Apolipoprotein C-II Mimetic Peptide Promotes the Plasma Clearance of Triglyceride-Rich Lipid Emulsion and the Incorporation of Fatty Acids into Peripheral Tissues of Mice.

AIM: Plasma apolipoprotein C-II (apoC-II) activates lipoprotein lipase (LPL) and thus lowers plasma triglycerides (TG). We previously reported that a human apoC-II mimetic peptide (C-II-a) decreased plasma TG in apoC-II mutant mice, as well as in apoE-knockout mice. Because it is unknown what tissues take up free fatty acids (FFAs) released from TG after C-II-a peptide administration, we investigated in mice TG plasma clearance and tissue incorporation, using 3H-triolein as a tracer, with and without C-II-a treatment. METHODS AND RESULTS: Intralipid® fat emulsion was labeled with 3H-triolein and then mixed with or without C-II-a. Addition of the peptide did not alter mean particle size of the lipid emulsion particles (298 nm) but accelerated their plasma clearance. After intravenous injection into C57BL/6N mice, the plasma half-life of the 3H-triolein for control and C-II-a treated emulsions was 18.3 ± 2.2 min and 14.8 ± 0.1 min, respectively. In apoC-II mutant mice, the plasma half-life of 3H-triolein for injected control and C-II-a treated emulsions was 30.1 ± 0.1 min and 14.8 ± 0.1 min, respectively. C57BL/6N and apoC-II mutant mice at 120 minutes after the injection showed increased tissue incorporation of radioactivity in white adipose tissue when C-II-a treated emulsion was used. Higher radiolabeled uptake of lipids from C-II-a treated emulsion was also observed in the skeletal muscle of C57BL/6N mice only. In case of apoC-II mutant mice, decreased uptake of radioactive lipids was observed in the liver and kidney after addition of C-II-a to the lipid emulsion. CONCLUSIONS: C-II-a peptide promotes the plasma clearance of TG-rich lipid emulsions in wild type and apoC-II mutant mice and promotes the incorporation of fatty acids from TG in the lipid emulsions into specific peripheral tissues.

Development of a novel fluorescent activity assay for lecithin:cholesterol acyltransferase.

Background Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol. Recombinant human LCAT (rhLCAT) is now being developed as an enzyme replacement therapy for familial LCAT deficiency and as a possible treatment for acute coronary syndrome. The current 'gold standard' assay for LCAT activity involves the use of radioisotopes, thus making it difficult for routine clinical use. Methods We have developed a novel and more convenient LCAT activity assay using fluorescence-labelled cholesterol (BODIPY-cholesterol), which is incorporated into proteoliposomes as a substrate instead of radiolabelled cholesterol. Results The apparent Km and Vmax were 31.5 µmol/L and 55.8 nmol/h/nmoL, rhLCAT, respectively, for the 3H-cholesterol method and 103.1 µmol/L and 13.4 nmol/h/nmol rhLCAT, respectively, for the BODIPY-cholesterol method. Although the two assays differed in their absolute units of LCAT activity, there was a good correlation between the two test assays ( r = 0.849, P < 1.6 × 10-7, y = 0.1378x + 1.106). The BODIPY-cholesterol assay had an intra-assay CV of 13.7%, which was superior to the intra-assay CV of 20.8% for the radioisotopic assay. The proteoliposome substrate made with BODIPY-cholesterol was stable to storage for at least 10 months. The reference range ( n = 20) for the fluorescent LCAT activity assay was 4.6-24.1 U/mL/h in healthy subjects. Conclusions In summary, a novel fluorescent LCAT activity assay that utilizes BODIPY-cholesterol as a substrate is described that yields comparable results to the radioisotopic method.

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.

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.

Lipoprotein X Causes Renal Disease in LCAT Deficiency.

Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.

A Short Synthetic Peptide Mimetic of Apolipoprotein A1 Mediates Cholesterol and Globotriaosylceramide Efflux from Fabry Fibroblasts.

Fabry disease is an X-linked sphingolipid storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (AGA, EC 3.2.1.22) resulting in the intracellular accumulation of globotriaosylceramide (Gb3). We found that Gb3 storage also correlates with accumulation of endosomal-lysosomal cholesterol in Fabry fibroblasts. This cholesterol accumulation may contribute to the phenotypic pathology of Fabry disease by slowing endosomal-lysosomal trafficking. We found that LDL receptor expression is not downregulated in Fabry fibroblasts resulting in accumulation of both cholesterol and Gb3. 5A-Palmitoyl oleoyl-phosphatidylcholine (5AP) is a phospholipid complex containing a short synthetic peptide that mimics apolipoprotein A1, the main protein component of high-density lipoprotein (HDL) that mediates the efflux of cholesterol from cells via the ATP-binding cassette transporter. We used 5AP and HDL to remove cholesterol from Fabry fibroblasts to examine the fate of accumulated cellular Gb3. Using immunostaining techniques, we found that 5AP is highly effective for depleting cholesterol and Gb3 in these cells. 5AP restores the ApoA-1-mediated cholesterol efflux leading to mobilization of cholesterol and reduction of Gb3 in Fabry fibroblasts.

Endothelial Expression of Scavenger Receptor Class B, Type I Protects against Development of Atherosclerosis in Mice.

The role of scavenger receptor class B, type I (SR-BI) in endothelial cells (EC) was examined in several novel transgenic mouse models expressing SR-BI in endothelium of mice with normal C57Bl6/N, apoE-KO, or Scarb1-KO backgrounds. Mice were also created expressing SR-BI exclusively in endothelium and liver. Endothelial expression of the Tie2-Scarb1 transgene had no significant effect on plasma lipoprotein levels in mice on a normal chow diet but on an atherogenic diet, significantly decreased plasma cholesterol levels, increased plasma HDL cholesterol (HDL-C) levels, and protected mice against atherosclerosis. In 8-month-old apoE-KO mice fed a normal chow diet, the Tie2-Scarb1 transgene decreased aortic lesions by 24%. Mice expressing SR-BI only in EC and liver had a 1.5 ± 0.1-fold increase in plasma cholesterol compared to mice synthesizing SR-BI only in liver. This elevation was due mostly to increased HDL-C. In EC culture studies, SR-BI was found to be present in both basolateral and apical membranes but greater cellular uptake of cholesterol from HDL was found in the basolateral compartment. In summary, enhanced expression of SR-BI in EC resulted in a less atherogenic lipoprotein profile and decreased atherosclerosis, suggesting a possible role for endothelial SR-BI in the flux of cholesterol across EC.

Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice.

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(-/-)] mice, which have a secondary defect in cholesterol esterification. Scarab(-/-)×LCAT-null [Lcat(-/-)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(-/-)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(-/-)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(-/-)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(-/-) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.

Elevated interleukin-10: a new cause of dyslipidemia leading to severe HDL deficiency.

BACKGROUND: Low high-density lipoprotein cholesterol (HDL-C) is a risk factor for coronary artery disease. Investigating mechanisms underlying acquired severe HDL deficiency in noncritically ill patients ("disappearing HDL syndrome") could provide new insights into HDL metabolism. OBJECTIVE: To determine the cause of low HDL-C in patients with severe acquired HDL deficiency. METHODS AND RESULTS: Patients with intravascular large B-cell lymphoma (n = 2), diffuse large B-cell lymphoma (n = 1), and autoimmune lymphoproliferative syndrome (n = 1) presenting with markedly decreased HDL-C, low low-density lipoprotein cholesterol (LDL-C), and elevated triglycerides were identified. The abnormal lipoprotein profile returned to normal after therapy in all 4 patients. All patients were found to have markedly elevated serum interleukin-10 (IL-10) levels that also normalized after therapy. In a cohort of autoimmune lymphoproliferative syndrome patients (n = 93), IL-10 showed a strong inverse correlation with HDL-C (R(2) = 0.3720, P < .0001). A direct causal role for increased serum IL-10 in inducing the observed changes in lipoproteins was established in a randomized, placebo-controlled clinical trial of recombinant human IL-10 in psoriatic arthritis patients (n = 18). Within a week of initiating subcutaneous recombinant human IL-10 injections, HDL-C precipitously decreased to near-undetectable levels. LDL-C also decreased by more than 50% (P < .0001) and triglycerides increased by approximately 2-fold (P < .005). All values returned to baseline after discontinuing IL-10 therapy. CONCLUSION: Increased IL-10 causes severe HDL-C deficiency, low LDL-C, and elevated triglycerides. IL-10 is thus a potent modulator of lipoprotein levels, a potential new biomarker for B-cell disorders, and a novel cause of disappearing HDL syndrome.

LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice.

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer's disease, including facilitating ß-amyloid (Aß) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aß clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aß or amyloid in APP/PS1 LCAT(-/-) mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer's-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aß clearance.

Endothelial expression of human ABCA1 in mice increases plasma HDL cholesterol and reduces diet-induced atherosclerosis.

The role of endothelial ABCA1 expression in reverse cholesterol transport (RCT) was examined in transgenic mice, using the endothelial-specific Tie2 promoter. Human ABCA1 (hABCA1) was significantly expressed in endothelial cells (EC) of most tissues except the liver. Increased expression of ABCA1 was not observed in resident peritoneal macrophages. ApoA-I-mediated cholesterol efflux from aortic EC was 2.6-fold higher (P < 0.0001) for cells from transgenic versus control mice. On normal chow diet, Tie2 hABCA1 transgenic mice had a 25% (P < 0.0001) increase in HDL-cholesterol (HDL-C) and more than a 2-fold increase of eNOS mRNA in the aorta (P < 0.04). After 6 months on a high-fat, high-cholesterol (HFHC) diet, transgenic mice compared with controls had a 40% increase in plasma HDL-C (P < 0.003) and close to 40% decrease in aortic lesions (P < 0.02). Aortas from HFHC-fed transgenic mice also showed gene expression changes consistent with decreased inflammation and apoptosis. Beneficial effects of the ABCA1 transgene on HDL-C levels or on atherosclerosis were absent when the transgene was transferred onto ApoE or Abca1 knockout mice. In summary, expression of hABCA1 in EC appears to play a role in decreasing diet-induced atherosclerosis in mice and is associated with increased plasma HDL-C levels and beneficial gene expression changes in EC.

5A apolipoprotein mimetic peptide promotes cholesterol efflux and reduces atherosclerosis in mice.

Intravenous administration of apolipoprotein (apo) A-I complexed with phospholipid has been shown to rapidly reduce plaque size in both animal models and humans. Short synthetic amphipathic peptides can mimic the antiatherogenic properties of apoA-I and have been proposed as alternative therapeutic agents. In this study, we investigated the atheroprotective effect of the 5A peptide, a bihelical amphipathic peptide that specifically effluxes cholesterol from cells by ATP-binding cassette transporter 1 (ABCA1). 5A stimulated a 3.5-fold increase in ABCA1-mediated efflux from cells and an additional 2.5-fold increase after complexing it with phospholipid (1:7 mol/mol). 5A-palmitoyl oleoyl phosphatidyl choline (POPC), but not free 5A, was also found to promote cholesterol efflux by ABCG1. When incubated with human serum, 5A-POPC bound primarily to high-density lipoprotein (HDL) but also to low-density lipoprotein (LDL) and promoted the transfer of cholesterol from LDL to HDL. Twenty-four hours after intravenous injection of 5A-POPC (30 mg/kg) into apoE-knockout (KO) mice, both the cholesterol (181%) and phospholipid (219%) content of HDL significantly increased. By an in vivo cholesterol isotope dilution study and monitoring of the flux of cholesterol from radiolabeled macrophages to stool, 5A-POPC treatment was observed to increase reverse cholesterol transport. In three separate studies, 5A when complexed with various phospholipids reduced aortic plaque surface area by 29 to 53% (n = 8 per group; p < 0.02) in apoE-KO mice. No signs of toxicity from the treatment were observed during these studies. In summary, 5A promotes cholesterol efflux both in vitro and in vivo and reduces atherosclerosis in apoE-KO mice, indicating that it may be a useful alternative to apoA-I for HDL therapy.

Immunoassay interference by a commonly used blood collection tube additive, the organosilicone surfactant silwet L-720.

BACKGROUND: A small number of immunoassays on several different types of analyzers were recently adversely affected by tube additives in Becton Dickinson (BD) Vacutainer SST, SST II, and Microtainer blood collection tubes. We examined the effect of a commonly used tube surfactant, Silwet L-720, on immunoassays and the mechanism for the interference. METHODS: Immunoassays were performed on serum supplemented with Silwet L-720 on the IMMULITE 2500 and AxSYM analyzers. Direct effects of the surfactant on the chemiluminescent detection step of immunoassays and on antibody immobilization on the solid phase were examined. RESULTS: Increasing the final surfactant concentration from 0 to 400 mg/L in serum significantly increased (approximately 51%) the apparent total triiodothyronine (TT3) concentrations measured on the IMMULITE 2500 but not the AxSYM analyzer. Several other competitive, but not noncompetitive, assays were also significantly affected by the surfactant on the IMMULITE 2500 analyzer. The effect was independent of serum components, and the surfactant had no direct effect on chemiluminescence reactions. The capture antibody, however, was displaced from the solid phase by incubation with solutions containing surfactant under conditions similar to the IMMULITE TT3 assay. CONCLUSIONS: The Silwet L-720 surfactant, which is used to coat the inner surfaces of tubes, appears to account for previously reported immunoassay interference by BD Vacutainer SST blood collection tubes. One of the mechanisms for the interference is the desorption of antibodies from the solid phase by the surfactant. The results identify an important factor in the selection of suitable blood collection tube surfactants and provide an approach for solving similar tube-assay interference problems in the future.

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.

Low-density lipoprotein receptor and apolipoprotein A-I and B expression in human enterocytes.

Low-density lipoprotein receptor (LDL-R) was found to be expressed in human small intestine epithelial cells, enterocytes. The relative abundance of LDL-R mRNA and protein was compared with that of apolipoproteins A-I (apoA-I) and B (apoB) in enterocytes and two other cell types: CaCo-2 and HepG2. The LDL-R mRNA content was comparable in three cell types. Human enterocytes expressed 5.2- to 14-fold more apoA-I mRNA than the other cells. In contrast, HepG2 cells expressed 10-to 19-fold more apoB mRNA than CaCo-2 cells and human enterocytes. Immunoprecipitation of [(35)S]methionine pulse-labeled intracellular proteins from these cell types demonstrated that human enterocytes synthesize more apoA-I and apoB, while HepG2 cells synthesize a slightly higher amount of LDL-R.