Effects of FXR in foam-cell formation and atherosclerosis development.

Farnesoid X receptor (FXR), a bile-acid-activated member of the nuclear receptor superfamily, is essential in regulating bile-acid, cholesterol, and triglyceride homeostasis. Disruption of the FXR gene in mice results in a proatherosclerotic lipid profile with increased serum cholesterols and triglycerides. However, the role of FXR in foam-cell formation and atherosclerosis development remains unclear. The current study showed that the peritoneal macrophages isolated from FXR-null mice took up less oxidized LDL-cholesterol (oxLDL-C), which was accompanied by a marked reduction in CD36 expression in these cells. This result appears to be FXR-independent, as FXR was not detected in the peritoneal macrophages. To assess to what extent FXR modulates atherosclerosis development, FXR/ApoE double-null mice were generated. Female mice were used for atherosclerosis analysis. Compared to ApoE-null mice, the FXR/ApoE double-null mice were found to have less atherosclerotic lesion area in the aorta, despite a further increase in the serum cholesterols and triglycerides. Our results indicate that disruption of the FXR gene could attenuate atherosclerosis development, most likely resulting from reduced oxLDL-C uptake by macrophages. Our study cautions the use of serum lipid levels as a surrogate marker to determine the efficiency of FXR modulators in treating hyperlipidemia.

Hepatic lipase expression in macrophages contributes to atherosclerosis in apoE-deficient and LCAT-transgenic mice.

Hepatic lipase (HL) has a well-established role in lipoprotein metabolism. However, its role in atherosclerosis is poorly understood. Here we demonstrate that HL deficiency raises the proatherogenic apoB-containing lipoprotein levels in plasma but reduces atherosclerosis in lecithin cholesterol acyltransferase (LCAT) transgenic (Tg) mice, similar to results previously observed with HL-deficient apoE-KO mice. These findings suggest that HL has functions that modify atherogenic risk that are separate from its role in lipoprotein metabolism. We used bone marrow transplantation (BMT) to generate apoE-KO and apoE-KO x HL-KO mice, as well as LCAT-Tg and LCAT-Tg x HL-KO mice, chimeric for macrophage HL gene expression. Using in situ RNA hybridization, we demonstrated localized production of HL by donor macrophages in the artery wall. We found that expression of HL by macrophages enhances early aortic lesion formation in both apoE-KO and LCAT-Tg mice, without changing the plasma lipid profile, lipoprotein lipid composition, or HL and lipoprotein lipase activities. HL does, however, enhance oxidized LDL uptake by peritoneal macrophages. These combined data demonstrate that macrophage-derived HL significantly contributes to early aortic lesion formation in two independent mouse models and identify a novel mechanism, separable from the role of HL in plasma lipoprotein metabolism, by which HL modulates atherogenic risk in vivo.

Study of ABCA1 function in transgenic mice.

The ATP-binding cassette transporter A1 (ABCA1), identified in 1999 as the gene defective in Tangier disease, promotes efflux of cellular cholesterol from macrophages and other peripheral tissues to apolipoprotein acceptors. These ABCA1-mediated processes are anticipated to have antiatherogenic properties, prompting the development of pharmacological agents that increase ABCA1 gene expression as well as the establishment of ABCA1-transgenic mouse lines. Preliminary studies of ABCA1-Tg mice seem to validate the selection of this transporter as a therapeutic target for the treatment of low HDL syndromes and cardiovascular disease but have also raised new questions regarding the function of ABCA1. In particular, the relative contribution of hepatic and peripheral ABCA1 to plasma HDL levels and to reverse cholesterol transport, as well as the potential role of ABCA1 in modulating the plasma concentrations of the apolipoprotein B-containing lipoproteins and protecting against atherosclerosis, seem to be promising areas of investigation. The present review summarizes the most recent studies and discusses insights provided by these transgenic mouse models.

Hepatic lipase, lipoprotein metabolism, and atherogenesis.

The role of hepatic lipase as a multifunctional protein that modulates lipoprotein metabolism and atherosclerosis has been extensively documented over the last decade. Hepatic lipase functions as a lipolytic enzyme that hydrolyzes triglycerides and phospholipids present in circulating plasma lipoproteins. Hepatic lipase also serves as a ligand that facilitates lipoprotein uptake by cell surface receptors and proteoglycans, thereby directly affecting cellular lipid delivery. Recently, another process by which hepatic lipase modulates atherogenic risk has been identified. Bone marrow transplantation studies demonstrate that hepatic lipase present in aortic lesions markedly alters aortic lesion formation even in the absence of changes in plasma lipids. These multiple functions of hepatic lipase, which facilitate not only plasma lipid metabolism but also cellular lipid uptake, can be anticipated to have a major and complex impact on atherogenesis. Consistently, human and animal studies support proatherogenic and antiatherogenic roles for hepatic lipase. The concept of hepatic lipase as mainly a lipolytic enzyme that reduces atherogenic risk has evolved into that of a complex protein with multiple functions that, depending on genetic background and sites of expression, can have a variable effect on atherosclerosis.

Clinical significance of high-density lipoproteins and the development of atherosclerosis: focus on the role of the adenosine triphosphate-binding cassette protein A1 transporter.

Low levels of high-density lipoprotein (HDL) cholesterol constitute a risk factor for coronary artery disease, and there is evidence that increasing HDL cholesterol levels reduces cardiovascular risk. The phenotype of low HDL cholesterol with or without elevated triglycerides is at least as common in patients hospitalized for cardiovascular disease as is hypercholesterolemia, and it is characteristic of diabetes and the metabolic syndrome, conditions associated with increased cardiovascular risk. Recent studies have elucidated mechanisms by which HDL acts to reduce cardiovascular risk, bolstering the rationale for targeting of HDL in lipid-modifying therapy. In particular, HDL (1) carries excess cholesterol from peripheral cells to the liver for removal in the process termed reverse cholesterol transport, (2) reduces oxidative modification of low-density lipoproteins (LDL), and (3) inhibits cytokine-induced expression of cellular adhesion molecules on endothelial cells. Studies of the newly described adenosine triphosphate-binding cassette protein A1 (ABCA1) transporter have established a crucial role for this transporter in modulating the levels of plasma HDL and intracellular cholesterol in the liver as well as in peripheral cells. Elevated levels of intracellular cholesterol stimulate the liver X receptor pathway, enhancing the expression of ABCA1, which increases intracellular trafficking of excess cholesterol to the cell surface for interaction with lipid-poor apolipoprotein A-I to form nascent HDL. Nascent HDL facilitates the removal of additional excess cellular cholesterol, which is esterified by lecithin-cholesterol acyltransferase with conversion of the nascent HDL to mature spherical HDL. Overexpression of ABCA1 in mice on a regular chow or Western diet results in a marked increase in plasma HDL, increased LDL, and increased transport of cholesterol to the liver. On a high cholesterol/cholate diet, transgenic mice overexpressing ABCA1 have increased HDL, reduced LDL, increased HDL-mediated cholesterol flux to the liver, and reduced atherosclerosis. Ongoing investigation of mechanisms by which HDL acts to reduce the risk of atherosclerosis will provide several new targets for the development of drugs to decrease the risk of atherosclerosis.

New insights into the role of the adenosine triphosphate-binding cassette transporters in high-density lipoprotein metabolism and reverse cholesterol transport.

Four adenosine triphosphate-binding cassette (ABC) transporters-ABCA1, ABCG1, ABCG5, and ABCG8-have been identified and shown to modulate cholesterol and lipoprotein metabolism. Recent analyses of ABCA1 indicate that upregulation of ABCA1 in the liver and macrophages of transgenic mice is associated with increased plasma high-density lipoprotein (HDL) cholesterol levels, increased net flux of cholesterol to the liver, and reduced diet-induced atherosclerosis. In ABCA1 transgenic mice, the enhanced expression of hepatic ABCA1 transporters is associated with increased plasma HDL cholesterol levels, suggesting that the liver plays an important role in the levels of plasma HDL cholesterol. Overexpression of ABCG1 in the liver of mice using recombinant ABCG1 vectors results in decreased plasma HDL levels and indicates that ABCG1 can modulate plasma lipoprotein levels in vivo. The potential importance of ABCG1 in reverse cholesterol transport has not been definitively established. Studies in patients with sitosterolemia have identified 2 major new transporters, ABCG5 and ABCG8, that play a pivotal role in the regulation of intestinal cholesterol, plant, and shellfish absorption. Modulation of the expression of ABCG5 and ABCG8 represents an important new mechanism in the regulation of cholesterol absorption in the intestine. The ABC transporters currently represent excellent targets for the development of new drugs for the treatment of patients with increased risk of premature cardiovascular disease.

Adenoviral expression of human lecithin-cholesterol acyltransferase in nonhuman primates leads to an antiatherogenic lipoprotein phenotype by increasing high-density lipoprotein and lowering low-density lipoprotein.

Lecithin-cholesterol acyltransferase (LCAT), a key enzyme in high-density lipoprotein (HDL) metabolism, has been proposed to have atheroprotective properties by promoting reverse cholesterol transport. Overexpression of LCAT in various animal models, however, has led to conflicting results on its overall effect on lipoproteins and atherosclerosis. In this study, the effect of overexpression of LCAT in nonhuman primates on lipoprotein metabolism is examined. Human LCAT was expressed with adenovirus in squirrel monkeys (n = 8), resulting on day 4 in a 22-fold increase of LCAT activity (257 +/- 23 vs 5618 +/- 799 nmol mL(-1) h(-1), P < .0001). At its peak, LCAT was found to nearly double the level of HDL cholesterol from baseline (113 +/- 7 vs 260 +/- 24 mg/dL, P < .01). High-density lipoprotein formed after treatment with the adenovirus was larger in size, as assessed by fast protein liquid chromatography (FPLC) analysis. By kinetic studies, it was determined that there was a decrease in apolipoprotein (Apo) A-I resident time (0.373 +/- 0.027 vs 0.685 +/- 0.045 d(-1), P < .0001) and almost a doubling in the ApoA-I synthetic rate (22 +/- 2 vs 41 +/- 3 mg kg(-1) d(-1), P < .0001), but no overall change in ApoA-I levels. In addition, increased expression of LCAT was associated with a 37% reduction of ApoB levels (12 +/- 1 vs 19 +/- 1 mg/dL, P < .05) due to increased low-density lipoprotein catabolism (fractional catabolic rate = 1.7 +/- 0.1 d(-1) in controls vs 4.2 +/- 0.3 d(-1) in LCAT-treated group, P < .05). In summary, overexpression of LCAT in nonhuman primates leads to an antiatherogenic lipoprotein profile by increasing HDL cholesterol and lowering ApoB, thus making LCAT a potential drug target for reducing atherosclerosis.

Lipolytic and ligand-binding functions of hepatic lipase protect against atherosclerosis in LDL receptor-deficient mice.

To elucidate the separate contributions of the lipolytic versus ligand-binding functions of hepatic lipase (HL) to lipoprotein metabolism and atherosclerosis, and to investigate the role of the low density lipoprotein receptor (LDLr) in these processes, we compared mice expressing catalytically active HL (HL-WT) with mice expressing inactive HL (HL-S145G) in a background lacking endogenous HL and the LDLr (LDLr-KOxHL-KO). HL-WT and HL-S145G reduced (P < 0.05 for all) cholesterol (55% vs. 20%), non-HDL-cholesterol (63% vs. 22%), and apolipoprotein B (apoB; 34% vs. 16%) by enhancing the catabolism of autologous (125)I-apoB-intermediate density lipoprotein (IDL)/LDL (fractional catabolic rate in day(-1): 6.07 +/- 0.25, LDLr-KOxHL-WT; 4.76 +/- 0.30, LDLr-KOxHL-S145G; 3.70 +/- 0.13, LDLr-KOxHL-KO); HL-WT had a greater impact on the concentration, composition, particle size, and catabolism of apoB-containing lipoproteins (apoB-Lps) and HDL. Importantly, consistent with the changes in apoB-Lps, atherosclerosis in LDLr-KOxHL-KO mice fed a regular chow diet (RCD) was reduced by both HL-WT and HL-S145G (by 71% and 51% in cross-sectional analysis, and by 85% and 67% in en face analysis; P < 0.05 for all). These data identify physiologically relevant but distinct roles for the lipolytic versus ligand-binding functions of HL in apoB-Lp metabolism and atherosclerosis and demonstrate that their differential effects on these processes are mediated by changes in catabolism via non-LDLr pathways. These changes, evident even in the presence of apoE, establish an antiatherogenic role of the ligand-binding function of HL in LDLr-deficient mice.

Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited.

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.

Enhanced ABCG1 expression increases atherosclerosis in LDLr-KO mice on a western diet.

ABCG1 promotes cholesterol efflux from cells, but ABCG1(-/-) bone marrow transplant into ApoE(-/-) and LDLr(-/-) mice reduces atherosclerosis. To further investigate the role of ABCG1 in atherosclerosis, ABCG1 transgenic mice were crossed with LDLr-KO mice and placed on a high-fat western diet. Increased expression of ABCG1 mRNA was detected in liver (1.8-fold) and macrophages (2.7-fold), and cholesterol efflux from macrophages to HDL was also increased (1.4-fold) in ABCG1xLDLr-KO vs. LDLr-KO mice. No major differences were observed in total plasma lipids. However, cholesterol in the IDL-LDL size range was increased by approximately 50% in ABCG1xLDLr-KO mice compared to LDLr-KO mice. Atherosclerosis increased by 39% (10.1+/-0.8 vs 6.1+/-0.9% lesion area, p=0.02), as measured by en face analysis, and by 53% (221+/-98 vs 104+/-58x10(3)microm(2), p =0.01), as measured by cross-sectional analysis in ABCG1xLDLr-KO mice. Plasma levels for MCP-1 (1.5-fold) and TNF-alpha (1.2-fold) were also increased in ABCG1xLDLr-KO mice. In summary, these findings suggest that enhanced expression of ABCG1 increases atherosclerosis in LDLr-KO mice, despite its role in promoting cholesterol efflux from cells.

ABCA1 overexpression in the liver of LDLr-KO mice leads to accumulation of pro-atherogenic lipoproteins and enhanced atherosclerosis.

The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg x LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.8-fold increase in cholesterol associated with a large pool of apoB-lipoproteins. ApoB synthesis was unchanged but the catabolism of (125)I-apoB-VLDL and -LDL were significantly delayed, accounting for the 1.35-fold increase in plasma apoB levels in ABCA1-Tg mice. We also found rapid in vivo transfer of free cholesterol from HDL to apoB-lipoproteins in ABCA1-Tg mice, associated with a significant 2.7-fold increase in the LCAT-derived cholesteryl linoleate content found primarily in apoB-lipoproteins. ABCA1-Tg mice had 1.4-fold increased hepatic cholesterol concentrations, leading to a compensatory 71% decrease in de novo hepatic cholesterol synthesis, as well as enhanced biliary cholesterol, and bile acid secretion. CAV-1, CYP2b10, and ABCG1 were significantly induced in ABCA1-overexpressing livers; however, no differences were observed in the hepatic expression of CYP7alpha1, CYP27alpha1, or ABCG5/G8 between ABCA1-Tg and control mice. As expected from the pro-atherogenic plasma lipid profile, aortic atherosclerosis was increased 10-fold in ABCA1-Tg mice. In summary, hepatic overexpression of ABCA1 in LDLr-KO mice leads to: 1) expansion of the pro-atherogenic apoB-lipoprotein cholesterol pool size via enhanced transfer of HDL-cholesterol to apoB-lipoproteins and delayed catabolism of cholesterol-enriched apoB-lipoproteins; 2) increased cholesterol concentration in the liver, resulting in up-regulated hepatobiliary sterol secretion; and 3) significantly enhanced aortic atherosclerotic lesions.

The human ABCG1 gene: identification of LXR response elements that modulate expression in macrophages and liver.

The ABC transporter ABCG1 (ATP binding cassette transporter G1), expressed in macrophages, liver, and other tissues, has been implicated in the efflux of cholesterol to high density lipoprotein. The ABCG1 gene is transcriptionally activated by cholesterol loading and activators of liver X receptors (LXRs) and retinoid X receptors (RXRs) through genomic sequences that have not been fully characterized. Here we show that ABCG1 mRNA is induced by LXR agonists in RAW264.7 macrophage cells, HepG2 hepatoma cells, and primary mouse hepatocytes. We identify two evolutionarily highly conserved LXR response elements (LXREs), LXRE-A and LXRE-B, located in the first and second introns of the human ABCG1 gene. Each element conferred robust LXR-agonist responsiveness to ABCG1 promoter-directed luciferase gene constructs in RAW264.7 and HepG2 cells. Overexpression of LXR/RXR activated the ABCG1 promoter in the presence of LXRE-A or LXRE-B sequences. In gel-shift assays, LXR/RXR heterodimers bound to wild-type but not to mutated LXRE-A and LXRE-B sequences. In chromatin immunoprecipitation assays, LXR and RXR were detected at LXRE-A and -B regions of DNA of human THP-1 macrophages. These studies clarify the mechanism of transcriptional upregulation of the ABCG1 gene by oxysterols in macrophages and liver, two key tissues where ABCG1 expression may affect cholesterol balance and atherogenesis.

Identification of mouse and human macrophages as a site of synthesis of hepatic lipase.

Hepatic lipase (HL) is synthesized by the liver and is also present in steroidogenic tissues. As both a lipolytic enzyme and a ligand that facilitates the cellular uptake of lipoproteins, HL plays a major role in lipoprotein metabolism and may modulate atherogenic risk. However, HL has not been directly implicated in lesion development. In the present study we demonstrate that HL is also synthesized by mouse and human macrophages. Northern analysis and real time RT-PCR showed that HL mRNA is present in mouse peritoneal macrophages, RAW-264.7, and IC-21 cells. The levels of HL mRNA in mouse peritoneal macrophages were approximately 10-30% that of mouse liver. HL protein was identified by Western blot analyses in human monocyte-derived macrophages, THP, RAW-264.7, and mouse peritoneal macrophages following fractionation by heparin-sepharose affinity chromatography. These combined findings establish that HL is synthesized de novo by macrophages as well as liver, and raises the possibility that HL may have a direct role in the pathogenesis of atherosclerosis.

The orphan nuclear receptor LRH-1 activates the ABCG5/ABCG8 intergenic promoter.

The ATP binding cassette (ABC) half-transporters ABCG5 and ABCG8 facilitate biliary and intestinal removal of neutral sterols. Here, we identify a binding site for the orphan nuclear receptor liver receptor homolog-1 (LRH-1) at nt 134-142 of the ABCG5/ABCG8 intergenic region necessary for the activity of both the ABCG5 and ABCG8 promoters. Mutating this LRH-1 binding site reduced promoter activity of the human ABCG5/ABCG8 intergenic region more than 7-fold in HepG2 and Caco2 cells. Electrophoretic mobility shift assays with HepG2 nuclear extracts demonstrated specific binding of LRH-1 to the LRH-1 binding motif in the human ABCG5/ABCG8 intergenic region. LRH-1 overexpression increased promoter activity up to 1.6-fold and 3-fold in Caco2 and 293 cells, respectively. Finally, deoxycholic acid repressed the ABCG5 and ABCG8 promoters, consistent with bile acid regulation via the farnesoid X receptor-small heterodimeric partner-LRH-1 pathway. These results demonstrate that LRH-1 is a positive transcription factor for ABCG5 and ABCG8 and, in conjunction with studies on LRH-1 activation of other promoters, identify LRH-1 as a "master regulator" for genes involved in sterol and bile acid secretion from liver and intestine.

The ABCA1 transporter functions on the basolateral surface of hepatocytes.

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.

The ligand-binding function of hepatic lipase modulates the development of atherosclerosis in transgenic mice.

To investigate the separate contributions of the lipolytic versus ligand-binding function of hepatic lipase (HL) to plasma lipoprotein metabolism and atherosclerosis, we compared mice expressing catalytically active wild-type HL (HL-WT) and inactive HL (HL-S145G) with no endogenous expression of mouse apoE or HL (E-KO x HL-KO, where KO is knockout). HL-WT and HL-S145G reduced plasma cholesterol (by 40 and 57%, respectively), non-high density lipoprotein cholesterol (by 48 and 61%, respectively), and apoB (by 36 and 44%, respectively) (p < 0.01), but only HL-WT decreased high density lipoprotein cholesterol (by 67%) and apoA-I (by 54%). Compared with E-KO x HL-KO mice, both active and inactive HL lowered the pro-atherogenic lipoproteins by enhancing the catabolism of autologous (125)I-apoB very low density/intermediate density lipoprotein (VLDL/IDL) (fractional catabolic rates of 2.87 +/- 0.04/day for E-KO x HL-KO, 3.77 +/- 0.03/day for E-KO x HL-WT, and 3.63 +/- 0.09/day for E-KO x HL-S145G mice) and (125)I-apoB-48 low density lipoprotein (LDL) (fractional catabolic rates of 5.67 +/- 0.34/day for E-KO x HL-KO, 18.88 +/- 1.72/day for E-KO x HL-WT, and 9.01 +/- 0.14/day for E-KO x HL-S145G mice). In contrast, the catabolism of apoE-free, (131)I-apoB-100 LDL was not increased by either HL-WT or HL-S145G. Infusion of the receptor-associated protein (RAP), which blocks LDL receptor-related protein function, decreased plasma clearance and hepatic uptake of (131)I-apoB-48 LDL induced by HL-S145G. Despite their similar effects on lowering pro-atherogenic apoB-containing lipoproteins, HL-WT enhanced atherosclerosis by up to 50%, whereas HL-S145G markedly reduced aortic atherosclerosis by up to 96% (p < 0.02) in both male and female E-KO x HL-KO mice. These data identify a major receptor pathway (LDL receptor-related protein) by which the ligand-binding function of HL alters remnant lipoprotein uptake in vivo and delineate the separate contributions of the lipolytic versus ligand-binding function of HL to plasma lipoprotein size and metabolism, identifying an anti-atherogenic role of the ligand-binding function of HL in vivo.

The E-box motif in the proximal ABCA1 promoter mediates transcriptional repression of the ABCA1 gene.

To identify regulatory elements in the proximal human ATP-binding cassette transporter A1 (hABCA1) gene promoter we transfected RAW cells with plasmids containing mutations in the E-box, AP1, and liver X receptor (LXR) elements as well as the two Sp1 motifs. Point mutations in either Sp1 site or in the AP1 site had only a minor effect whereas mutation of the LXR element decreased promoter activity. In contrast, mutation or deletion of the E-box motif caused a 3-fold increase in transcriptional activity under basal conditions. Gel shift and DNaseI footprint analysis showed binding of a protein or protein complex to this region. Preincubation of nuclear extracts with antibodies established that USF1, USF2, and fos related antigen (Fra) 2 bind to DNA sequences in the human ABCA1 promoter that contains the intact E-box but not the mutant or deleted E-box. Co-transfection of USF1 and USF2 enhanced, but Fra2 repressed, ABCA1 promoter activity. Thus, a complex consisting of USF1, USF2, and Fra2 binds the E-box motif 147 bp upstream of the transcriptional start site and facilitates repression of the human ABCA1 promoter. These combined studies identify a novel site in the human ABCA1 promoter involved in the regulation of ABCA1 gene expression.

Structural and functional consequences of missense mutations in exon 5 of the lipoprotein lipase gene.

Missense mutations in exon 5 of the LPL gene are the most common reported cause of LPL deficiency. Exon 5 is also the region with the strongest homology to pancreatic and hepatic lipase, and is conserved in LPL from different species. Mutant LPL proteins from post-heparin plasma from patients homozygous for missense mutations at amino acid positions 176, 188, 194, 205, and 207, and from COS cells transiently transfected with the corresponding cDNAs were quantified and characterized, in an attempt to determine which aspect of enzyme function was affected by each specific mutation. All but one of the mutant proteins were present, mainly as partially denatured LPL monomer, rendering further detailed assessment of their catalytic activity, affinity to heparin, and binding to lipoprotein particles difficult. However, the fresh unstable Gly(188)-->Glu LPL and the stable Ile(194)-->Thr LPL, although in native conformation, did not express lipase activity. It is proposed that many of the exon 5 mutant proteins are unable to achieve or maintain native dimer conformation, and that the Ile(194)-->Thr substitution interferes with access of lipid substrate to the catalytic pocket. These results stress the importance of conformational evaluation of mutant LPL. Absence of catalytic activity does not necessarily imply that the substituted amino acid plays a specific direct role in catalysis.

The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease.

We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.

Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster.

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.