Tangier Disease Subjects Research Articles

Hepatic ABCA1 deficiency is associated with delayed apolipoprotein B secretory trafficking and augmented VLDL triglyceride secretion

ATP binding cassette transporter A1 (ABCA1) is a membrane transporter that facilitates nascent HDL formation. Tangier disease subjects with complete ABCA1 deficiency have <5% of normal levels of plasma HDL, elevated triglycerides (TGs), and defective vesicular trafficking in fibroblasts and macrophages. Hepatocyte-specific ABCA1 knockout mice (HSKO) have a similar lipid phenotype with 20% of normal plasma HDL levels and a two-fold elevation of plasma TGs due to hepatic overproduction of large, triglyceride-enriched VLDL. We hypothesized that enhanced VLDL TG secretion in the absence of hepatocyte ABCA1 is due to altered intracellular trafficking of apolipoprotein B (apoB), resulting in augmented TG addition to nascent VLDL. We found that trafficking of newly synthesized apoB through the secretory pathway was delayed in ABCA1-silenced rat hepatoma cells and HSKO primary hepatocytes, relative to controls. Endoglycosidase H treatment of cellular apoB revealed a likely delay in apoB trafficking in post-ER compartments. The reduced rate of protein trafficking was also observed for an adenoviral-expressed GPI-linked fluorescent fusion protein, but not albumin, suggesting a selective delay of secretory cargoes in the absence of hepatocyte ABCA1. Our results suggest an important role for hepatic ABCA1 in regulating secretory trafficking and modulating VLDL expansion during the TG accretion phase of hepatic lipoprotein particle assembly.

Targeted Deletion of Hepatocyte ABCA1 Leads to Very Low Density Lipoprotein Triglyceride Overproduction and Low Density Lipoprotein Hypercatabolism

Loss of ABCA1 activity in Tangier disease (TD) is associated with abnormal apoB lipoprotein (Lp) metabolism in addition to the complete absence of high density lipoprotein (HDL). We used hepatocyte-specific ABCA1 knock-out (HSKO) mice to test the hypothesis that hepatic ABCA1 plays dual roles in regulating Lp metabolism and nascent HDL formation. HSKO mice recapitulated the TD lipid phenotype with postprandial hypertriglyceridemia, markedly decreased LDL, and near absence of HDL. Triglyceride (TG) secretion was 2-fold higher in HSKO compared with wild type mice, primarily due to secretion of larger TG-enriched VLDL secondary to reduced hepatic phosphatidylinositol 3-kinase signaling. HSKO mice also displayed delayed clearance of postprandial TG and reduced post-heparin plasma lipolytic activity. In addition, hepatic LDLr expression and plasma LDL catabolism were increased 2-fold in HSKO compared with wild type mice. Last, adenoviral repletion of hepatic ABCA1 in HSKO mice normalized plasma VLDL TG and hepatic phosphatidylinositol 3-kinase signaling, with a partial recovery of HDL cholesterol levels, providing evidence that hepatic ABCA1 is involved in the reciprocal regulation of apoB Lp production and HDL formation. These findings suggest that altered apoB Lp metabolism in TD subjects may result from hepatic VLDL TG overproduction and increased hepatic LDLr expression and highlight hepatic ABCA1 as an important regulatory factor for apoB-containing Lp metabolism.

POPC/apoA-I discs as a potent lipoprotein modulator in Tangier disease

Tangier disease (TD) is a rare familial disorder with mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. It results in extremely low levels of HDL cholesterol. Since TD is a genetic disorder, a therapeutic approach to TD has not been established. We report a typical case of TD with a homozygous novel point mutation in the ABCA1 gene by using genomic DNA sequencing. Primary monocyte-derived macrophages of blood from a patient with TD and normolipidemic subjects were compared for cholesterol efflux. The macrophages from the TD patient showed no apoA-I-mediated cholesterol efflux. In contrast, POPC/apoA-I discs were able to take up cholesterol from macrophages from both the TD and normolipidemic subject. Capillary isotachophoresis (cITP), which separates lipoprotein into subfractions according to electrophoretic mobility, was used to characterize plasma lipoprotein subfractions. Both slow-migrating HDL (sHDL) and slow-migrating LDL (sLDL; unmodified LDL) subfractions were extremely low in the patient with TD. After incubation of plasma from the TD patient with POPC/apoA-I discs, sHDL and sLDL subfractions rapidly appeared. In conclusion, POPC/apoA-I discs not only have beneficial effects on cholesterol efflux, but also have potential as a lipoprotein modulator in patients with TD.

Insight Into ABCG1-Mediated Cholesterol Efflux

Macrophages in the arterial intima take up modified LDL and become cholesterol-ester laden foam cells, which are the primary cell type in newly formed fatty streak lesions, and which play an important role throughout lesion progression and plaque vulnerability. Macrophages can unload their excess cellular cholesterol stores via lipid efflux, the first step in the protective reverse cholesterol transport pathway. In this pathway, efflux of cellular cholesterol to extracellular acceptors, such as HDL and lipid-poor apolipoproteins, targets this cholesterol for delivery to the liver for metabolism and direct excretion into the bile. The mechanisms of cellular cholesterol efflux are the focus of intensive research, and this field was advanced greatly by the discovery of ABCA1 as the Tangier disease gene in 1999.1 Tangier disease subjects have almost no plasma HDL, and their cells have a complete deficiency in cholesterol and phospholipid efflux to apolipoprotein A-I (apoAI) and a partial defect in lipid efflux to HDL.2 Thus, it was apparent that there is more than one pathway for lipid efflux to HDL. In 2001, Schmitz and colleagues demonstrated that ABCG1, another member of the ABC gene superfamily, was upregulated by cholesterol in human macrophages, and that an ABCG1 antisense oligonucleotide that reduced ABCG1 expression also decreased lipid efflux to HDL.3 The role of ABCG1 in lipid efflux was confirmed in 2004 when Wang and colleagues and Edwards and colleagues showed that ABCG1 transfected cells had increased cholesterol efflux to HDL.4,5 See page 1310 In the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Wang et al provide novel insights both into the regulation of ABCG1 activity and into aspects of the mechanism of ABCG1-mediated lipid efflux,6 which differentiate it from the mechanism of ABCA1-mediated lipid efflux. Their essential discovery is that treatment of cells with …

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.

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.

Familial HDL deficiency due to ABCA1 gene mutations with or without other genetic lipoprotein disorders

Mutations in ABCA1 have been shown to be the cause of Tangier disease (TD) and some forms of familial hypoalphalipoproteinemia (HA), two genetic disorders characterized by low plasma HDL levels. Here we report six subjects with low HDL, carrying seven ABCA1 mutations, six of which are previously unreported. Two mutations (R557X and H160FsX173) were predicted to generate short truncated proteins; two mutations (E284K and Y482C) were located in the first extracellular loop and two (R1901S and Q2196H) in the C-terminal cytoplasmic domain of ABCA1. Two subjects found to be compound heterozygotes for ABCA1 mutations did not have overt clinical manifestations of TD. Three subjects, all with premature coronary artery disease (pCAD), had a combination of genetic defects. Besides being heterozygotes for ABCA1 mutations, two of them were also carriers of the R3500Q substitution in apolipoprotein B and the third was a carrier of N291S substitution in lipoprotein lipase. By extending family studies we identified 17 heterozygotes for ABCA1 mutations. Plasma HDL-C and Apo A-I values in these subjects were 38.3 and 36.9% lower than in unaffected family members and similar to the values found in heterozygotes for Apo A-I gene mutations which prevent Apo A-I synthesis. This survey underlines the allelic heterogeneity of ABCA1 mutations and suggests that: (i) TD subjects, if asymptomatic, may be overlooked and (ii) there may be a selection bias in genotyping towards carriers of ABCA1 mutations who have pCAD possibly related to a combination of genetic and environmental cardiovascular risk factors.

Accumulation of cardiolipin and lysocardiolipin in fibroblasts from Tangier disease subjects

Tangier disease (TD) is an inherited disorder of lipid metabolism characterized by very low high density lipoprotein (HDL) plasma levels, cellular cholesteryl ester accumulation and reduced cholesterol excretion in response to HDL apolipoproteins. Molecular defects in the ATP binding cassette transporter 1 (ABCA1) have recently been identified as the cause of TD. ABCA1 plays a key role in the translocation of cholesterol across the plasma membrane, and defective ABCA1 causes cholesterol storage in TD cells. Not only cholesterol efflux, but also phospholipid efflux was shown to be impaired in TD cells. By use of thin layer chromatography, high performance liquid chromatography and time-of-flight secondary ion mass spectrometry, we characterized the cellular phospholipid content in fibroblasts from three homozygous TD patients. The cellular content of the major phospholipids was not found to be significantly altered in TD fibroblasts. However, the two phospholipids cardiolipin and lysocardiolipin, which make up minute amounts in normal cells, were at least 3–5-fold enriched in fibroblasts from TD subjects. A structurally closely related phospholipid (lysobisphosphatidic acid) has recently been shown to be enriched in Niemann–Pick type C, another lipid storage disorder. Altogether these data may indicate that the role of these phospholipids is a regulatory one rather than that of a bulk mediator of cholesterol solubilization in sterol trafficking and efflux.

Accumulation of RhoA, RhoB, RhoG, and Rac1 in Fibroblasts from Tangier Disease Subjects Suggests a Regulatory Role of Rho Family Proteins in Cholesterol Efflux

Tangier disease (TD) is an inherited disorder of lipid metabolism characterized by very low high density lipoprotein (HDL) plasma levels, cellular cholesteryl ester accumulation and reduced cholesterol excretion in response to HDL apolipoproteins. Molecular defects in the ATP binding cassette transporter 1 (ABCA1) have recently been identified as the cause of TD. ABCA1 plays a key role in the translocation of cholesterol across the plasma membrane, and defective ABCA1 causes cholesterol storage in TD cells. However, the exact relationship of many of the biochemical and morphological abnormalities in TD to ABCA1 is unknown. Since small GTP-binding proteins are important regulators of many cellular functions, we characterized these proteins in normal and TD fibroblasts using the [α-32P]GTP overlay technique and Western blotting of SDS and isoelectric focusing gels. Our results indicate that GTP-binding proteins of the Rho family (RhoA, RhoB, RhoG, Rac-1) are enriched in fibroblasts from TD patients. The accumulation of small G proteins may have potential implications for the TD phenotype and the regulation of cholesterol excretion in TD cells.

The High Density Lipoprotein- and Apolipoprotein A-I-Induced Mobilization of Cellular Cholesterol Is Impaired in Fibroblasts from Tangier Disease Subjects

Tangier disease (also known as familial HDL-deficiency) is characterized by very low high density lipoprotein (HDL) plasma levels, splenomegaly, and massive cholesteryl ester accumulation in the cytoplasm of various cell types. Since this phenotype may in part be caused by a defect in the pathway mediating cholesterol efflux from peripheral cells, we investigated the HDL3-mediated mobilization of cholesterol synthesized de novo from [14C]-mevalonolactone in cultivated fibroblasts from two patients with Tangier disease. Our results indicate that the HDL3-induced translocation of [14C]-cholesterol from intracellular pools to the plasma membrane and its subsequent secretion into the extracellular medium was approximately 50 % less in the cells from the patients than in controls. The same result was also obtained with artificial apolipoprotein A-I-containing phospholipid vesicles. By contrast, no significant difference in HDL3-induced cholesterol efflux was observed when plasma membrane was labeled with exogenous [14C]-cholesterol. We conclude that inefficient cholesterol efflux in Tangier disease is primarily caused by impaired HDL3-induced activation of cholesterol translocation from intracellular pools to the plasma membrane.

In vivo metabolism of proapolipoprotein A-I in Tangier disease.

Tangier disease is a rare familial disorder characterized by extremely low levels of apolipoprotein A-I (apoA-I) and high density lipoproteins (HDL). In normal subjects, proapoA-I is secreted into plasma and converted to mature apoA-I by the cleavage of the amino-terminal six amino acids with the major isoprotein in plasma being mature apoA-I. In contrast, in Tangier disease there is a marked relative increase of proapoA-I as compared with mature apoA-I. ProapoA-I and mature apoA-I were isolated from normal and Tangier disease subjects, radio-labeled, and autologous apoA-I isoproteins injected into normal and Tangier subjects. The in vivo catabolism and conversion of proapoA-I and mature apoA-I in normal and Tangier disease subjects were quantitated. A comparison of the rate of catabolism of apoA-I isoproteins from plasma revealed a significantly faster rate of catabolism of both isoproteins of apoA-I in Tangier subjects when compared with normal subjects. The fractional conversion rate of proapoA-I to mature apoA-I was 3.9 d-1 in normal subjects and 3.6 d-1 in Tangier subjects. The results indicate that (a) apoA-I enters plasma as the pro isoprotein in both normal and Tangier subjects, (b) Tangier disease subjects have a normal fractional rate of conversion of proapoA-I to mature apoA-I, (c) proapoA-I is catabolized at the same rate as mature apoA-I in Tangier subjects, and (d) Tangier subjects catabolize both pro and mature apoA-I at a much greater rate than do normal subjects. Therefore, the relative increase in proapoA-I in Tangier disease is due to a marked decrease in mature apoA-I resulting from rapid catabolism of both pro- and mature apoA-I and not to defective conversion of proapoA-I to mature apoA-I.