HDL particle size is a critical determinant of ABCA1-mediated macrophage cellular cholesterol export.

RATIONALE: High-density lipoprotein (HDL) is a heterogeneous population of particles. Differences in the capacities of HDL subfractions to remove cellular cholesterol may explain variable correlations between HDL-cholesterol and cardiovascular risk and inform future targets for HDL-related therapies. The ATP binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux to lipid-free apolipoprotein A-I, but the majority of apolipoprotein A-I in the circulation is transported in a lipidated state and ABCA1-dependent efflux to individual HDL subfractions has not been systematically studied. OBJECTIVE: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process. METHODS AND RESULTS: We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions. CONCLUSIONS: Small, dense HDL subfractions are the most efficient mediators of cholesterol efflux, and ABCA1 mediates cholesterol efflux to small dense HDL and to lipid-free apolipoprotein A-I. HDL-directed therapies should target increasing the concentrations or the cholesterol efflux capacity of small, dense HDL species in vivo.

Specific mutations in ABCA1 have discrete effects on ABCA1 function and lipid phenotypes both in vivo and in vitro.

Mutations in ATP-binding cassette transporter A1 (ABCA1) cause Tangier disease and familial hypoalphalipoproteinemia, resulting in low to absent plasma high-density lipoprotein cholesterol levels. However, wide variations in clinical lipid phenotypes are observed in patients with mutations in ABCA1. We hypothesized that the various lipid phenotypes would be the direct result of discrete and differing effects of the mutations on ABCA1 function. To determine whether there is a correlation between the mutations and the resulting phenotypes, we generated in vitro 15 missense mutations that have been described in patients with Tangier disease and familial hypoalphalipoproteinemia. Using localization of ABCA1, its ability to induce cell surface binding of apolipoprotein A-I, and its ability to elicit efflux of cholesterol and phospholipids to apolipoprotein A-I we determined that the phenotypes of patients correlate with the severity and nature of defects in ABCA1 function.

ApoA-I(MALLORCA) impairs LCAT activation and induces dominant familial hypoalphalipoproteinemia.

Apolipoprotein (apo)A-I is the major protein component of HDL and the cofactor for LCAT. We describe a large Spanish kindred, living in the Mediterranean Island of Mallorca, that presents a dominant form of hypoalphalipoproteinemia. The lipid profile of this family was studied because the proband, a 40-year-old male presenting signs of coronary atherosclerosis, showed severe HDL deficiency. However, none of the other family members had a known history of cardiovascular disease. Sequence analysis of the apoA-I gene in affected members identified a 33-base pair deletion, corresponding to residues 165-175 of the mature protein, eliminating the first 11 amino acids of the internal repeat 7. ApoA-I(MALLORCA) is associated with HDL-cholesterol deficiency (concentration ranging from 8-48% of the value in non-carriers), and a 2- to 3-fold decrease in plasma concentrations of apoA-I and apoA-II and endogenous LCAT activity, concomitant with a slight decrease in serum cholesterol efflux capability. Impairment of LCAT activity in HDL particles containing only mutated forms of apoA-I would not explain a pattern of dominant inheritance. HDL particles containing wild type apoA-I and at least one mutant apoA-I may also present impaired LCAT activity and/or other alterations leading to defective HDL maturation, a situation that would increase HDL lipid catabolism. We conclude that amino acids 165-175 of apoA-I are critical for normal HDL metabolism, at least in part because of their role in LCAT activation. However, apoA-I(MALLORCA) is not necessarily associated with clinical signs of atherosclerosis.

Lecithin:cholesterol acyl transferase G30S: association with atherosclerosis, hypoalphalipoproteinemia and reduced in vivo enzyme activity.

OBJECTIVES: A 69 yr old male was referred for assessment of a very low plasma HDL cholesterol and apolipoprotein AI concentration. At age 65, he had undergone triple vessel coronary bypass graft surgery. He had a strong family history of early coronary heart disease. We analyzed the molecular basis of his clinical and biochemical abnormalities. DESIGN AND METHODS: We used DNA sequencing to determine whether mutations in LCAT were present. We also evaluated plasma biochemistry and LCAT activity. RESULTS: DNA sequencing revealed that the patient was a heterozygote for the G30S mutation in the gene encoding lecithin:cholesteol acyl transferase (LCAT). His plasma was found to have half-normal LCAT activity. CONCLUSIONS: The findings in this patient suggest that rare dysfunctional mutations in candidate genes, such as LCAT, can contribute to the spectrum of patients ascertained because of low HDL cholesterol.

Decreased expression of a member of the Rho GTPase family, Cdc42Hs, in cells from Tangier disease - the small G protein may play a role in cholesterol efflux.

Cholesterol efflux (CE) is the initial and important step of reverse cholesterol transport (RCT), a major protective system against atherosclerosis. However, most of the molecular mechanism for CE still remains to be clarified. In the present study, cDNA subtraction revealed that the expression of a member of the Rho GTPase family, Cdc42Hs, was markedly decreased in both passaged fibroblasts and macrophages (Mφ) from patients with Tangier disease (TD), a rare lipoprotein disorder with reduced CE. This small G protein is known to have many cell biological activities such as rearrangement of actin cytoskeleton and vesicular transport, however the association between this molecule and lipid transport has never been reported. We demonstrate that MDCK cells expressing the dominant negative form of Cdc42Hs had reduced CE, inversely ones expressing the dominant active form had increased CE. From these observations, we would like to raise a novel hypothesis that this type of small G protein may play a role in some steps of CE. To our knowledge, the present study is the first demonstration that the expression of this molecule is altered in cells from human disease.

Modulated serum activities and concentrations of paraoxonase in high density lipoprotein deficiency states.

Paraoxonase is a high density lipoprotein (HDL) associated enzyme with a hypothesised role in the protection of low density lipoproteins (LDL) from oxidative stress. The present study examined paraoxonase in several genetically distinct HDL deficiency states. Despite reduction or even absence of detectable HDL, enzyme activity was present in sera from A-I-Pisa, A-I-Helsinki, A-I-Milano and Tangier patients. Both enzyme activities and peptide concentrations were modulated (reduced) but specific activities were broadly similar to controls, suggesting an impact on peptide concentration rather than an inhibition of enzyme activity. Despite the absence of HDL in A-I-Pisa and Tangier subjects, there was no association of paraoxonase with very low density lipoproteins or LDL. Paraoxonase function is maintained in HDL deficient states. It implies that certain HDL-associated anti-atherogenic processes may not be entirely compromised by HDL deficiency. This has important implications for the cardiovascular risk associated with modulated HDL concentrations.

Molecular genetic study of Finns with hypoalphalipoproteinemia and hyperalphalipoproteinemia: a novel Gly230 Arg mutation (LCAT[Fin]) of lecithin:cholesterol acyltransferase (LCAT) accounts for 5% of cases with very low serum HDL cholesterol levels.

In an attempt to identify genetic factors underlying extreme alterations of serum HDL cholesterol (HDL-C) concentrations, we examined two probands with HDL-C levels <0.2 mmol/L and subsequently screened two large cohorts of smoking men, one with very low (0.2 to 0.7 mmol/L, n=156) and the other with elevated (1.9 to 3.6 mmol/L, n=160) HDL-C levels, for the newly detected mutations as well as some other mutations proposed to affect HDL-C levels. One of the probands had corneal opacities, microalbuminuria, hypertriglyceridemia, and reduced LDL apoprotein B concentration; the other had anemia and presented with stomatocytosis in his peripheral blood. The first proband was found to be homozygous for a novel LCAT Gly230Arg (LCAT[Fin]) mutation, and the second was homozygous for an Arg399Cys mutation we described previously. Transient expression of the mutant LCAT(Fin) cDNA in COS cells disclosed markedly diminished LCAT enzyme activity. In the low-HDL-C group of men (n=156), 8 carriers of LCAT(Fin) and 1 carrier of the LCAT Arg399Cys were identified. In addition, the frequency of the lipoprotein lipase (LPL) Asn291Ser mutation was significantly (P<.05) higher in the low-HDL-C group (4.8%) than in the high-HDL-C group (1.6%). In addition, we identified 1 carrier of the intron 14G-->A mutation of cholesterol ester transfer protein (CETP) in the high-HDL-C group and subsequently demonstrated cosegregation of the mutant allele with elevated HDL-C levels in the proband's family. In conclusion, we have identified a novel LCAT gene Gly230Arg mutation (LCAT[Fin]), which, together with the LPL Asn291Ser mutation, represents a relatively common genetic cause of diminishing HDL-C levels, at least among Finns. This article also reports occurrence of a CETP mutation in subjects having non-Japanese roots.

Activation of phosphatidylinositol-specific phospholipase C in response to HDL3 and LDL is markedly reduced in cultured fibroblasts from Tangier patients.

We compared HDL3- and LDL-induced signal transduction in normal and Tangier fibroblasts to elucidate whether impaired signal transduction responses to lipoproteins might contribute to disturbed cellular lipid and lipoprotein metabolism in Tangier disease, a rare autosomal disorder of cellular lipid and lipoprotein metabolism. In several cell types HDL and LDL activate a currently unknown isoform of phosphatidylinositol-specific phospholipase C (PI-PLC) that results in the generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Compared with normal fibroblasts, Tangier fibroblasts stimulated with HDL3 or LDL resulted in a significantly reduced accumulation of inositol phosphates and 1,2-diacylglycerol formation. Furthermore, in Tangier fibroblasts both lipoproteins failed to mobilize calcium from internal pools, and the cytosol-to-membrane redistribution of protein kinase C (in both the alpha and epsilon isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.

[Increase of gamma-glutamyltranspeptidase in patients with compensated hypoalphalipoproteinemia and type IIa dyslipidemias]. / Elevación de gammaglutamiltranspeptidasa en pacientes con hipoalfalipoproteinemia y dislipemias tipo IIa compensadas.

Twenty-one asymptomatic patients presenting isolated elevations of gamma-glutamyltranspeptidase (GGT) were studied over the previous 10 years with all the findings being accidental. No other analytical alterations were demonstrated. Ingestion of alcohol, drugs or another type of toxic substance, diabetes, neurologic disease or neoplasm were discarded. Echography of the liver and the biliary tract was normal. In the first nine patients studied, liver biopsy was performed being normal or with minimum unspecific alterations. In two patients endoscopic retrograde cholangiography was carried out with no alterations being observed. Lipid study was performed in all the patients with alpha hypolipoproteinemia being found in 15 patients, compensated type II lipid profile in 5 and a normal lipid profile in one. To the author's knowledge there have been no reports of this lipid disorder causing elevations in GGT. However, on being the only abnormality found in these patients the authors believe that this may be the cause although the mechanism is unknown.

Microsomal enzyme inducers raise plasma high-density lipoprotein cholesterol levels in healthy control subjects but not in patients with primary hypoalphalipoproteinemia.

In this study we compared the ability of phenytoin, a microsomal enzyme inducer, to raise plasma high-density lipoprotein (HDL) levels in normolipidemic subjects and patients with primary hypoalphalipoproteinemia. In healthy control subjects, phenytoin caused a dose-dependent increase of plasma HDL, HDL2, and HDL3 cholesterol levels, up to 40% to 50%. Minor changes were recorded in the plasma concentrations of apolipoprotein (apo) A-I and apo A-II; the plasma level of the cholesteryl ester transfer protein (CETP) decreased by 42%. In contrast, none of the patients with hypoalphalipoproteinemia had changes in plasma HDL, HDL2, or HDL3 cholesterol, apo A-I, apo A-II, or CETP levels. These findings indicate that microsomal enzyme inducers are unsuitable to increase plasma HDL levels in high-risk patients with primary hypoalphalipoproteinemia, and they disclose a new mechanism, that is, decreased CETP-mediated transfer of cholesterol out of HDL, for the HDL-raising effect of microsomal enzyme inducers in healthy individuals.

Characterization of metalloelastase-like activity from the plasma of a patient with Tangier disease.

An enzymatic activity with releases p-nitroaniline from 3-carboxypropionyl-trialanine p-nitroanilide (Suc[Ala]3NA) was characterized in blood plasma of patients with Tangier disease. This activity results from the sequential action of a metalloendopeptidase (MP) and an aminopeptidase (AP). These proteases were purified 134- (MP) and 82-fold (AP) from low density and very low density lipoproteins (LDL and VLDL) depleted Tangier plasma by DEAE-Trisacryl chromatography and gel filtration. MP and AP could be separated by polyacrylamide gel electrophoresis. MP shares some analogy with neutral endopeptidase (membrane metalloendopeptidase, EC 3.4.24.11) and is able to degrade human plasma fibronectin (mainly to fragments of 185, 168 and 128 kDa) as evidenced on Western blots. It cannot hydrolyse 3H-labelled insoluble elastin and apolipoprotein AII, but did cleave a dinitrophenyl-octapeptide as well as apolipoprotein AI to 25-kDa and 24-kDa fragments formed sequentially. It may therefore be partially responsible for the in vivo degradation of apoAI observed in Tangier disease.

Absence of "A"-esterase activity in the serum of a patient with Tangier disease.

The levels of apolipoprotein A-I, A-II and B in subjects who are homozygous or heterozygous for Tangier disease are reported and compared with the amount of "A"-esterase in the serum. The "A"-esterases hydrolyse toxic organophosphate pesticides and are currently classified by the nomenclature committee of the International Union of Biochemistry as arylesterases (EC 3.1.1.2) although recent evidence has cast doubt on this classification. The apolipoprotein data are consistent with previous data reported for a number of Tangier patients. The homozygote has a marked reduction in apo A-I and A-II levels and a 30% reduction in apo B. The heterozygotes have about a 50% reduction of apo A-I, a slight reduction in apo A-II and no change in apo B. These apolipoprotein values correspond to a marked reduction in HDL cholesterol for the homozygote and substantial reductions in the heterozygotes. The "A"-esterase activity is zero in one homozygote while heterozygotes have about 5% of the levels in control subjects. Arylesterase activity appears to be essentially normal. The data thus support previous observations that the HDL "A"-esterase activity is greatly reduced in those conditions where HDL apo A-I is markedly reduced, e.g., in "Fish-eye" Disease.

Lipoproteins alter the catalytic behavior of the platelet-activating factor acetylhydrolase in human plasma.

Platelet-activating factor (PAF) has been implicated as a mediator of inflammation, allergy, shock, and thrombosis. A specific degradative enzyme, PAF acetylhydrolase (EC 3.1.1.47), is found in plasma and could regulate the concentration of PAF in blood. In plasma, 70% of the PAF acetylhydrolase is found with low density lipoprotein (LDL), and the remainder is in high density lipoprotein (HDL). In previous studies we found that with subsaturating concentrations of PAF the activity in LDL seemed to be the relevant one; e.g., depletion of LDL slowed degradation of PAF, while removal of HDL accelerated the degradation slightly. We have pursued this observation by using plasma from humans with lipoprotein mutations. In abetalipoproteinemia, all of the PAF acetylhydrolase activity was in HDL, whereas in Tangier disease all of the activity was in LDL. In both conditions the total activity measured in an optimized assay was normal or increased. However, when we measured the t1/2 of PAF in plasma, we found that it was prolonged in subjects with abetalipoproteinemia compared to normal controls. Conversely, the t1/2 in Tangier plasma was shortened. We next demonstrated that the PAF acetylhydrolase in HDL was recognized by an antibody to the enzyme purified from LDL, establishing that the enzyme in the two particles is the same protein. Finally, we inactivated the PAF acetylhydrolase in isolated lipoprotein particles and then reconstituted them with enzyme from the opposite particle. The reconstituted particles were used to measure the t1/2 of PAF, and we again found that the LDL particle was more efficient. We conclude that the lipoprotein environment of the PAF acetylhydrolase markedly influences its catalytic behavior. This may be important in pathophysiology and will complicate attempts to assess the role of this enzyme in such circumstances.

Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma.

Lecithin-cholesterol acyltransferase (LCAT) mass, activity and endogenous cholesterol esterification rate were measured in plasma and apolipoprotein A-I-free (A-I-free) plasma from two normolipidemic and two hyperlipidemic subjects, and from a patient with Tangier disease. A-I was removed from plasma by an anti-A-I immunosorbent. LCAT activity was measured using an exogenous substrate. The plasma LCAT concentration of the four non-Tangier subjects was 4.63 +/- 0.64 micrograms/ml (mean +/- S.D.); means of 26 +/- 7% of total LCAT mass and 22 +/- 11% of plasma LCAT activity were found in their A-I-free plasma. The plasma LCAT concentration of the Tangier subject was 1.49 micrograms/ml. About 95% of LCAT mass and all LCAT activity were found in the A-I-free plasma. Thus, the LCAT mass (1.4 micrograms/ml) and activity (43.1 nmol/h per ml) in Tangier A-I-free plasma were not significantly different from that found in the four non-Tangier A-I-free plasmas (mass = 1.21 +/- 0.44 micrograms/ml; activity: 27.3 +/- 18.4 nmol/h per ml). Although the LCAT activity per unit mass of the enzyme in plasma and A-I-free plasma were comparable (24.9 +/- 2.8 vs. 22.8 +/- 7.8 nmol/h per micrograms LCAT, n = 5), the plasma cholesterol esterification rate of A-I-free plasma from all subjects was lower than that found in plasma (7.5 +/- 2.7 vs. 13.0 +/- 3.8 nmol/h per micrograms LCAT). In conclusion, although A-I-containing lipoproteins are the preferred substrates of LCAT, other LCAT substrates and cofactors are found in A-I-free plasma along with LCAT. Thus, non-A-I-containing particles can serve as physiological substrates for cholesterol esterification mediated by LCAT.

Normalization of high density lipoprotein in fish eye disease plasma by purified normal human lecithin: cholesterol acyltransferase.

Plasma from a patient with fish eye disease has been enriched with autologous high density lipoproteins (HDL) and supplemented with highly purified normal human plasma lecithin:cholesterol acyltransferase (LCAT). Incubation of such plasma at 37 C in vitro resulted in normalization of its low HDL cholesteryl ester percentage, from 23% to 79%, associated with a two-fold increase in both the cholesteryl ester and triglyceride contents of the HDL fraction, as compared to incubation experiments with absent or heat-inactivated purified normal LCAT. The normalization of the HDL cholesteryl ester percentage induced by incubation with purified normal LCAT also was accompanied by an increase in the size of the original fish eye disease HDL particles, which had a mean mass of 115 kd, to HDL particle populations with mean particle masses ranging from 130-220 kd, depending on the concentration of purified LCAT in the incubate. Both HDL cholesterol esterification and particle enlargement were abolished completely by the LCAT inhibitor DTNB and by heat inactivation of the purified normal LCAT. The results give further evidence that fish eye disease is an alpha-LCAT deficiency.

Lecithin:cholesterol acyltransferase in familial HDL deficiency (Tangier disease).

These studies were performed to investigate the relationship between the enzyme lecithin:cholesterol acyltransferase and plasma lipoproteins in Tangier disease, a condition characterized by a virtual absence of high-density lipoproteins (HDLs) and an accumulation of cholesteryl esters in peripheral tissues. Apolipoprotein A-I was nearly absent from the patient's plasma (1% of the normal levels were found). However, apolipoprotein A-I purified from the plasma of the Tangier disease patient, was found to activate both purified and the plasma enzyme. At lower apolipoprotein concentrations (up to 25 micrograms/ml), about twice the amount of Tangier apolipoprotein A-I was required to achieve a certain level of lecithin:cholesterol acyltransferase activity as compared with the activating potential of the normal apolipoprotein. Gel chromatography studies revealed that as in normal plasma, lecithin:cholesterol acyltransferase in Tangier plasma was associated with an HDL-size lipoprotein fraction. However, unlike in normal plasma, this lipoprotein complex (containing lecithin:cholesterol acyltransferase) was not removed from Tangier plasma by immunoaffinity chromatography utilizing immobilized anti-apolipoprotein A-I antibodies. Plasma incubation studies showed that free cholesterol was primarily supplied by LDL in normal plasma, whereas both LDL and VLDL donated the free cholesterol for lecithin:cholesterol acyltransferase reaction in Tangier plasma. The majority of the cholesteryl esters, generated during the incubation experiments, were transferred back to LDL in normal plasma, whereas in Tangier plasma both LDL and VLDL served as cholesteryl ester acceptors. The cholesteryl ester transfer from HDL to lower-density lipoproteins was lower in Tangier plasma as compared to this process in a normal control, suggesting that a minimal cholesteryl ester core may be required for the stability of HDL.

Net lipid transfer between lipoproteins in fish-eye disease plasma supplemented with normal high density lipoproteins.

Native fish-eye disease plasma, which is deficient of both high density lipoproteins (HDL) and lecithin-cholesterol acyltransferase activity (alpha-LCAT), processing the free cholesterol of these lipoproteins, has been supplemented with normal isolated HDL2 or HDL3 and incubated in vitro at 37 C. After incubation for 0, 7.5 and 24 hr the very low density (VLDL) and low density (LDL) lipoproteins as well as HDL were isolated, and their contents of triglycerides, phospholipids and free, esterified and total cholesterol were quantified. The resulting net mass transfer of the different lipids revealed a functioning transfer of cholesteryl esters and all other analyzed lipids between the lipoproteins, although no de novo esterification of the HDL cholesterol by LCAT in this plasma occurred. In accordance with previous findings there was a functioning esterification process of the free cholesterol of the combined VLDL and LDL of fish-eye disease plasma. The present reports make it reasonable to conclude that the lack of HDL cholesterol esterification in this disease is not a result of a deficiency of cholesteryl ester transfer or lipid transfer activities.

Different substrate specificities of plasma lecithin: cholesterol acyl transferase in fish eye disease and Tangier disease.

Esterification of plasma free cholesterol is mediated by lecithin:cholesterol acyl transferase (LCAT). The free cholesterol of plasma high density lipoproteins (HDL) is considered to be the preferred substrate for LCAT. It therefore appeared as a paradox that plasma cholesterol esterification, both in vivo and in vitro, is normal in fish eye disease and Tangier disease, two familial conditions with extremely low plasma HDL levels. Fish eye disease plasma, however, was shown to have LCAT activity primarily acting on combined very low (VLDL) and low (LDL) density lipoproteins, denominated beta-LCAT, while it lacked LCAT activity esterifying HDL cholesterol (alpha-LCAT). Here we show that Tangier plasma, in contrast, has both alpha- and beta-LCAT. Thus, in both fish eye and Tangier diseases it is beta-LCAT that explains the apparent normal plasma cholesterol esterification. We also show that Tangier plasma, having alpha-LCAT activity, normalizes the low cholesteryl ester content as well as the abnormally small size of fish eye disease HDL particles during incubation.