotein of VLDL and LDL. We conclude that administration of LPS to monkeys prevents normal intravascular metabolism of lipoproteins and results in the accumulation of relatively nascent forms of lipoproteins in plasma. These immature lipoproteins resemble those isolated from the recirculating perfusion of African green monkey livers, which are relatively deficient of LCAT activity and those isolated from the plasma of patients with familial LCAT deficiency.     

Differential involvement of thrombin receptors in Ca2+ release from two different intracellular stores in human platelets

In the present study we have used adenovirus-mediated gene transfer of apoA-I (apolipoprotein A-I) mutants in apoA-I-/- mice to investigate how structural mutations in apoA-I affect the biogenesis and the plasma levels of HDL (high-density lipoprotein). The natural mutants apoA-I(R151C)Paris, apoA-I(R160L)Oslo and the bioengineered mutant apoA-I(R149A) were secreted efficiently from cells in culture. Their capacity to activate LCAT (lecithin:cholesterol acyltransferase) in vitro was greatly reduced, and their ability to promote ABCA1 (ATP-binding cassette transporter A1)-mediated cholesterol efflux was similar to that of WT (wild-type) apoA-I. Gene transfer of the three mutants in apoA-I-/- mice generated aberrant HDL phenotypes. The total plasma cholesterol of mice expressing the apoA-I(R160L)Oslo, apoA-I(R149A) and apoA-I(R151C)Paris mutants was reduced by 78, 59 and 61% and the apoA-I levels were reduced by 68, 64 and 55% respectively, as compared with mice expressing the WT apoA-I. The CE (cholesteryl ester)/TC (total cholesterol) ratio of HDL was decreased and the apoA-I was distributed in the HDL3 region. apoA-I(R160L)Oslo and apoA-I(R149A) promoted the formation of prebeta1 and alpha4-HDL subpopulations and gave a mixture of discoidal and spherical particles. apoA-I(R151C)Paris generated subpopulations of different sizes that migrate between prebeta and alpha-HDL and formed mostly spherical and a few discoidal particles. Simultaneous treatment of mice with adenovirus expressing any of the three mutants and human LCAT normalized plasma apoA-I, HDL cholesterol levels and the CE/TC ratio. It also led to the formation of spherical HDL particles consisting mostly of alpha-HDL subpopulations of larger size. The correction of the aberrant HDL phenotypes by treatment with LCAT suggests a potential therapeutic intervention for HDL abnormalities that result from specific mutations in apoA-I.     

Characterization of four lipoprotein classes in human cerebrospinal fluid.

Lipoprotein metabolism in brain has not yet been fully elucidated, although there are a few reports concerning lipids in the brain and lipoproteins and apolipoproteins in the cerebrospinal fluid (CSF). To establish normal levels of lipoproteins in human CSF, total cholesterol, phospholipids, and fatty acids as well as apolipoprotein E (apoE) and apoA-I levels were determined in CSF samples from 216 individuals. For particle characterization, lipoproteins from human CSF were isolated by affinity chromatography and analyzed for size, lipid and apolipoprotein composition. Two consecutive immunoaffinity columns with antibodies, first against apoE and subsequently against apoA-I, were used to define four distinct lipoprotein classes. The major lipoprotein fraction consisted of particles of 13;-20 nm containing apoE and apoA-I as well as apoA-IV, apoD, apoH, and apoJ. In the second particle class (13;-18 nm) mainly apoA-I and apoA-II but no apoE was detected. Third, there was a small number of large particles (18;-22 nm) containing no apoA-I but apoE associated with apoA-IV, apoD, and apoJ. In the unbound fraction we detected small particles (10;-12 nm) with low lipid content containing apoA-IV, apoD, apoH, and apoJ. In summary, we established lipid and apolipoprotein levels in CSF in a large group of individuals and described four distinct lipoprotein classes in human CSF, differing in their apolipoprotein pattern, lipid composition, and size. On the basis of our own data and previous findings from other groups, we propose a classification of CSF lipoproteins.     

Association Between Lipids, Lipoproteins Composition of HDL Particles and Triglyceride-Rich Lipoproteins, and LCAT and CETP Activity in Post-renal Transplant Patients

High-density lipoprotein (HDL) remodeling within the plasma compartment and the association between lecithin-cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) activity, and lipid, lipoprotein concentrations and composition were investigated. The aim was to examine the high sensitivity of C-reactive protein (hsCRP), lipid, apolipoprotein B (apoB), apoAI, total apoAII, apoAIInonB, apoB-containing apoAII (apoB:AII), total apoCIII, apoCIIInonB, apoB-containing apoCIII (apoB:CIII) concentration and LCAT and CETP activity to gain an insight into the association between them and LCAT and CETP, 57 post-renal transplant (Tx) patients with and without statin therapy and in 15 healthy subjects. Tx patients had moderate hypertriglyceridemia, hypercholesterolemia, and dyslipoproteinemia, disturbed triglyceride-rich lipoproteins (TRLs) and HDL composition, decreased LCAT, and slightly increased hsCRP but no CETP activity. Spearman’s correlation test showed the association between lipids and lipoproteins and LCAT or CETP, and multiple ridge stepwise forward regression showed that immunosuppressive therapy in Tx patients can disturb HDL and TRLs composition. The results suggest that inhibition or activation of LCAT is due, in part, to HDL-associated lipoprotein. Lipoprotein composition of apoAI, apoAIInonB, and apoCIIInonB in HDL particle and apoB:AII TRLs can contribute to decrease LCAT mass in Tx patients. Tx patients without statin and with lower triglycerides but higher HDL cholesterol concentration and disturbed lipoprotein composition of ApoAI and apoAII in HDL particle can decrease LCAT, increase LDL cholesterol, aggravate renal graft, and accelerate atherosclerosis and chronic heart diseases.     

Action of lecithin-cholesterol acyltransferase on low-density lipoproteins in native pig plasma

The action of lecithin-cholesterol acyltransferase (LCAT, EC 2.3.1.43) on the different pig lipoprotein classes was investigated with emphasis on low-density lipoproteins (LDL). It was demonstrated previously that LDL can serve as substrate for LCAT, probably because they contain sufficient amounts of apoA-I and other non-apoB proteins, known as LCAT activators. Upon a 24-h incubation of pig plasma in vitro in the presence of active LCAT, both pig LDL subclasses, LDL-1 and LDL-2, fused together, forming one fraction, as revealed by analytical ultracentrifugation. This fusion was time dependent, becoming visible after 3 h and complete after 18 h of incubation. Concomitantly, free cholesterol and phospholipids decreased and cholesteryl esters increased. When isolated LDL-1 and LDL-2 were incubated with purified pig LCAT for 24 h, LDL-1 floated toward higher densities and LDL-2 toward lower densities, although this effect was not as pronounced as in incubations of whole serum. In further experiments, pig serum was incubated for various periods of time in the presence and absence of the LCAT inhibitor sodium iodoacetate. The individual lipoproteins then were separated by density gradient ultracentrifugation or by specific immunoprecipitation and chemically analyzed. Both methods revealed that in the absence of active LCAT there was a transfer of free cholesterol from LDL to high-density lipoproteins (HDL) and a small transfer of cholesteryl esters in the opposite direction. In the presence of LCAT the loss of free cholesterol started immediately in all three lipoprotein classes, was most prominent in LDL, and was proportional to the newly synthesized cholesteryl esters incorporated in each fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation by estrogen of synthesis and secretion of apolipoprotein A-I in the chicken hepatoma cell line,LMH-2A

The synthesis and secretion of apolipoprotein A-I (apoA-I) in response to the treatment with estrogen were investigated in the chicken hepatoma cell line, LMH-2A. Exposure of these cells to exogenous estrogen for up to 48 h results in a decrease of apoA-I production, as evident from Western blotting, immunoprecipitation, and immunofluorescence experiments. Likewise, the secretion of apoA-I is also decreased in estrogen-treated cells when compared to controls. However, under both conditions, the disappearance of the apoprotein from the cells occurs very rapidly and with similar kinetics. The bulk of apoA-I secreted from LMH-2A cells is recovered on lipoprotein particles with a buoyant density of > or =1.10 g/ml, corresponding to HDL and heavy LDL. Interestingly, apoA-I is detectable on apoB-containing lipoproteins by sequential immunoprecipitation, suggesting that the two apoproteins co-reside at least on a subfraction of the secreted particles, or that apoB- and apoA-I-containing particles interact. These interactions are more pronounced in estrogen-treated cells, most likely due to the dramatic estrogen-mediated induction of apoB synthesis and secretion.     

Deletions of helices 2 and 3 of human apoA-I are associated with severe dyslipidemia following adenovirus-mediated gene transfer in apoA-I-deficient mice

The objective of this study was to determine the effect of two amino-terminal apolipoprotein A-I (apoA-I) deletions on high-density lipoprotein (HDL) biosynthesis and lipid homeostasis. Adenovirus-mediated gene transfer showed that the apoA-I[Delta(89-99)] deletion mutant caused hypercholesterolemia, characterized by increased plasma cholesterol and phospholipids, that were distributed in the very low density/intermediate density/low-density lipoprotein (VLDL/IDL/LDL) region, and normal triglycerides. The capacity of the mutant protein to promote ATP-binding cassette transporter A1- (ABCA1-) mediated cholesterol efflux and to activate lecithin:cholesterol acyltranserase (LCAT) was approximately 70-80% of the wild-type (WT) control. The phospholipid transfer protein (PLTP) activity of plasma containing the apoA-I[Delta(89-99)] mutant was decreased to 32% of the WT control. Similar analysis showed that the apoA-I[Delta(62-78)] deletion mutant in apoA-I-deficient mice caused combined hyperlipidemia characterized by increased triglycerides, cholesterol, and phospholipids in the VLDL/IDL region. There was enrichment of the VLDL/IDL with mutant apoA-I that resulted in reduction of in vitro lipolysis. The capacity of this mutant to promote ABCA1-mediated cholesterol efflux was normal, and the capacity to activate LCAT in vitro was reduced by 53%. The WT apoA-I and the apoA-I[Delta(62-78)] mutant formed spherical HDL particles, whereas the apoA-I[Delta(89-99)] mutant formed discoidal HDL particles. We conclude that alterations in apoA-I not only may have adverse effects on HDL biosynthesis but also may promote dyslipidemia due to interference of the apoA-I mutants on the overall cholesterol and triglycerides homeostasis.