Maxime Denis

Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency

Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) levels. Here we have identified two Tangier disease (TD) families, confirmed 9q31 linkage and refined the disease locus to a limited genomic region containing the gene encoding the ATP-binding cassette transporter (ABC1). Familial HDL deficiency (FHA) is a more frequent cause of low HDL levels. On the basis of independent linkage and meiotic recombinants, we localized the FHA locus to the same genomic region as the TD locus. Mutations in ABC1 were detected in both TD and FHA, indicating that TD and FHA are allelic. This indicates that the protein encoded by ABC1 is a key gatekeeper influencing intracellular cholesterol transport, hence we have named it cholesterol efflux regulatory protein (CERP).

Mutations in the ABC1 gene in familial HDL deficiency with defective cholesterol efflux

BACKGROUND A low concentration of HDL cholesterol is the most common lipoprotein abnormality in patients with premature atherosclerosis. We have shown that Tangier disease, a rare and severe form of HDL deficiency characterised by a biochemical defect in cellular cholesterol efflux, is caused by mutations in the ATP-binding-cassette (ABC1) gene. This gene codes for the cholesterol-efflux regulatory protein (CERP). We investigated the presence of mutations in this gene in patients with familial HDL deficiency. METHODS Three French-Canadian families and one Dutch family with familial HDL deficiency were studied. Fibroblasts from the proband of each family were defective in cellular cholesterol efflux. Genomic DNA of each proband was used for mutation detection with primers flanking each exon of the ABC1 gene, and for sequencing of the entire coding region of the gene. PCR and restriction-fragment length polymorphism assays specific to each mutation were used to investigate segregation of the mutation in each family, and to test for absence of the mutation in DNA from normal controls. FINDINGS A different mutation was detected in ABC1 in each family studied. Each mutation either created a stop codon predicted to result in truncation of CERP, or altered a conserved aminoacid residue. Each mutation segregated with low concentrations of HDL-cholesterol in the family, and was not observed in more than 500 control chromosomes tested. INTERPRETATION These data show that mutations in ABC1 are the major cause of familial HDL deficiency associated with defective cholesterol efflux, and that CERP has an essential role in the formation of HDL. Our findings highlight the potential of modulation of ABC1 as a new route for increasing HDL concentrations.

ATP-binding Cassette Transporter A1 Expression Disrupts Raft Membrane Microdomains through Its ATPase-related Functions

ATP-binding cassette transporter A1 (ABCA1) is known to mediate cholesterol efflux to lipid-poor apolipoprotein A-I. In addition, ABCA1 has been shown to influence functions of the plasma membrane, such as endocytosis and phagocytosis. Here, we report that ABCA1 expression results in a significant redistribution of cholesterol and sphingomyelin from rafts to non-rafts. Caveolin, a raft/caveolae marker also redistributes from punctate caveolae-like structures to the general area of the plasma membrane upon ABCA1 expression. Furthermore, we observed significant reduction of Akt activation in ABCA1-expressing cells, consistent with raft disruption. Cholesterol content in the plasma membrane is, however, not altered. Moreover, we provide evidence that a non-functional ABCA1 with mutation in an ATP-binding domain, A937V, fails to redistribute cholesterol, sphingomyelin, or caveolin. A937V also fails to influence Akt activation. Finally, we show that apolipoprotein A-I preferentially associates with non-raft membranes in ABCA1-expressing cells. Our results thus demonstrate that ABCA1 causes a change in overall lipid packing of the plasma membrane, likely through its ATPase-related functions. Such reorganization by ABCA1 effectively expands the non-raft membrane fractions and, consequentially, pre-conditions cells for cholesterol efflux.

Gene Inactivation of Proprotein Convertase Subtilisin/Kexin Type 9 Reduces Atherosclerosis in Mice

Background— The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ≈7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results— We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E–deficient, and LDL receptor–deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E–deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor–deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions— Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective.Background —The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the LDL receptor (LDLR). PCSK9 gain-of-function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ~7-fold lower levels of LDL-cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results —We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E (apoE)-deficient and LDLR-deficient mouse models. Circulating cholesterol levels, FPLC profiles, aortic cholesteryl esters (CE) and plaque size were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (KO), normal (WT) or high (Tg) levels of PCSK9 were fed a 12 month-Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next, we generated apoE-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e) or high (Tg/e) levels of PCSK9. Following a 6 month-regular diet, KO/e mice showed a 39% reduction compared to WT/e mice in aortic CE accumulation, while Tg/e mice showed a 137% increase. Finally, LDLR-deficient mice expressing no (KO/L), normal (WT/L) or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited similar levels of plasma cholesterol and CE accumulation to WT/L, suggesting that PCSK9 modulates atherosclerosis mainly via the LDLR. Conclusions —Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is pro-atherogenic, while its absence is protective.

Gene Inactivation of PCSK9 Reduces Atherosclerosis in Mice

Background— The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ≈7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results— We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E–deficient, and LDL receptor–deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E–deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor–deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions— Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective.Background —The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the LDL receptor (LDLR). PCSK9 gain-of-function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ~7-fold lower levels of LDL-cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results —We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E (apoE)-deficient and LDLR-deficient mouse models. Circulating cholesterol levels, FPLC profiles, aortic cholesteryl esters (CE) and plaque size were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (KO), normal (WT) or high (Tg) levels of PCSK9 were fed a 12 month-Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next, we generated apoE-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e) or high (Tg/e) levels of PCSK9. Following a 6 month-regular diet, KO/e mice showed a 39% reduction compared to WT/e mice in aortic CE accumulation, while Tg/e mice showed a 137% increase. Finally, LDLR-deficient mice expressing no (KO/L), normal (WT/L) or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited similar levels of plasma cholesterol and CE accumulation to WT/L, suggesting that PCSK9 modulates atherosclerosis mainly via the LDLR. Conclusions —Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is pro-atherogenic, while its absence is protective.

Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis

It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated “high-capacity binding site” (HCBS). Glyburide drastically reduced 125I-apoA-I binding to the HCBS, whereas 125I-apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (Δ187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of 125I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis.

ATP-binding Cassette A1-mediated Lipidation of Apolipoprotein A-I Occurs at the Plasma Membrane and Not in the Endocytic Compartments

ATP-binding cassette transporter (ABC) A1 is required for the lipidation of apolipoprotein A-I to generate high density lipoprotein (HDL). This process is proposed to occur through a retro-endocytosis pathway in which apoA-I internalizes with ABCA1 and generates HDL from the endosomal compartments before resecretion. The aim of this study was to determine the route of apoA-I endocytosis and whether endocytosis contributes to HDL biogenesis. Using confocal microscopy, we found that internalized apoA-I only transiently colocalized with transferrin, a retro-endocytosis marker. Instead, apoA-I perfectly colocalized with a bulk phase uptake marker (fluorescein isothiocyanate-dextran) and, at later time points, with LysoTracker in several cell models including macrophages, fibroblasts, and baby hamster kidney cells. ABCA1 colocalized poorly with internalized apoA-I. To determine the contribution of internalized apoA-I to HDL biogenesis, we specifically removed apoA-I from the cell surface and analyzed the fate of internalized apoA-I. We found that 23% of cell-associated apoA-I was internalized at steady state. Of internalized apoA-I, only 20% was converted to HDL, and the rest was degraded, consistent with a lysosomal destination. We also found that apoA-I was released approximately five times faster from the plasma membrane than from the intracellular compartments. From these kinetic parameters, we estimated that ∼5.6% of apoA-I that interacts with cells is degraded and that internalized apoA-I contributes to ∼1.4% of total HDL production. We also found that blocking endocytosis with sucrose or cytochalasin D did not decrease cholesterol efflux or HDL biogenesis. We therefore conclude that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs.

Molecular interactions between apoE and ABCA1 impact on apoE lipidation

Apolipoprotein E (apoE)/ABCA1 interactions were investigated in human intact fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Here, we show that purified human plasma apoE3 forms a complex with ABCA1 in normal fibroblasts. Lipid-free apoE3 inhibited the binding of 125I-apoA-I to ABCA1 more efficiently than reconstituted HDL particles (IC50 = 2.5 ± 0.4 μg/ml vs. 12.3 ± 1.3 μg/ml). ApoE isoforms showed similar binding for ABCA1 and exhibited identical kinetics in their abilities to induce ABCA1-dependent cholesterol efflux. Mutation of ABCA1 associated with Tangier disease (C1477R) abolished both apoE3 binding and apoE3-mediated cholesterol efflux. Analysis of apoE3-containing particles generated during the incubation of lipid-free apoE3 with stimulated normal cells showed nascent apoE3/cholesterol/phospholipid complexes that exhibited preβ-electrophoretic mobility with a particle size ranging from 9 to 15 nm, whereas lipid-free apoE3 incubated with ABCA1 mutant (C1477R) cells was unable to form such particles. These results demonstrate that 1) apoE association with lipids reduced its ability to interact with ABCA1; 2) apoE isoforms did not affect apoE binding to ABCA1; 3) apoE-mediated ABCA1-dependent cholesterol efflux was not affected by apoE isoforms in fibroblasts; and 4) the lipid translocase activity of ABCA1 generates apoE-containing high density-sized lipoprotein particles. Thus, ABCA1 is essential for the biogenesis of high density-sized lipoprotein containing only apoE particles in vivo.

Expression, regulation, and activity of ABCA1 in human cell lines

Mutations in the ATP-binding cassette transporter A1 (ABCA1) gene cause familial high-density lipoprotein deficiency and Tangier disease. ABCA1 plays a crucial role in active apolipoprotein A-I (apoA-I) lipidation, a key step in reverse cholesterol transport. We compared ABCA1 transcriptional regulation and cholesterol efflux in human skin fibroblasts, monocyte-derived macrophages and hepatocytes (HepG2). 8-Br-cAMP did not increase ABCA1 transcription in these tissues compared to mouse macrophages. We found that ABCA1 is differentially regulated among tissues. While transcription in HepG2 appears to be constitutive, sterols stimulate ABCA1 transcription in fibroblasts and monocyte-derived macrophages. ApoA-I promoted cholesterol efflux in fibroblasts, macrophages, and HepG2. Cholesterol homeostasis in fibroblasts is tightly regulated, and ABCA1 mRNA closely follows the cellular mass of free cholesterol (dose- and time-dependent manner). To further determine the mechanism used by fibroblasts to maintain sterol balance, we used a competitive inhibition approach with geranylgeranyl pyrophosphate (GGPP) to block the LXR induction pathway. GGPP blocked basal, 22-(R)-hydroxycholesterol- and cholesterol-induced ABCA1 expression. Taken together, these results demonstrate that: (1) ABCA1 expression varies among tissues, and (2) cholesterol conversion to hydroxycholesterol is an important mechanism for the maintenance of cholesterol homeostasis in fibroblasts.

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 (∼50–100%). Analysis of LCAT kinetics parameters (Vmax and Km) 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 [3H]SM-labeled LpA-I and HDL3. Furthermore, expression of mutant SMase (ΔR608) in CHO cells revealed that ΔR608 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.