A High-Throughput NMR Method for Lipoprotein-X Quantification.

Lipoprotein X (LP-X) is an abnormal cholesterol-rich lipoprotein particle that accumulates in patients with cholestatic liver disease and familial lecithin-cholesterol acyltransferase deficiency (FLD). Because there are no high-throughput diagnostic tests for its detection, a proton nuclear magnetic resonance (NMR) spectroscopy-based method was developed for use on a clinical NMR analyzer commonly used for the quantification of lipoproteins and other cardiovascular biomarkers. The LP-X assay was linear from 89 to 1615 mg/dL (cholesterol units) and had a functional sensitivity of 44 mg/dL. The intra-assay coefficient of variation (CV) varied between 1.8 and 11.8%, depending on the value of LP-X, whereas the inter-assay CV varied between 1.5 and 15.4%. The assay showed no interference with bilirubin levels up to 317 mg/dL and was also unaffected by hemolysis for hemoglobin values up to 216 mg/dL. Samples were stable when stored for up to 6 days at 4 °C but were not stable when frozen. In a large general population cohort (n = 277,000), LP-X was detected in only 50 subjects. The majority of LP-X positive cases had liver disease (64%), and in seven cases, had genetic FLD (14%). In summary, we describe a new NMR-based assay for LP-X, which can be readily implemented for routine clinical laboratory testing.

Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen.

A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.

Novel lecithin: cholesterol acyltransferase-based therapeutic approaches.

PURPOSE OF REVIEW: To review recent lecithin:cholesterol acyltransferas (LCAT)-based therapeutic approaches for atherosclerosis, acute coronary syndrome, and LCAT deficiency disorders. RECENT FINDINGS: A wide variety of approaches to using LCAT as a novel therapeutic target have been proposed. Enzyme replacement therapy with recombinant human LCAT is the most clinically advanced therapy for atherosclerosis and familial LCAT deficiency (FLD), with Phase I and Phase 2A clinical trials recently completed. Liver-directed LCAT gene therapy and engineered cell therapies are also another promising approach. Peptide and small molecule activators have shown efficacy in early-stage preclinical studies. Finally, lifestyle modifications, such as fat-restricted diets, cessation of cigarette smoking, and a diet rich in antioxidants may potentially suppress lipoprotein abnormalities in FLD patients and help preserve LCAT activity and renal function but have not been adequately tested. SUMMARY: Preclinical and early-stage clinical trials demonstrate the promise of novel LCAT therapies as HDL-raising agents that may be used to treat not only FLD but potentially also atherosclerosis and other disorders with low or dysfunctional HDL.

Incorporation of α-methylated amino acids into Apolipoprotein A-I mimetic peptides improves their helicity and cholesterol efflux potential.

Apolipoprotein A-I (ApoA-I) mimetic peptides are potential therapeutic agents for promoting the efflux of excess cellular cholesterol, which is dependent upon the presence of an amphipathic helix. Since α-methylated Ala enhances peptide helicity, we hypothesized that incorporating other types of α-methylated amino acids into ApoA-I mimetic peptides may also increase their helicity and cholesterol efflux potential. The last helix of apoA-I, peptide 'A' (VLESFKVSFLSALEEYTKKLNT), was used to design peptides containing a single type of α-methylated amino acid substitution (Ala/Aα, Glu/Dα, Lys/Kα, Leu/Lα), as well as a peptide containing both α-methylated Lys and Leu (6α). Depending on the specific residue, the α-helical content as measured by CD-spectroscopy and calculated hydrophobic moments were sometimes higher for peptides containing other types of α-methylated amino acids than those with α-methylated Ala. In ABCA1-transfected cells, cholesterol efflux to the peptides showed the following order of potency: 6α>Kα≈Lα≈Aα≫Dα≈A. In general, α-methylated peptides were resistant to proteolysis, but this varied depending on the type of protease and specific amino acid substitution. In summary, increased helicity and amphilicity due to α-methylated amino acid substitutions in ApoA-I mimetic peptides resulted in improved cholesterol efflux capacity and resistance to proteolysis, indicating that this modification may be useful in the future design of therapeutic ApoA-I mimetic peptides.

Plasma lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment ex vivo.

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20-200 mg/dl FC; coefficient of variation <20%) and sensitively (lower limit of quantitation <1 mg/ml FC), enabling LP-X detection in FLD, cholestatic, and even fish-eye disease patients. rhLCAT incubation with FLD plasma ex vivo reduced LP-X dose dependently, generated HDL, and decreased lipoprotein FC content. Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.

LCAT Enzyme Replacement Therapy Reduces LpX and Improves Kidney Function in a Mouse Model of Familial LCAT Deficiency.

Familial LCAT deficiency (FLD) is due to mutations in lecithin:cholesterol acyltransferase (LCAT), a plasma enzyme that esterifies cholesterol on lipoproteins. FLD is associated with markedly reduced levels of plasma high-density lipoprotein and cholesteryl ester and the formation of a nephrotoxic lipoprotein called LpX. We used a mouse model in which the LCAT gene is deleted and a truncated version of the SREBP1a gene is expressed in the liver under the control of a protein-rich/carbohydrate-low (PRCL) diet-regulated PEPCK promoter. This mouse was found to form abundant amounts of LpX in the plasma and was used to determine whether treatment with recombinant human LCAT (rhLCAT) could prevent LpX formation and renal injury. After 9 days on the PRCL diet, plasma total and free cholesterol, as well as phospholipids, increased 6.1 ± 0.6-, 9.6 ± 0.9-, and 6.7 ± 0.7-fold, respectively, and liver cholesterol and triglyceride concentrations increased 1.7 ± 0.4- and 2.8 ±0.9-fold, respectively, compared with chow-fed animals. Transmission electron microscopy revealed robust accumulation of lipid droplets in hepatocytes and the appearance of multilamellar LpX particles in liver sinusoids and bile canaliculi. In the kidney, LpX was found in glomerular endothelial cells, podocytes, the glomerular basement membrane, and the mesangium. The urine albumin/creatinine ratio increased 30-fold on the PRCL diet compared with chow-fed controls. Treatment of these mice with intravenous rhLCAT restored the normal lipoprotein profile, eliminated LpX in plasma and kidneys, and markedly decreased proteinuria. The combined results suggest that rhLCAT infusion could be an effective therapy for the prevention of renal disease in patients with FLD.

Safety and Tolerability of ACP-501, a Recombinant Human Lecithin:Cholesterol Acyltransferase, in a Phase 1 Single-Dose Escalation Study.

RATIONALE: Low high-density lipoprotein-cholesterol (HDL-C) in patients with coronary heart disease (CHD) may be caused by rate-limiting amounts of lecithin:cholesterol acyltransferase (LCAT). Raising LCAT may be beneficial for CHD, as well as for familial LCAT deficiency, a rare disorder of low HDL-C. OBJECTIVE: To determine safety and tolerability of recombinant human LCAT infusion in subjects with stable CHD and low HDL-C and its effect on plasma lipoproteins. METHODS AND RESULTS: A phase 1b, open-label, single-dose escalation study was conducted to evaluate safety, tolerability, pharmacokinetics, and pharmacodynamics of recombinant human LCAT (ACP-501). Four cohorts with stable CHD and low HDL-C were dosed (0.9, 3.0, 9.0, and 13.5 mg/kg, single 1-hour infusions) and followed up for 28 days. ACP-501 was well tolerated, and there were no serious adverse events. Plasma LCAT concentrations were dose-proportional, increased rapidly, and declined with an apparent terminal half-life of 42 hours. The 0.9-mg/kg dose did not significantly change HDL-C; however, 6 hours after doses of 3.0, 9.0, and 13.5 mg/kg, HDL-C was elevated by 6%, 36%, and 42%, respectively, and remained above baseline ≤4 days. Plasma cholesteryl esters followed a similar time course as HDL-C. ACP-501 infusion rapidly decreased small- and intermediate-sized HDL, whereas large HDL increased. Pre-ß-HDL also rapidly decreased and was undetectable ≤12 hours post ACP-501 infusion. CONCLUSIONS: ACP-501 has an acceptable safety profile after a single intravenous infusion. Lipid and lipoprotein changes indicate that recombinant human LCAT favorably alters HDL metabolism and support recombinant human LCAT use in future clinical trials in CHD and familial LCAT deficiency patients. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01554800.

Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive ß-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.

The Changing Face of HDL and the Best Way to Measure It.

BACKGROUND: HDL cholesterol (HDL-C) is a commonly used lipid biomarker for assessing cardiovascular health. While a central focus has been placed on the role of HDL in the reverse cholesterol transport (RCT) process, our appreciation for the other cardioprotective properties of HDL continues to expand with further investigation into the structure and function of HDL and its specific subfractions. The development of novel assays is empowering the research community to assess different aspects of HDL function, which at some point may evolve into new diagnostic tests. CONTENT: This review discusses our current understanding of the formation and maturation of HDL particles via RCT, as well as the newly recognized roles of HDL outside RCT. The antioxidative, antiinflammatory, antiapoptotic, antithrombotic, antiinfective, and vasoprotective effects of HDL are all discussed, as are the related methodologies for assessing these different aspects of HDL function. We elaborate on the importance of protein and lipid composition of HDL in health and disease and highlight potential new diagnostic assays based on these parameters. SUMMARY: Although multiple epidemiologic studies have confirmed that HDL-C is a strong negative risk marker for cardiovascular disease, several clinical and experimental studies have yielded inconsistent results on the direct role of HDL-C as an antiatherogenic factor. As of yet, our increased understanding of HDL biology has not been translated into successful new therapies, but will undoubtedly depend on the development of alternative ways for measuring HDL besides its cholesterol content.

High-density lipoprotein reduces inflammation from cholesterol crystals by inhibiting inflammasome activation.

Interleukin-1ß (IL-1ß), a potent pro-inflammatory cytokine, has been implicated in many diseases, including atherosclerosis. Activation of IL-1ß is controlled by a multi-protein complex, the inflammasome. The exact initiating event in atherosclerosis is unknown, but recent work has demonstrated that cholesterol crystals (CC) may promote atherosclerosis development by activation of the inflammasome. High-density lipoprotein (HDL) has consistently been shown to be anti-atherogenic and to have anti-inflammatory effects, but its mechanism of action is unclear. We demonstrate here that HDL is able to suppress IL-1ß secretion in response to cholesterol crystals in THP-1 cells and in human-monocyte-derived macrophages. HDL is able to blunt inflammatory monocyte cell recruitment in vivo following intraperitoneal CC injection in mice. HDL appears to modulate inflammasome activation in several ways. It reduces the loss of lysosomal membrane integrity following the phagocytosis of CC, but the major mechanism for the suppression of inflammasome activation by HDL is decreased expression of pro-IL-1ß and NLRP3, and reducing caspase-1 activation. In summary, we have described a novel anti-inflammatory effect of HDL, namely its ability to suppress inflammasome activation by CC by modulating the expression of several key components of the inflammasome.

Creation of Apolipoprotein C-II (ApoC-II) Mutant Mice and Correction of Their Hypertriglyceridemia with an ApoC-II Mimetic Peptide.

Apolipoprotein C-II (apoC-II) is a cofactor for lipoprotein lipase, a plasma enzyme that hydrolyzes triglycerides (TGs). ApoC-II deficiency in humans results in hypertriglyceridemia. We used zinc finger nucleases to create Apoc2 mutant mice to investigate the use of C-II-a, a short apoC-II mimetic peptide, as a therapy for apoC-II deficiency. Mutant mice produced a form of apoC-II with an uncleaved signal peptide that preferentially binds high-density lipoproteins (HDLs) due to a 3-amino acid deletion at the signal peptide cleavage site. Homozygous Apoc2 mutant mice had increased plasma TG (757.5 ± 281.2 mg/dl) and low HDL cholesterol (31.4 ± 14.7 mg/dl) compared with wild-type mice (TG, 55.9 ± 13.3 mg/dl; HDL cholesterol, 55.9 ± 14.3 mg/dl). TGs were found in light (density < 1.063 g/ml) lipoproteins in the size range of very-low-density lipoprotein and chylomicron remnants (40-200 nm). Intravenous injection of C-II-a (0.2, 1, and 5 µmol/kg) reduced plasma TG in a dose-dependent manner, with a maximum decrease of 90% occurring 30 minutes after the high dose. Plasma TG did not return to baseline until 48 hours later. Similar results were found with subcutaneous or intramuscular injections. Plasma half-life of C-II-a is 1.33 ± 0.72 hours, indicating that C-II-a only acutely activates lipolysis, and the sustained TG reduction is due to the relatively slow rate of new TG-rich lipoprotein synthesis. In summary, we describe a novel mouse model of apoC-II deficiency and show that an apoC-II mimetic peptide can reverse the hypertriglyceridemia in these mice, and thus could be a potential new therapy for apoC-II deficiency.

Scavenger receptor-BI is a receptor for lipoprotein(a).

Scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that binds a variety of lipoproteins, including high density lipoprotein (HDL) and low density lipoprotein (LDL), but lipoprotein(a) [Lp(a)] has not been investigated as a possible ligand. Stable cell lines (HEK293 and HeLa) expressing human SR-BI were incubated with protein- or lipid-labeled Lp(a) to investigate SR-BI-dependent Lp(a) cell association. SR-BI expression enhanced the association of both (125)I- and Alexa Fluor-labeled protein from Lp(a). By confocal microscopy, SR-BI was also found to promote the internalization of fluorescent lipids (BODIPY-cholesteryl ester (CE)- and DiI-labeled) from Lp(a), and by immunocytochemistry the cellular internalization of apolipoprotein(a) and apolipoprotein B. When dual-labeled ((3)H-cholesteryl ether,(125)I-protein) Lp(a) was added to cells expressing SR-BI, there was a greater relative increase in lipid uptake over protein, indicating that SR-BI mediates selective lipid uptake from Lp(a). Compared with C57BL/6 control mice, transgenic mice overexpressing human SR-BI in liver were found to have increased plasma clearance of (3)H-CE-Lp(a), whereas mouse scavenger receptor class B type I knockout (Sr-b1-KO) mice had decreased plasma clearance (fractional catabolic rate: 0.63 ± 0.08/day, 1.64 ± 0.62/day, and 4.64 ± 0.40/day for Sr-b1-KO, C57BL/6, and human scavenger receptor class B type I transgenic mice, respectively). We conclude that Lp(a) is a novel ligand for SR-BI and that SR-BI mediates selective uptake of Lp(a)-associated lipids.

CD49d Expression Identifies a Biologically Distinct Subtype of Chronic Lymphocytic Leukemia with Inferior Progression-Free Survival on BTK Inhibitor Therapy.

PURPOSE: To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: In patients treated with acalabrutinib (n = 48), CD49d expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients. RESULTS: In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4-mediated inside-out activation. Transcriptomes of CD49d+ and CD49d- cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK-STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d- CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d- CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d- were estimated progression-free at 8 years (P = 0.0004). CONCLUSIONS: CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression. See related commentary by Tissino et al., p. 3560.

Cell-free, high-density lipoprotein-specific phospholipid efflux assay predicts incident cardiovascular disease.

BACKGROUNDCellular cholesterol efflux capacity (CEC) is a better predictor of cardiovascular disease (CVD) events than HDL-cholesterol (HDL-C) but is not suitable as a routine clinical assay.METHODSWe developed an HDL-specific phospholipid efflux (HDL-SPE) assay to assess HDL functionality based on whole plasma HDL apolipoprotein-mediated solubilization of fluorescent phosphatidylethanolamine from artificial lipid donor particles. We first assessed the association of HDL-SPE with prevalent coronary artery disease (CAD): study I included NIH severe-CAD (n = 50) and non-CAD (n = 50) participants, who were frequency matched for sex, BMI, type 2 diabetes mellitus, and smoking; study II included Japanese CAD (n = 70) and non-CAD (n = 154) participants. We also examined the association of HDL-SPE with incident CVD events in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study comparing 340 patients with 340 controls individually matched for age, sex, smoking, and HDL-C levels.RESULTSReceiver operating characteristic curves revealed stronger associations of HDL-SPE with prevalent CAD. The AUCs in study I were as follows: HDL-SPE, 0.68; apolipoprotein A-I (apoA-I), 0.62; HDL-C, 0.63; and CEC, 0.52. The AUCs in study II were as follows: HDL-SPE, 0.83; apoA-I, 0.64; and HDL-C, 0.53. Also longitudinally, HDL-SPE was significantly associated with incident CVD events independent of traditional risk factors with ORs below 0.2 per SD increment in the PREVEND study (P < 0.001).CONCLUSIONHDL-SPE could serve as a routine clinical assay for improving CVD risk assessment and drug discovery.TRIAL REGISTRATIONClinicalTrials.gov NCT01621594.FUNDINGNHLBI Intramural Research Program, NIH (HL006095-06).

A novel loop-mediated isothermal amplification-based genotyping method and its application for identifying proprotein convertase subtilisin/kexin type 9 variants in familial hypercholesterolemia.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating low-density lipoprotein levels in plasma. While PCSK9 variants are causatively associated with familial hypercholesterolemia (FH), additional genotyping methods for FH targeting PCSK9 variants are required in a clinical setting. Loop-mediated isothermal amplification (LAMP) is a unique amplification method that amplifies a target gene under isothermal conditions (60-65 °C). However, a robust standardized method has not yet been established for LAMP-based genetic screening tests for genetic diseases, including FH. The present study aimed to develop a novel modification of the LAMP method designed to genotype single nucleotide variants (SNVs) and to apply it for the detection of PCSK9 variants. METHODS: Using short quenching probes (≤ 10 nucleotides) for the loop structures of LAMP amplicons, accurate detection of SNVs was verified separately for each allele, without any additional procedures, within 3 h. The diagnostic performance of this method in detecting PCSK9 variants was validated in FH patients. RESULTS: All PCSK9 variants tested via conventional sequencing in FH patients were successfully detected using this novel LAMP method. CONCLUSIONS: We developed a LAMP-based genotyping method to detect PCSK9 variants in FH. Compared to conventional sequencing, our genotyping method is relatively convenient and time-efficient and is suitable for the screening of FH in clinical settings. Future studies on various genes are also warranted.

A Simple Fluorescent Cholesterol Labeling Method to Cryoprotect and Detect Plasma Lipoprotein-X.

Lipoprotein-X (LpX) are abnormal nephrotoxic lipoprotein particles enriched in free cholesterol and phospholipids. LpX with distinctive lipid compositions are formed in patients afflicted with either familial LCAT deficiency (FLD) or biliary cholestasis. LpX is difficult to detect by standard lipid stains due to the absence of a neutral lipid core and because it is unstable upon storage, particularly when frozen. We have recently reported that free cholesterol-specific filipin staining after agarose gel electrophoresis sensitively detects LpX in fresh human plasma. Herein, we describe an even more simplified qualitative method to detect LpX in both fresh and frozen-thawed human FLD or cholestatic plasma. Fluorescent cholesterol complexed to fatty-acid-free BSA was used to label LpX and was added together with trehalose in order to cryopreserve plasma LpX. The fluorescent cholesterol bound to LpX was observed with high sensitivity after separation from other lipoproteins by agarose gel electrophoresis. This methodology can be readily developed into a simple assay for the clinical diagnosis of FLD and biliary liver disease and to monitor the efficacy of treatments intended to reduce plasma LpX in these disease states.

Interleukin 10 promotes macrophage uptake of HDL and LDL by stimulating fluid-phase endocytosis.

OBJECTIVE: Highly elevated plasma levels of interleukin-10 (IL-10) are causally associated with "Disappearing HDL Syndrome" and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages. METHODS AND RESULTS: Macrophages (RAW264.7, Kupffer and human) were incubated with vehicle (PBS) or IL-10 (20 ng/ml) for 7 days. Uptake of HDL, LDL, and/or fluid-phase endocytosis probes (albumin-Alexa680®, 70 kDa FITC-Dextran and Lucifer Yellow, LY) was evaluated by FACS. Intracellular cofilin and phosphorylated cofilin (p-cofilin) levels were determined by immunoblotting. Macrophage uptake of lipoproteins and probes was non-saturable and increased after IL-10 incubation (p < 0.0001). Furthermore, pre-incubation with fluid-phase endocytosis inhibitors (LY294002, Latrunculin A, and Amiloride) significantly reduced uptake (p < 0.05). IL-10 increased the cofilin/p-cofilin ratio (p = 0.021), signifying increased cofilin activation and hence filamentous actin. Consistently, phalloidin staining revealed increased filamentous actin in macrophages after IL-10 treatment (p = 0.0018). Finally, RNA-seq analysis demonstrated enrichment of gene sets related to actin filament dynamics, membrane ruffle formation and endocytosis in IL-10-treated macrophages (p < 0.05). IL-10 did not alter mRNA levels of Ldlr, Vldlr, Scarb1, Cd36 or Lrp1. In primary human monocyte-derived macrophages and murine Kupffer cells, IL-10 incubation also increased uptake of lipoproteins, albumin and LY (p < 0.01). CONCLUSIONS: Interleukin-10 induces the uptake of HDL and LDL by fluid-phase endocytosis by increasing actin-filament rearrangement in macrophages, thus providing a plausible mechanism contributing to "Disappearing HDL Syndrome".

LCAT protects against Lipoprotein-X formation in a murine model of drug-induced intrahepatic cholestasis.

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by low HDL-C levels, low plasma cholesterol esterification, and the formation of Lipoprotein-X (Lp-X), an abnormal cholesterol-rich lipoprotein particle. LCAT deficiency causes corneal opacities, normochromic normocytic anemia, and progressive renal disease due to Lp-X deposition in the glomeruli. Recombinant LCAT is being investigated as a potential therapy for this disorder. Several hepatic disorders, namely primary biliary cirrhosis, primary sclerosing cholangitis, cholestatic liver disease, and chronic alcoholism also develop Lp-X, which may contribute to the complications of these disorders. We aimed to test the hypothesis that an increase in plasma LCAT could prevent the formation of Lp-X in other diseases besides FLD. We generated a murine model of intrahepatic cholestasis in LCAT-deficient (KO), wild type (WT), and LCAT-transgenic (Tg) mice by gavaging mice with alpha-naphthylisothiocyanate (ANIT), a drug well known to induce intrahepatic cholestasis. Three days after the treatment, all mice developed hyperbilirubinemia and elevated liver function markers (ALT, AST, Alkaline Phosphatase). The presence of high levels of LCAT in the LCAT-Tg mice, however, prevented the formation of Lp-X and other plasma lipid abnormalities in WT and LCAT-KO mice. In addition, we demonstrated that multiple injections of recombinant human LCAT can prevent significant accumulation of Lp-X after ANIT treatment in WT mice. In summary, LCAT can protect against the formation of Lp-X in a murine model of cholestasis and thus recombinant LCAT could be a potential therapy to prevent the formation of Lp-X in other diseases besides FLD.

A dual apolipoprotein C-II mimetic-apolipoprotein C-III antagonist peptide lowers plasma triglycerides.

Recent genetic studies have established that hypertriglyceridemia (HTG) is causally related to cardiovascular disease, making it an active area for drug development. We describe a strategy for lowering triglycerides (TGs) with an apolipoprotein C-II (apoC-II) mimetic peptide called D6PV that activates lipoprotein lipase (LPL), the main plasma TG-hydrolyzing enzyme, and antagonizes the TG-raising effect of apoC-III. The design of D6PV was motivated by a combination of all-atom molecular dynamics simulation of apoC-II on the Anton 2 supercomputer, structural prediction programs, and biophysical techniques. Efficacy of D6PV was assessed ex vivo in human HTG plasma and was found to be more potent than full-length apoC-II in activating LPL. D6PV markedly lowered TG by more than 80% within a few hours in both apoC-II-deficient mice and hAPOC3-transgenic (Tg) mice. In hAPOC3-Tg mice, D6PV treatment reduced plasma apoC-III by 80% and apoB by 65%. Furthermore, low-density lipoprotein (LDL) cholesterol did not accumulate but rather was decreased by 10% when hAPOC3-Tg mice lacking the LDL-receptor (hAPOC3-Tg × Ldlr-/- ) were treated with the peptide. D6PV lowered TG by 50% in whole-body inducible Lpl knockout (iLpl-/- ) mice, confirming that it can also act independently of LPL. D6PV displayed good subcutaneous bioavailability of about 80% in nonhuman primates. Because it binds to high-density lipoproteins, which serve as a long-term reservoir, it also has an extended terminal half-life (42 to 50 hours) in nonhuman primates. In summary, D6PV decreases plasma TG by acting as a dual apoC-II mimetic and apoC-III antagonist, thereby demonstrating its potential as a treatment for HTG.

DENND5B Regulates Intestinal Triglyceride Absorption and Body Mass.

Regulation of lipid absorption by enterocytes can influence metabolic status in humans and contribute to obesity and related complications. The intracellular steps of chylomicron biogenesis and transport from the Endoplasmic Reticulum (ER) to the Golgi complex have been described, but the mechanisms for post-Golgi transport and secretion of chylomicrons have not been identified. Using a newly generated Dennd5b-/- mouse, we demonstrate an essential role for this gene in Golgi to plasma membrane transport of chylomicron secretory vesicles. In mice, loss of Dennd5b results in resistance to western diet induced obesity, changes in plasma lipids, and reduced aortic atherosclerosis. In humans, two independent exome sequencing studies reveal that a common DENND5B variant, p.(R52K), is correlated with body mass index. These studies establish an important role for DENND5B in post-Golgi chylomicron secretion and a subsequent influence on body composition and peripheral lipoprotein metabolism.