HDLs in apoA-I transgenic Abca1 knockout mice are remodeled normally in plasma but are hypercatabolized by the kidney

Patients homozygous for Tangier disease have a near absence of plasma HDL as a result of mutations in ABCA1 and hypercatabolize normal HDL particles. To determine the relationship between ABCA1 expression and HDL catabolism, we investigated intravascular remodeling, plasma clearance, and organ-specific uptake of HDL in mice expressing the human apolipoprotein A-I (apoA-I) transgene in the Abca1 knockout background. Small HDL particles (7.5 nm), radiolabeled with (125)I-tyramine cellobiose, were injected into recipient mice to quantify plasma turnover and the organ uptake of tracer. Small HDL tracer was remodeled to 8.2 nm diameter particles within 5 min in human apolipoprotein A-I transgenic (hA-I(Tg)) mice (control) and knockout mice. Decay of tracer from plasma was 1.6-fold more rapid in knockout mice (P < 0.05) and kidney uptake was twice that of controls, with no difference in liver uptake. We also observed 2-fold greater hepatic expression of ABCA1 protein in hA-I(Tg) mice compared with nontransgenic mice, suggesting that overexpression of human apoA-I stabilized hepatic ABCA1 protein in vivo. We conclude that ABCA1 is not required for in vivo remodeling of small HDLs to larger HDL subfractions and that the hypercatabolism of normal HDL particles in knockout mice is attributable to a selective catabolism of HDL apoA-I by the kidney.     

An apolipoprotein A-I mimetic dose-dependently increases the formation of prebeta1 HDL in human plasma

Prebeta1 HDL is the initial plasma acceptor of cell-derived cholesterol in reverse cholesterol transport. Recently, small amphipathic peptides composed of D-amino acids have been shown to mimic apolipoprotein A-I (apoA-I) as a precursor for HDL formation. ApoA-I mimetic peptides have been proposed to stimulate the formation of prebeta1 HDL and increase reverse cholesterol transport in apoE-null mice. The existence of a monoclonal antibody (MAb 55201) and a corresponding ELISA method that is selective for the detection of the prebeta(1) subclass of HDL provides a means of establishing a correlation between apoA-I mimetic dose and prebeta1 HDL formation in human plasma. Using this prebeta1 HDL ELISA, we demonstrate marked apoA-I mimetic dose-dependent prebeta1 HDL formation in human plasma. These results correlated with increases in band density of the plasma prebeta1 HDL, when observed by Western blotting, as a function of increased apoA-I mimetic concentration. Increased prebeta1 HDL formation was observed after as little as 1 min and was maximal within 1 h. Together, these data suggest that a high-throughput prebeta1 HDL ELISA provides a way to quantitatively measure a key component of the reverse cholesterol transport pathway in human plasma, thus providing a possible method for the identification of apoA-I mimetic molecules.     

Initial interaction of apoA-I with ABCA1 impacts in vivo metabolic fate of nascent HDL

We investigated the in vivo metabolic fate of pre-beta HDL particles in human apolipoprotein A-I transgenic (hA-I (Tg)) mice. Pre-beta HDL tracers were assembled by incubation of [(125)I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-beta HDLs of increasing size (pre-beta1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-I (Tg)-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-beta2, -3, and -4 had similar plasma die-away rates, whereas pre-beta1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-beta HDL size increased. In plasma, pre-beta1 and -2 were rapidly (<5 min) remodeled into larger HDLs, whereas pre-beta3 and -4 were remodeled into smaller HDLs. Pre-beta HDLs were similarly remodeled in vitro with control or LCAT-immunodepleted plasma, but not when incubated with phospholipid transfer protein knockout plasma. Our results suggest that initial interaction of apoA-I with ABCA1 imparts a unique conformation that partially determines the in vivo metabolic fate of apoA-I, resulting in increased liver and decreased kidney catabolism as pre-beta HDL particle size increases.     

Role of the kidney in regulating the metabolism of HDL in rabbits: evidence that iodination alters the catabolism of apolipoprotein A-I by the kidney

To evaluate the factors that regulate HDL catabolism in vivo, we have measured the clearance of human apoA-I from rabbit plasma by following the isotopic decay of (125)I-apoA-I and the clearance of unlabeled apoA-I using a radioimmunometric assay (RIA). We show that the clearance of unlabeled apoA-I is 3-fold slower than that of (125)I-apoA-I. The mass clearance of iodinated apoA-I, as determined by RIA, is superimposable with the isotopic clearance of (125)I-apoA-I. The data demonstrate that iodination of tyrosine residues alters the apoA-I molecule in a manner that promotes an accelerated catabolism. The clearance from rabbit plasma of unmodified apoA-I on HDL(3) and a reconstituted HDL particle (LpA-I) were very similar and about 3-4-fold slower than that for (125)I-apoA-I on the lipoproteins. Therefore, HDL turnover in the rabbit is much slower than that estimated from tracer kinetic studies. To determine the role of the kidney in HDL metabolism, the kinetics of unmodified apoA-I and LpA-I were reevaluated in animals after a unilateral nephrectomy. Removal of one kidney was associated with a 40-50% reduction in creatinine clearance rates and a 34% decrease in the clearance rate of unlabeled apoA-I and LpA-I particles. In contrast, the clearance of (125)I-labeled molecules was much less affected by the removal of a kidney; FCR for (125)I-LpA-I was reduced by <10%. The data show that the kidneys are responsible for most (70%) of the catabolism of apoA-I and HDL in vivo, while (125)I-labeled apoA-I and HDL are rapidly catabolized by different tissues. Thus, the kidney is the major site for HDL catabolism in vivo. Modification of tyrosine residues on apoA-I may increase its plasma clearance rate by enhancing extra-renal degradation pathways.     

Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.     

Apolipoprotein A-I charge and conformation regulate the clearance of reconstituted high density lipoprotein in vivo

While low apolipoprotein A-I (apoA-I) levels are primarily associated with increased high density lipoprotein (HDL) fractional catabolic rate (FCR), the factors that regulate the clearance of HDL from the plasma are unclear. In this study, the effect of lipid composition of reconstituted HDL particles (LpA-I) on their rate of clearance from rabbit plasma has been investigated. Sonicated LpA-I containing 1 to 2 molecules of purified human apoA-I and 5 to 120 molecules of palmitoyl-oleoyl phosphatidylcholine (POPC) exhibit similar charge and plasma FCR to that for lipid free apoA-I, 2.8 pools/day. Inclusion of 1 molecule of apoA-II to an LpA-I complex increases the FCR to 3.5 pools/day, a value similar to that observed for exchanged-labeled HDL3. In contrast, addition of 40 molecules of triglyceride, diglyceride, or cholesteryl ester to a sonicated LpA-I containing 120 moles of POPC and 2 molecules of apoA-I increases the negative charge of the particle and reduces the FCR to 1.8 pools/day. Discoidal LpA-I are the most positively charged lipoprotein particles and also have the fastest clearance rates, 4.5 pools/day. Immunochemical characterization of the different LpA-I particles shows that the exposure of an epitope at residues 98 to 121 of the apoA-I molecule is associated with an increased negative particle charge and a slower clearance from the plasma.We conclude that the charge and conformation of apoA-I are sensitive to the lipid composition of LpA-I and play a central role in regulating the clearance of these lipoproteins from plasma. conformation regulate the clearance of reconstituted high density lipoprotein in vivo.     

Increased sphingomyelin content impairs HDL biogenesis and maturation in human Niemann-Pick disease type B

We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL. In this study, we investigated the pathophysiology of this HDL deficiency by examining both HDL samples from NPD-B patients and nascent high density lipoprotein (LpA-I) generated by incubation of lipid-free apolipoprotein A-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-I and HDL isolated from patient plasma had a significant increase in sphingomyelin (SM) mass ( approximately 50-100%). Analysis of LCAT kinetics parameters (V(max) and K(m)) revealed that either LpA-I or plasma HDL from NPD-B, as well as reconstituted HDL enriched with SM, exhibited severely decreased LCAT-mediated cholesterol esterification. Importantly, we documented that SM enrichment of NPD-B LpA-I was not attributable to increased cellular mass transfer of SM or unesterified cholesterol to lipid-free apoA-I. Finally, we obtained evidence that the conditioned medium from HUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts, contained active secretory sphingomyelinase (S-SMase) that mediated the hydrolysis of [(3)H]SM-labeled LpA-I and HDL(3). Furthermore, expression of mutant SMase (DeltaR608) in CHO cells revealed that DeltaR608 was synthesized normally but had defective secretion and activity. Our data suggest that defective S-SMase in NPD leads to SM enrichment of HDL that impairs LCAT-mediated nascent HDL maturation and contributes to HDL deficiency. Thus, S-SMase and LCAT may act in concert and play a crucial role in the biogenesis and maturation of nascent HDL particles.     

Deletion of the propeptide of apolipoprotein A-I reduces protein expression but stimulates effective conversion of prebeta-high density lipoprotein to alpha-high density lipoprotein

The properties of the mature and pro-forms of recombinant apolipoprotein A-I (apoA-I) were compared with those of apoA-I isolated from human plasma. When the synthesis and secretion of pro- and mature forms of apoA-I from a baculovirus/insect cell expression system were compared in parallel experiments, the amount of the pro-form of apoA-I synthesized and secreted was severalfold higher than that of the mature form of apoA-I. A comparison of the properties of the pro- and mature forms of recombinant apoA-I and human plasma apoA-I showed no difference between all three in their secondary structure, their ability to self-associate, lipid-binding capacity, lecithin: cholesterol acyltransferase activation, and binding to the phospholipid transfer protein. The properties of reconstituted high density lipoprotein (HDL) particles formed from the proteins and their ability to promote cholesterol and phospholipid efflux from human skin fibroblasts were also similar. However, their ability to bind to plasma HDL subfractions differed, because twice as much proapoA-I associated with prebeta(1)-HDL and prebeta(2)-HDL subfractions compared with both mature recombinant and plasma apoA-I. Correspondingly, the amount of proapoA-I in alpha-HDL subfractions, especially in alpha(1)-HDL and alpha(2)-HDL, was decreased. We conclude that while the propeptide of apoA-I is required for the effective synthesis and secretion of apoA-I, cleavage of this peptide is a requisite for the effective interconversion of HDL subfractions.     

Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase. Implications for HDL function

When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL(3) by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL(3) revealed that proteolysis for up to 24 h did not alter the integrity of the alpha-migrating HDL, whereas a minor peak containing particles of smaller size with prebeta mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the alphaHDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL(3) with chymase inhibited binding of Mab A-I-9 (specific for prebeta(1)HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL(3) to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL(3) by chymase failed to reduce the ability of HDL(3) to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation. This differential sensitivity of the two key functions of HDL(3) to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for prebeta(1)HDL to promote the high-affinity efflux of cellular cholesterol, but not for the alpha-migrating HDL particles to activate LCAT.     

Binding characteristics of high density lipoprotein subclasses to porcine liver, adrenal and skeletal muscle plasma membranes

1. We compared binding characteristics of 125I-labeled high density lipoprotein (HDL) subclasses to porcine liver, adrenal and skeletal muscle plasma membranes. 2. HDL subclasses were discriminated by their buoyant densities (HDL2 and HDL3) or by their apolipoprotein (apo) content (Lp-AI (particles containing apoA-I but no apoA-II) and LpA-I/A-II (particles containing both apoA-I and apoA-II)). 3. HDL2 and HDL3 showed saturable binding to the three types of membrane preparations. 4. No differences were found in the Kds within one HDL subclass. 5. Kds and maximal binding of HDL2 were lower than these of HDL3. Unlabeled HDL2 and HDL3, but not LDL, effectively displaced 125I-HDL2 and 125I-HDL3. 6. Binding of HDL was independent of the concentration of NaCl and did not require calcium. 7. These results suggest a process mediated by a single specific receptor in porcine liver, adrenal and skeletal muscle plasma membranes. 8. We also studied binding characteristics of HDL subclasses Lp-AI and LpA-I/A-II to porcine liver membranes. LpA-I showed the highest Kd and maximal binding. 9. All types of HDL subclasses studied (i.e. HDL2, HDL3, LpA-I and LpA-I/A-II) effectively competed for binding of both Lp-AI and LpA-I/A-II, suggesting that the HDL subclasses studied bind to the same receptor by their apoA-I moiety.     

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.     

Dietary n-3 polyunsaturated fat increases the fractional catabolic rate of medium-sized HDL particles in African green monkeys

We have previously described a novel pathway for the metabolism of HDL subfractions in which small [2 apolipoprotein (apoA-I) molecules per particle] HDL particles are converted in a unidirectional manner outside the plasma compartment to medium (3 apoA-I molecules per particle) or large (4 apoA-I molecules per particle) HDL particles, which are subsequently removed from the circulation by the liver (Colvin et al. 1999. J. Lipid Res. 40: 1782;-1792; Huggins et al. 2000. J. Lipid Res. 41: 384;-394). The purpose of the present study was to determine whether the reduction in concentration of medium HDL in African green monkeys consuming n-3 polyunsaturated versus saturated fat diets resulted from decreased in vivo production or increased catabolism. Tracer small LpA-I (HDL containing only apoA-I) were isolated, without ultracentrifugation, by gel filtration and immunoaffinity chromatography and radiolabeled. After injection, the specific activity of apoA-I in small, medium, and large HDL was determined, and the kinetic data were analyzed using our previously published multicompartmental model for HDL subfraction metabolism. We found a significant reduction of apoA-I concentration in medium HDL in the animals fed n-3 polyunsaturated fat (31.2 +/- 0.7 mg/dl) compared with animals fed saturated fat (85.4 +/- 11.9 mg/dl; P = 0.002). The production rates of apoA-I in small, medium, and large HDL were similar in both diet groups; however, there was a significant increase in the fractional catabolic rate of apoA-I in medium HDL in the animals fed n-3 polyunsaturated fat (2.188 +/- 0.501 pools/day) compared with animals fed saturated fat (0.714 +/- 0.191 pools/day; P = 0.02).We conclude that n-3 polyunsaturated fat reduces HDL cholesterol concentration by increasing the fractional catabolic rate of medium-sized HDL particles in African green monkeys.     

In vivo metabolism of apolipoprotein A-I on high density lipoprotein particles LpA-I and LpA-I,A-II.

Apolipoprotein (apo) A-I is the major protein in high density lipoproteins (HDL) and is found in two major subclasses of lipoproteins, those containing apolipoprotein A-II (termed LpA-I,A-II) and those without apoA-II (termed LpA-I). The in vivo kinetics of apoA-I on LpA-I and LpA-I,A-II were investigated in normolipidemic human subjects. In the first series of studies, radiolabeled apoA-I and apoA-II were reassociated with autologous plasma lipoproteins and injected into normal subjects. LpA-I and LpA-I,A-II were isolated from plasma at selected time points by immunoaffinity chromatography. By 24 h after injection, only 52.8 +/- 1.0% of the apoA-I in LpA-I remained, whereas 66.9 +/- 2.7% of apoA-I in LpA-I,A-II remained (P less than 0.01). In the second series of studies, purified apoA-I was labeled with either 131I or 125I and reassociated with autologous plasma. Isolated LpA-I and LpA-I,A-II particles differentially labeled with 131I-labeled apoA-I and 125I-labeled apoA-I, respectively, were simultaneously injected into study subjects. The plasma residence time of apoA-I injected on LpA-I (mean 4.39 days) was substantially shorter than that of apoA-I injected on LpA-I,A-II (mean 5.17 days), with a mean difference in residence times of 0.79 +/- 0.08 days (P less than 0.001). These data demonstrate that apoA-I injected on LpA-I is catabolized more rapidly than apoA-I injected on LpA-I,A-II. The results are consistent with the concept that LpA-I and LpA-I,A-II have divergent metabolic pathways.     

Serum amyloid A protein generates pre beta 1 high-density lipoprotein from alpha-migrating high-density lipoprotein

Serum amyloid A protein (SAA), an acute-phase reactant in reactive amyloidosis, has high affinity for high-density lipoprotein (HDL). When SAA is added to HDL, SAA displaces apolipoprotein A-I (apoA-I) and phospholipid from the HDL particles. These dissociated components may form prebeta1-HDL because free apoA-I can associate with phospholipid to become a lipoprotein having prebeta mobility. To determine whether SAA generates prebeta1-HDL from alpha-migrating HDL, we investigated the effects of recombinant SAA on HDL subfraction concentration using nondenaturing two-dimensional gradient gel electrophoresis. When we added SAA (0.5 mg/mL) to plasma, the prebeta1-HDL concentration increased by 164% (from 4.7% +/- 1.3% to 12.4% +/- 3.2% of apoA-I, p < 0.005). The increase in prebeta1-HDL was proportional to the dose of SAA. When we added SAA to a column of Sepharose beads coupled to the isolated HDL (alpha-migrating HDL), prebeta1-HDL was dissociated from the column together with the SAA-associated HDL. In summary, we demonstrate that SAA generates prebeta1-HDL from alpha-migrating HDL. We speculate that SAA-mediated HDL remodeling may take place in inflammation.     

Determination of the tissue sites responsible for the catabolism of large high density lipoprotein in the African green monkey

In vivo multicompartmental modeling of the turnover of HDL subfractions has suggested that HDL containing four molecules of apoA-I per particle and no other apolipoproteins (large LpA-I) are terminal particles in plasma. We hypothesized that these terminal particles were the end product of HDL metabolism and, as such, would be cleared preferentially by the liver. Thus, the purpose of this study was to determine: 1) the tissue sites of catabolism of large LpA-I in African green monkeys, and 2) whether saturated versus n;-6 polyunsaturated dietary fat affected tissue accumulation. Large LpA-I were isolated, without ultracentrifugation, by size exclusion and immunoaffinity chromatography and radiolabeled with either the residualizing compound, (125)I-labeled tyramine cellobiose (TC), or with (131)I. After injection into recipient animals, the plasma die-away of the radiolabels was followed for 12 or 24 h, after which the animals were killed and tissues were collected for determining radiolabel sites of catabolism. The plasma die-away of the (125)I-labeled TC-LpA-I and (131)I-labeled LpA-I doses was similar suggesting that the TC radiolabeling did not modify the metabolism of the large LpA-I dose. The liver, adrenal, kidney, and spleen had the greatest accumulation of large LpA-I degradation products on a per gram tissue basis. On a whole organ basis, the liver was the major site of large LpA-I degradation in both the 12-h (15.4 +/- 0.3% of injected dose) and 24-h (9.1 +/- 0.6% of injected dose) catabolic studies. The kidney, compared to the liver, had less uptake of large LpA-I radioactivity in either study (1.3 +/- 0.4% and 1.2 +/- 0.3% of injected dose). There was no apparent influence of dietary fat type on the tissue accumulation of large LpA-I. We conclude that the liver is the primary site of catabolism of large LpA-I in the African green monkey.     

Influence of apolipoprotein A-I and apolipoprotein A-II availability on nascent HDL heterogeneity

It is important to understand HDL heterogeneity because various subspecies possess different functionalities. To understand the origins of HDL heterogeneity arising from the existence of particles containing only apoA-I (LpA-I) and particles containing both apoA-I and apoA-II (LpA-I+A-II), we compared the abilities of both proteins to promote ABCA1-mediated efflux of cholesterol from HepG2 cells and form nascent HDL particles. When added separately, exogenous apoA-I and apoA-II were equally effective in promoting cholesterol efflux, although the resultant LpA-I and LpA-II particles had different sizes. When apoA-I and apoA-II were mixed together at initial molar ratios ranging from 1:1 to 16:1 to generate nascent LpA-I+A-II HDL particles, the particle size distribution altered, and the two proteins were incorporated into the nascent HDL in proportion to their initial ratio. Both proteins formed nascent HDL particles with equal efficiency, and the relative amounts of apoA-I and apoA-II incorporation were driven by mass action. The ratio of lipid-free apoA-I and apoA-II available at the surface of ABCA1-expressing cells is a major factor in determining the contents of these proteins in nascent HDL. Manipulation of this ratio provides a means of altering the relative distribution of LpA-I and LpA-I+A-II HDL particles.     

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.     

Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.     

Interconversion of prebeta-migrating lipoproteins containing apolipoprotein A-I and HDL

Mouse plasma from strains C57BL/6J and C3H/HeJ includes a high density lipoprotein (HDL) fraction containing apolipoprotein A-I which migrates in the prebeta region upon agarose gel electrophoresis, similar to the prebeta HDL previously reported in humans. This prebeta A-I lipoprotein species has a buoyant density of 1.080-1.210 g/ml and has two molecular weight species, 65,000 and 71,000. It is lipid-poor and deficient in apolipoprotein E. When mice are fed a high fat and high cholesterol diet, the quantity of prebeta A-I increases in both strains as determined by quantitative densitometry of agarose gel immunoblots. Prebeta A-I species are highly unstable in plasma at 37 degrees C. Initially (0-1 h) levels decreased and with further incubation (1-8 h) levels increased. Nondenaturing polyacrylamide gel electrophoresis (PAGE) demonstrated that the prebeta HDL formed during prolonged incubation (1-8 h) was identical in size to HDL in unincubated samples. The initial decrease of prebeta HDL observed during the first hour of incubation, phase I, was inhibited by DTNB, suggesting that phase I is dependent on lecithin:cholesterol acyltransferase (LCAT); however, the subsequent increase, phase II, was unaffected by DTNB and appears LCAT-independent. The prebeta A-I species formed in plasma containing DTNB after a 4-h incubation resulted in a polydisperse particle size distribution. The two strains, the atherosclerosis-susceptible C57BL/6 and -resistant C3H, displayed a similar elevation and induction of prebeta HDL during a dietary switch from laboratory chow to an atherogenic diet with a transient peak occurring at 7 days even when total HDL in the susceptible strain was greatly reduced.     

Speciated human high-density lipoprotein protein proximity profiles

It is expected that the attendant structural heterogeneity of human high-density lipoprotein (HDL) complexes is a determinant of its varied metabolic functions. To determine the structural heterogeneity of HDL, we determined major apolipoprotein stoichiometry profiles in human HDL. First, HDL was separated into two main populations, with and without apolipoprotein (apo) A-II, LpA-I and LpA-I/A-II, respectively. Each main population was further separated into six individual subfractions using size exclusion chromatography (SEC). Protein proximity profiles (PPPs) of major apolipoproteins in each individual subfraction was determined by optimally cross-linking apolipoproteins within individual particles with bis(sulfosuccinimidyl) suberate (BS(3)), a bifunctional cross-linker, followed by molecular mass determination by MALDI-MS. The PPPs of LpA-I subfractions indicated that the number of apoA-I molecules increased from two to three to four with an increase in the LpA-I particle size. On the other hand, the entire population of LpA-I/A-II demonstrated the presence of only two proximal apoA-I molecules per particle, while the number of apoA-II molecules varied from one dimeric apoA-II to two and then to three. For most of the PPPs described above, an additional population that contained a single molecule of apoC-III in addition to apoA-I and/or apoA-II was detected. Upon composition analyses of individual subpopulations, LpA-I/A-II exhibited comparable proportions for total protein (～58%), phospholipids (～21%), total cholesterol (～16%), triglycerides (～5%), and free cholesterol (～4%) across subfractions. LpA-I components, on the other hand, showed significant variability. This novel information about HDL subfractions will form a basis for an improved understanding of particle-specific functions of HDL.