Human Lipoprotein Research Articles

Exon Skipping of Hepatic APOB Pre-mRNA With Splice-switching Oligonucleotides Reduces LDL Cholesterol In Vivo

Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting very low-density lipoprotein (VLDL) assembly and increasing LDL clearance. We demonstrate that these "APO-skip " SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for >6 days. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.

Identification of a loss-of-function inducible degrader of the low-density lipoprotein receptor variant in individuals with low circulating low-density lipoprotein

Recent genome-wide association studies suggest that IDOL (also known as MYLIP) contributes to variation in circulating levels of low-density lipoprotein cholesterol (LDL-C). IDOL, an E3-ubiquitin ligase, is a recently identified post-transcriptional regulator of LDLR abundance. Briefly, IDOL promotes degradation of the LDLR thereby limiting LDL uptake. Yet the exact role of IDOL in human lipoprotein metabolism is unclear. Therefore, this study aimed at identifying and functionally characterizing IDOL variants in the Dutch population and to assess their contribution to circulating levels of LDL-C. We sequenced the IDOL coding region in 677 individuals with LDL-C above the 95th percentile adjusted for age and gender (high-LDL-C cohort) in which no mutations in the LDLR, APOB, and PCSK9 could be identified. In addition, IDOL was sequenced in 560 individuals with baseline LDL-C levels below the 20th percentile adjusted for age and gender (low-LDL-C cohort). We identified a total of 14 IDOL variants (5 synonymous, 8 non-synonymous, and 1 non-sense). Functional characterization of these variants demonstrated that the p.Arg266X variant represents a complete loss of IDOL function unable to promote ubiquitylation and subsequent degradation of the LDLR. Consistent with loss of IDOL function, this variant was identified in individuals with low circulating LDL-C. Our results support the notion that IDOL contributes to variation in circulating levels of LDL-C. Strategies to inhibit IDOL activity may therefore provide a novel therapeutic venue to treating dyslipidaemia.

Top–down lipidomic analysis of human lipoproteins by chip-type asymmetrical flow field-flow fractionation–electrospray ionization-tandem mass spectrometry

This study demonstrates the potential utility of on-line chip-type asymmetrical flow field-flow fractionation (cAF4) and electrospray ionization tandem mass spectrometry (ESI-MS-MS) for the top–down lipidomic analysis of human lipoproteins. Utilizing a cAF4, which is a miniaturized AF4 channel operated with a micro flow rate regime, enabled high density lipoprotein (HDL) and low density lipoprotein (LDL) to be separated by hydrodynamic diameter in an aqueous solution with the simultaneous desalting of lipoproteins. On-line desalting was found to enhance the ionization of lipoproteinic lipid molecules during the feeding of cAF4 effluent to ESI-MS when compared to the direct infusion of lipoproteins to MS. An evaluation of top–down lipidomic analysis was performed to test the efficiency of in-source fragmentation during cAF4–ESI-MS in the dissociation of lipoprotein particles into individual lipid molecules. This study demonstrates the structural identification of the following lipid classes: phosphatidylcholines (PCs), cholesteryl esters (CEs), and regioisomers of triacylglycerols (TAGs) having an identical mass but different acyl chains and dimeric forms of TAGs in the positive ion mode, and phosphatidylglycerols (PGs), phosphatidic acids (PAs), phosphatidyinositols (PIs), and their lyso species in the negative ion mode. The developed method was applied to plasma samples from patients with coronary artery disease (CAD) for the separation of HDL and LDL and for the simultaneous analysis of lipoproteinic lipids, resulting in the identification of 11 PCs, 9 PGs, 4 PAs, 2 PIs, 2 PEs, 18 TAGs, and 6 CEs.

Distinct composition of human fetal HDL attenuates its anti-oxidative capacity

In human high-density lipoprotein (HDL) represents the major cholesterol carrying lipoprotein class in cord blood, while cholesterol is mainly carried by low-density lipoprotein in maternal serum. Additionally, to carrying cholesterol, HDL also associates with a range of proteins as cargo. We tested the hypothesis that fetal HDL carries proteins qualitatively and quantitatively different from maternal HDL. These differences then contribute to distinct HDL functionality in both circulations. Shotgun proteomics and biochemical analyses were used to assess composition/function of fetal and maternal HDL isolated from uncomplicated human pregnancies at term of gestation. The pattern of analyzed proteins that were statistically elevated in fetal HDL (apoE, proteins involved in coagulation, transport processes) suggests a particle characteristic for the light HDL2 sub-fraction. In contrast, proteins that were enriched in maternal HDL (apoL, apoF, PON1, apoD, apoCs) have been described almost exclusively in the dense HDL3 fraction and relevant to its anti-oxidative function and role in innate immunity. Strikingly, PON1 mass and activity were 5-fold lower (p<0.01) in the fetus, which was accompanied by attenuation of anti-oxidant capacity of fetal HDL. Despite almost equal quantity of CETP in maternal and fetal HDL, its enzymatic activity was 55% lower (p<0.001) in the fetal circulation, whereas LCAT activity was not altered. These findings indicate that maternally derived HDL differs from fetal HDL with respect to its proteome, size and function. Absence of apoA-1, apoL and PON1 on fetal HDL is associated with decreased anti-oxidative properties together with deficiency in innate immunity collectively indicating distinct HDLs in fetuses.

Human Apolipoprotein E is necessary and sufficient for production of infectious HCV particles in human non-liver cell lines

HCV has a narrow tissue tropism. Accordingly, efficient replication is only observed in the liver and in cultured human liver cell lines. The molecular determinants that are responsible for the restricted tropism of HCV are incompletely understood. Recent evidence indicates that HCV assembly is tightly coupled to the synthesis of human lipoproteins that occurs in liver cells. Specifically, Apolipoprotein B (ApoB), ApoE, and microsomal triglyceride transfer protein (MTP), which are all essential for production of very low density lipoproteins (VLDL) have been implicated in HCV assembly. To define the minimal liver cell-specific requirements for production of infectious HCV progeny, we attempted to reconstitute assembly of HCV particles in human non-liver cell lines. To this end, we created stable HeLa (human cervical cancer) and Huh-7.5 (human hepatocarcinoma) cells replicating selectable subgenomic HCV RNA replicons. Co-expression of viral structural proteins reconstituted particle production only in Huh-7.5 replicon cells, but not in HeLa replicon cells. Importantly, ectopic expression of ApoE was necessary and sufficient to allow production of infectious HCV trans-complemented particles (HCVtcp) in HeLa cells. Comparison between Huh-7.5 and HeLa derived particles revealed no gros differences between the density distribution of intra- and extracellular particle. Using a similar approach, we could also observe ApoE-dependent production of infectious HCVtcp in several other non-liver cell lines. Enhancement of HCV replication by introduction of miR-122 into ApoE-expressing 293T and HeLa cells was sufficient for production of full-length HCV particles. More detailed analyses of particle composition and receptor usage of these particle types are currently ongoing. Collectively, these data indicate that ApoE may be the sole liver cell-specific cofactor for assembly of infectious HCV particles. In turn, ApoE may be major determinant limiting HCV tissue tropism.

Rapid regression of atherosclerosis with MTP inhibitor treatment

ObjectiveRegression of atherosclerosis is a vital treatment goal of atherosclerotic vascular disease. Inhibitors of the microsomal triglyceride transfer protein (MTP) have been shown to reduce apolipoprotein B (apoB)-containing lipoproteins in animals and humans effectively. Therefore, the major aim of our study is to evaluate the effect of MTP inhibition on atherosclerotic plaque regression. MethodsLDL-receptor-deficient (LDLr−/−) mice were fed a Western diet for 16 weeks and then harvested for baseline (n = 8), switched to chow diet (n = 8) or chow diet containing MTP inhibitor (BMS 212122; n = 8) for 2 weeks before harvesting. ResultsTreatment with MTP inhibitor led to rapid reduction in plasma lipid levels, which were accompanied by a significant decrease in lipid content and monocyte-derived (CD68+) cells in atherosclerotic plaques compared to baseline and chow diet control groups. MTP inhibitor-treated mice had increased collagen content, a marker associated with increased stability in human plaques. Furthermore, plaques of these mice showed a significant decrease in tissue factor and pro-inflammatory M1 macrophage marker monocyte chemoattractant protein-1 (MCP-I) and an increase in anti-inflammatory M2 macrophage markers arginase-I and mannose receptor 1 compared to mice in the baseline group. ConclusionReversal of hyperlipidemia in atherosclerotic mice by inhibition of MTP leads to rapid and beneficial changes in the composition and inflammatory state of the plaque.

Acute Cardiovascular Toxicity of Sterilizers, PHMG, and PGH: Severe Inflammation in Human Cells and Heart Failure in Zebrafish

In 2011, dozens of children and pregnant women in Korea died by exposure to sterilizer for household humidifier, such as Oxy(®) and Cefu(®). Until now, however, it remains unknown how the sterilizer affect the human health to cause the acute deaths. To find its toxicity for organ, we investigated the putative toxicity of the sterilizer in the cardiovascular system. The sterilizers, polyhexamethylene guanidine phosphate (PHMG, Cefu(®)), and oligo-[2-(2-ethoxy)-ethoxyethyl)-guanidinium-chloride (PGH, Oxy(®)) were treated to human lipoproteins, macrophages, and dermal fibroblast cells. The PGH and PHMG at normal dosages caused severe atherogenic process in human macrophages, cytotoxic effect, and aging in human dermal cell. Zebrafish embryos, which were exposed to the sterilizer, showed early death with acute inflammation and attenuated developmental speed. All zebrafish exposed to the working concentration of PHMG (final 0.3 %) and PGH (final 10 mM) died within 70 min and displayed acute increases in serum triacylglycerol level and fatty liver induction. The dead zebrafish showed severe accumulation of fibrous collagen in the bulbous artery of the heart with elevation of reactive oxygen species. In conclusion, the sterilizers showed acute toxic effect in blood circulation system, causing by severe inflammation, atherogenesis, and aging, with embryo toxicity.

Proteomic Analysis of Aortae from Human Lipoprotein(a) Transgenic Mice

Proteomic Analysis of Aortae from Human Lipoprotein(a) Transgenic Mice

血清リポ蛋白のアポ蛋白に関する研究第2報: アポリポ蛋白A-IIの精製及びその免疫化学的性質について

血清リポ蛋白のアポ蛋白に関する研究第2報: アポリポ蛋白A-IIの精製及びその免疫化学的性質について

Abstract 18904: High-denisty Lipoprotein Distribution in Obesity Favors Apolipoprotein E and C-III-containing Subfractions Associated with Risk for Future Coronary Heart Disease

Human high-density lipoprotein (HDL) particles are highly heterogeneous in composition. Both the plasma concentration of HDL with apoC-III and the concentration of apoE in HDL predict higher incidence of coronary heart disease (CHD). Little is known about the concentration of HDL apoA-I associated with apoE or apoC-III, whether apoE and apoC-III are present in smaller or larger cholesterol ester-rich HDL, and whether the distribution is affected in obesity. We studied 20 normal weight subjects (BMI 23-25) and 20 obese subjects (BMI 30-35) matched by age (mean age, 51 years). Sixteen distinct HDL subpopulations were separated from plasma samples by first using anti-apoAI, anti-apoE and anti-apoC-III immunoaffinity chromatography in sequence followed by size separation using nondenaturing electrophoresis. The mean HDL-cholesterol and plasma apoA-I concentrations in the normal weight group were 65 and 179mg/dL respectively, compared to 50 and 170mg/dL in the obese group. Mean apoA-I concentrations of HDL fractions without apoE or apoC-III was 160mg/dL in the normal weight group, compared to 136mg/dL in the obese; with apoE only 9 and 12mg/dL respectively; with apoC-III only 7.4 and 14.7 mg/dL; and with both apoE and apoC-III 2.6 and 7.5mg/dL (p&lt;0.01 for a difference in apoA-I concentration and distribution across HDL types). All told, about 10% of HDL apoA-I in normal weight subjects contained apoE, apoC-III or both, compared to 20% in obese subjects. The concentrations of apoE and apoC-III in HDL in the obese were about double that of the normal weight group (p&lt;0.01). In the obese group, the high concentration of HDL with apoE was distributed to small HDL. More so, obese had a higher percent of large HDL containing apoC-III and no apoE. In conclusion, despite similar plasma total apoA-I levels in obese and normal weight groups, the obese had higher concentrations of HDL containing apoE or apoC-III, subfractions which are associated with CHD, whereas the normal weight group had higher concentration of HDL without apoE or apoC-III, a subtraction with protective association against CHD. Mechanistically, the increased presence of apoC-III in large size HDL and apoE in small size HDL in obese may indicate a reduced capacity of HDL for RCT.

Discovery of candidate phospholipid biomarkers in human lipoproteins with coronary artery disease by flow field-flow fractionation and nanoflow liquid chromatography–tandem mass spectrometry

In this study, an analytical method is demonstrated to identify and develop potential phospholipid (PL) biomarkers of high density lipoprotein (HDL) and low density lipoprotein (LDL) in plasma from individuals with coronary artery disease (CAD) by employing a combination of off-line multiplexed hollow fiber flow field-flow fractionation (MxHF5) and nanoflow liquid chromatography–electrospray ionization-tandem mass spectrometry (nLC–ESI-MS–MS). HDL and LDL particles of human plasma were sorted by size at a semi-preparative scale using MxHF5, after which PL extracts of each lipoprotein fraction were qualitatively and quantitatively analyzed by nLC–ESI-MS–MS. Experiments were performed using plasma samples from 10 CAD patients and 10 controls. Quantitative analysis of the 93 PL species identified yielded a selection of 19 species from HDL fractions and 10 from LDL fractions exhibiting at least a five fold change in average concentration in CAD patients. Among the selected species, only a few were found exclusively in patient HDL fractions (18:3-LPA and 20:2/16:0-PG), control HDL fractions (16:0/16:1-PC, 20:1/20:4-PE, and 16:1-LPA), and control LDL fractions (16:0/22:3-PG). Moreover, 16:1/18:2-PC was detected from both HDL and LDL fractions of controls but disappeared in CAD patients. Although the typical change in lipoproteins for CAD is well known, with decreased levels of HDLs and reduced LDL particle size, the current study provides fundamental information on the molecular level of lipoprotein variation which can be utilized for diagnostic and therapeutic tracking.

Effect of weight loss, independent of change in diet composition, on apolipoprotein AI kinetic in men with metabolic syndrome

We investigated the effect of weight loss, independent of change in diet composition, on HDL and apoAI metabolism in men with metabolic syndrome (MetS). Subjects (19 men with MetS [NCEP-ATPIII]) were fed an isoenergetic Mediterranean-style diet for 5 weeks (all foods provided). Participants then underwent a 20-week free-living period during which they were counseled to restrict energy intake, after which they were again fed an isoenergetic Mediterranean-style diet for 5 weeks. At the end of the two controlled diets, participants received a single bolus of [5,5,5-(2)H(3)] (L)-leucine, and fasting blood samples were collected over a 96 h period. ApoAI kinetic was assessed using multicompartmental modeling of the tracer enrichment data. Participants achieved a 9.1 ± 2.8% reduction in body weight (P < 0.001). Weight loss resulted in an increase in plasma HDL-cholesterol (HDL-C) concentrations of 6.0% (P = 0.059) and HDL(3)-C of 7.9% (P = 0.045), attributable to a reduction in apoAI fractional catabolic rate (-7.8%; P = 0.046) with no change in apoAI production rate (2.2%; P = 0.58). These data indicate that weight loss, independent of variation in diet composition, increases plasma HDL primarily by delaying the catabolism of apoAI.

Tumor necrosis factor-α, insulin resistance, the lipoprotein metabolism and obesity in humans

In the obese adipose tissue produces proinflammatory molecules as tumor necrosis factor-α, which has local effects on adipocyte physiology and systemic effects in other organs. Many studies linking TNF-α, obesity, insulin resistance and lipid metabolism have been conducted in rats, rabbits and dogs, but the results observed in several of these studies have been conflicting and many of them have not been able to reproduce in humans, which on human makes difficult the interpretation of the effect of TNF-α on human metabolism. To conduct a systematic review of human studies which relates, TNF-α insulin resistance and lipoprotein metabolism. We searched the PubMed database for studies in humans, human tissue and human cell lines linking TNF-α, obesity, insulin resistance and lipoprotein. There is a increased production of TNF-α on adipose tissue of obese. TNF-α decreases the cellular response to insulin in adipocytes, hepatocytes and human muscle cells. There is an increase of TNF-α in patients with dyslipidemia, and inactivation of TNF-α affects lipid metabolism. In human hepatocytes, TNF-α inhibits expression of APO AI, which may decrease the secretion of high density lipoproteins. TNF-α affects the excretion of cholesterol by inhibiting the enzyme cholesterol-7α-hydroxylase in hepatocytes. TNF-α decreases the cellular response to insulin, and has effects on the metabolism of cholesterol and lipoproteins in humans. A better understanding of the mechanisms of the inflammatory response induced obesity in humans, can lead to identifying new therapeutic targets that can prevent the complications associated with obesity.

Physiological role of hepatic NPC1L1 in human cholesterol and lipoprotein metabolism: New perspectives and open questions

The importance of the cholesterol transporter Niemann-Pick C1 like protein 1 (NPC1L1)in the small intestine for efficient absorption of cholesterol is well documented and its physiological importance is clear (1). Similarly, the drug ezetimibe (Zetia™), which blocks function of this transporter, reduces plasma cholesterol, primarily LDL, by dramatically reducing intestinal absorption of dietary and biliary cholesterol (2). There are controversies about the exact mechanism by which NPC1L1 functions: whether the protein is on the apical cell membrane or intracellular vesicles or both (3–5), and whether ezetimibe directly blocks NPC1L1 or interrupts its association with other proteins in the sterol absorption and transport pathway (6–8). Nevertheless, it is now accepted that this protein is the key player in sterol absorption, although other transporters may play ancillary roles (9, 10).

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.

1,8-cineole protected human lipoproteins from modification by oxidation and glycation and exhibited serum lipid-lowering and anti-inflammatory activity in zebrafish

We recently reported that a water extract of laurel or turmeric, 1,8-cineole enriched fractions, showed hypolipidemic activity in the zebrafish model. Therefore, the present study investigated the cineole's anti-oxidant and anti-inflammatory activities in lipoprotein metabolism in vitro and in vivo. Cineole had inhibitory effects on cupric ion-mediated oxidation of lipoproteins in general, while simultaneously enhancing ferric ion removal ability in high-density lipoprotein (HDL). Hypercholesterolemia was induced in zebrafish using cholesterol-feeding treatment, 4% cholesterol, for 3 weeks. After feeding with or without the addition of cineole, the results revealed that cineole possessed lipid-lowering and anti-inflammatory activities in hypercholesterolemic zebrafish. In addition, serum amyloid A and interleukin-6 levels were lowered and lipid accumulation was decreased in the liver. Conclusively, 1,8-cineole was found to have anti-oxidant activities in lipoprotein metabolism both in vitro and in vivo with simultaneous reduction of lipid accumulation in the liver of zebrafish.

Cholesterol Determines and Limits rHDL Formation from Human Plasma Apolipoprotein A-II and Phospholipid Membranes

Apolipoprotein (apo) A-II, the second most abundant protein after apo A-I of human plasma high-density lipoproteins (HDL), is the most lipophilic of the exchangeable apolipoproteins. The rate of microsolubilization of dimyristoylphosphatidylcholine (DMPC) membranes by apo A-I to give rHDL increases as the level of membrane free cholesterol (FC) increases up to 20 mol % when the level of reaction decreases to nil. Given its greater lipophilicity, we tested the hypothesis that apo A-II and its reduced and carboxymethylated monomer (rcm apo A-II) would form rHDL at a membrane FC content of >20 mol %. According to turbidimetric titrations, the DMPC/apo A-II stoichiometry is 65/1 (moles to moles). At this stoichiometry, apo A-II forms rHDL from DMPC and FC. Contrary to our hypothesis, apo A-II, like apo A-I, reacts poorly with DMPC containing ≥20 mol % FC. The rate of formation of rHDL from rcm apo A-II and DMPC at all FC mole percentages is faster than that of apo A-II but nil at 20 mol % FC. In parallel reactions, monomeric and dimeric apo A-II form large FC-rich rHDL coexisting with smaller FC-poor rHDL; increasing the FC mole percentage increases the number and size of FC-rich rHDL. On the basis of the compositions of coexisting large and small rHDL, the free energy of transfer of FC from the smallest to the largest particle is approximately −1.2 kJ. On the basis of our data, we propose a model in which apo A-I and apo A-II bind to DMPC via surface defects that disappear at 20 mol % FC. These data suggest apo A-II-containing HDL formed intrahepatically are likely cholesterol-rich compared to the smaller intracellular lipid-poor apo A-I HDL.

High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Functional Characterization of Newly-Discovered Mutations in Human SR-BI

In rodents, SR-BI has been firmly established as a physiologically relevant HDL receptor that mediates removal of HDL-cholesteryl esters (CE). However, its role in human lipoprotein metabolism is less defined. Recently, two unique point mutations in human SR-BI — S112F or T175A — were identified in subjects with high HDL-cholesterol (HDL-C) levels. We hypothesized that mutation of these conserved residues would compromise the cholesterol-transport functions of SR-BI. To test this hypothesis, S112F- and T175A-SR-BI were generated by site-directed mutagenesis. Cell surface expression was confirmed for both mutant receptors in COS-7 cells upon transient transfection, albeit at lower levels for T175A-SR-BI. Both mutant receptors displayed defective HDL binding, selective uptake of HDL-CE and release of free cholesterol (FC) from cells to HDL. Mutant receptors were also unable to re-organize plasma membrane pools of FC. While these impaired functions were independent of receptor oligomerization, inability of T175A-SR-BI to mediate cholesterol-transport functions could be related to altered N-linked glycosylation status. In conclusion, high HDL-C levels observed in carriers of S112F- or T175A-SR-BI mutant receptors are consistent with the inability of these SR-BI receptors to mediate efficient selective uptake of HDL-CE, and suggest that increased plasma HDL concentrations in these settings may not be associated with lower risk of cardiovascular disease.

Apolipoprotein B100 is a suppressor of Staphylococcus aureus‐induced innate immune responses in humans and mice

Plasma lipoproteins such as LDL (low-density lipoprotein) are important therapeutic targets as they play a crucial role in macrophage biology and metabolic disorders. The impact of lipoprotein profiles on host defense pathways against Gram-positive bacteria is poorly understood. In this report, we discovered that human serum lipoproteins bind to lipoteichoic acid (LTA) from Staphylococcus aureus and thereby alter the immune response to these bacteria. Size-exclusion chromatography and solid-phase-binding analysis of serum revealed the direct interaction of LTA with apolipoproteins (Apo) B100, ApoA1, and ApoA2. Only ApoB100 and the corresponding LDL exerted biological effects as this binding significantly inhibited LTA-induced cytokine releases from human and murine immune cells. Serum from hypercholesterolemic mice or humans significantly diminished cytokine induction in response to S. aureus or its LTA. Sera taken from the patients with familial hypercholesterolemia before and after ApoB100-directed immuno-apheresis confirmed that ApoB100 inhibited LTA-induced inflammation in humans. In addition, mice in which LDL secretion was pharmacologically inhibited, displayed significantly increased serum cytokine levels upon infection with S. aureus in vivo. The present study identifies ApoB100 as an important suppressor of innate immune activation in response to S. aureus and its LTA.