Effect of niacin monotherapy on high density lipoprotein composition and function.

BACKGROUND: Niacin has modest but overall favorable effects on plasma lipids by increasing high density lipoprotein cholesterol (HDL-C) and lowering triglycerides. Clinical trials, however, evaluating niacin therapy for prevention of cardiovascular outcomes have returned mixed results. Recent evidence suggests that the HDL proteome may be a better indicator of HDL's cardioprotective function than HDL-C. The objective of this study was to evaluate the effect of niacin monotherapy on HDL protein composition and function. METHODS: A 20-week investigational study was performed with 11 participants receiving extended-release niacin (target dose = 2 g/day) for 16-weeks followed by a 4-week washout period. HDL was isolated from participants at weeks: 0, 16, and 20. The HDL proteome was analyzed at each time point by mass spectrometry and relative protein quantification was performed by label-free precursor ion intensity measurement. RESULTS: In this cohort, niacin therapy had typical effects on routine clinical lipids (HDL-C + 16%, q < 0.01; LDL-C - 20%, q < 0.01; and triglyceride - 15%, q = 0.1). HDL proteomics revealed significant effects of niacin on 5 proteins: serum amyloid A (SAA), angiotensinogen (AGT), apolipoprotein A-II (APOA2), clusterin (CLUS), and apolipoprotein L1 (APOL1). SAA was the most prominently affected protein, increasing 3-fold in response to niacin (q = 0.008). Cholesterol efflux capacity was not significantly affected by niacin compared to baseline, however, stopping niacin resulted in a 9% increase in efflux (q < 0.05). Niacin did not impact HDL's ability to influence endothelial function. CONCLUSION: Extended-release niacin therapy, in the absence of other lipid-modifying medications, can increase HDL-associated SAA, an acute phase protein associated with HDL dysfunction.

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.

Human SR-BI and SR-BII Potentiate Lipopolysaccharide-Induced Inflammation and Acute Liver and Kidney Injury in Mice.

The class B scavenger receptors BI (SR-BI) and BII (SR-BII) are high-density lipoprotein receptors that recognize various pathogens, including bacteria and their products. It has been reported that SR-BI/II null mice are more sensitive than normal mice to endotoxin-induced inflammation and sepsis. Because the SR-BI/II knockout model demonstrates multiple immune and metabolic disorders, we investigated the role of each receptor in the LPS-induced inflammatory response and tissue damage using transgenic mice with pLiv-11-directed expression of human SR-BI (hSR-BI) or human SR-BII (hSR-BII). At 6 h after i.p. LPS injection, transgenic hSR-BI and hSR-BII mice demonstrated markedly higher serum levels of proinflammatory cytokines and 2- to 3-fold increased expression levels of inflammatory mediators in the liver and kidney, compared with wild-type (WT) mice. LPS-stimulated inducible NO synthase expression was 3- to 6-fold higher in the liver and kidney of both transgenic strains, although serum NO levels were similar in all mice. Despite the lower high-density lipoprotein plasma levels, both transgenic strains responded to LPS by a 5-fold increase of plasma corticosterone levels, which were only moderately lower than in WT animals. LPS treatment resulted in MAPK activation in tissues of all mice; however, the strongest response was detected for hepatic extracellular signal-regulated protein kinase 1 and 2 and kidney JNK of both transgenic mice. Histological examination of hepatic and renal tissue from LPS-challenged mice revealed more injury in hSR-BII, but not hSR-BI, transgenic mice versus WT controls. Our findings demonstrate that hSR-BII, and to a lesser extent hSR-BI, significantly increase LPS-induced inflammation and contribute to LPS-induced tissue injury in the liver and kidney, two major organs susceptible to LPS toxicity.

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.

Randomized double blind clinical trial on the effect of oral α-cyclodextrin on serum lipids.

BACKGROUND: This single center, double-blinded, cross-over, placebo controlled clinical trial investigated the effect of oral α-cyclodextrin (α-CD), a soluble dietary fiber, on blood lipid and lipoprotein levels in healthy human subjects. α-CD, a cyclical polymer containing 6 glucose subunits, is currently sold as an over the counter food supplement and is also a common additive in many foods. α-CD forms a hydrophobic central cavity that binds lipids and has been shown in animal studies and in previous clinical trials to alter plasma lipid levels. METHODS: We screened for healthy subjects, males and females, between ages 18 to 75. Out of total 103 subjects interviewed, 75 subjects completed the study. Qualified individuals in each gender group were randomized into two groups in terms of which treatment arm they received first (placebo vs. α-CD, receiving 6 grams P.O. a day, for 12-14 weeks with a 7 day wash out between arms). The primary outcome variable, plasma total cholesterol, as well as other tests related to lipids and lipoprotein and glucose metabolism, were measured at baseline and at the end of each arm of the study. RESULTS: α-CD was well tolerated; no serious adverse events related to α-CD were observed. Approximately 8 % of the subjects on α-CD complained of minor gastrointestinal symptoms versus 3 % on placebo (p = 0.2). Small-LDL particle number decreased 10 % (p < 0.045) for subjects on α-CD versus placebo. Fasting plasma glucose (1.6 %, p < 0.05) and Insulin resistance index (11 %, p < 0.04) were also decreased when on α-CD versus placebo. CONCLUSION: α-CD treatment appears to be safe and well tolerated in healthy individuals and showed a modest reduction in small LDL particles, and an improvement in glucose related parameters. TRIAL REGISTRATION: NCT01131299.

A novel apolipoprotein C-II mimetic peptide that activates lipoprotein lipase and decreases serum triglycerides in apolipoprotein E-knockout mice.

Apolipoprotein A-I (apoA-I) mimetic peptides are currently being developed as possible new agents for the treatment of cardiovascular disease based on their ability to promote cholesterol efflux and their other beneficial antiatherogenic properties. Many of these peptides, however, have been reported to cause transient hypertriglyceridemia due to inhibition of lipolysis by lipoprotein lipase (LPL). We describe a novel bihelical amphipathic peptide (C-II-a) that contains an amphipathic helix (18A) for binding to lipoproteins and stimulating cholesterol efflux as well as a motif based on the last helix of apolipoprotein C-II (apoC-II) that activates lipolysis by LPL. The C-II-a peptide promoted cholesterol efflux from ATP-binding cassette transporter ABCA1-transfected BHK cells similar to apoA-I mimetic peptides. Furthermore, it was shown in vitro to be comparable to the full-length apoC-II protein in activating lipolysis by LPL. When added to serum from a patient with apoC-II deficiency, it restored normal levels of LPL-induced lipolysis and also enhanced lipolysis in serum from patients with type IV and V hypertriglyceridemia. Intravenous injection of C-II-a (30 mg/kg) in apolipoprotein E-knockout mice resulted in a significant reduction of plasma cholesterol and triglycerides of 38 ± 6% and 85 ± 7%, respectively, at 4 hours. When coinjected with the 5A peptide (60 mg/kg), the C-II-a (30 mg/kg) peptide was found to completely block the hypertriglyceridemic effect of the 5A peptide in C57Bl/6 mice. In summary, C-II-a is a novel peptide based on apoC-II, which promotes cholesterol efflux and lipolysis and may therefore be useful for the treatment of apoC-II deficiency and other forms of hypertriglyceridemia.

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.

Hydrophobic amino acids in the hinge region of the 5A apolipoprotein mimetic peptide are essential for promoting cholesterol efflux by the ABCA1 transporter.

The bihelical apolipoprotein mimetic peptide 5A effluxes cholesterol from cells and reduces inflammation and atherosclerosis in animal models. We investigated how hydrophobic residues in the hinge region between the two helices are important in the structure and function of this peptide. By simulated annealing analysis and molecular dynamics modeling, two hydrophobic amino acids, F-18 and W-21, in the hinge region were predicted to be relatively surface-exposed and to interact with the aqueous solvent. Using a series of 5A peptide analogs in which F-18 or W-21 was changed to either F, W, A, or E, only peptides with hydrophobic amino acids in these two positions were able to readily bind and solubilize phospholipid vesicles. Compared with active peptides containing F or W, peptides containing E in either of these two positions were more than 10-fold less effective in effluxing cholesterol by the ABCA1 transporter. Intravenous injection of 5A in C57BL/6 mice increased plasma-free cholesterol (5A: 89.9 ± 13.6 mg/dl; control: 38.7 ± 4.3 mg/dl (mean ± S.D.); P < 0.05) and triglycerides (5A: 887.0 ± 172.0 mg/dl; control: 108.9 ± 9.9 mg/dl; P < 0.05), whereas the EE peptide containing E in both positions had no effect. Finally, 5A increased cholesterol efflux approximately 2.5-fold in vivo from radiolabeled macrophages, whereas the EE peptide was inactive. These results provide a rationale for future design of therapeutic apolipoprotein mimetic peptides and provide new insights into the interaction of hydrophobic residues on apolipoproteins with phospholipids in the lipid microdomain created by the ABCA1 transporter during the cholesterol efflux process.

Zinc finger protein tristetraprolin interacts with CCL3 mRNA and regulates tissue inflammation.

Zinc finger protein tristetraprolin (TTP) modulates macrophage inflammatory activity by destabilizing cytokine mRNAs. In this study, through a screen of TTP-bound mRNAs in activated human macrophages, we have identified CCL3 mRNA as the most abundantly bound TTP target mRNA and have characterized this interaction via conserved AU-rich elements. Compared to the wild-type cells, TTP(-/-) macrophages produced higher levels of LPS-induced CCL3. In addition, the plasma level of CCL3 in TTP(-/-) mice was markedly higher than that in wild-type mice. To determine the in vivo significance of TTP-regulated CCL3, we generated CCL3(-/-)TTP(-/-) double-knockout mice. Along with decreased proinflammatory cytokines in their paw joints, there were significant functional and histologic improvements in the inflammatory arthritis of TTP(-/-) mice when CCL3 was absent, although cachexia, reflecting systemic inflammation, was notably unaffected. Furthermore, the marked exacerbation of aortic plaque formation caused by TTP deficiency in the APOE(-/-) mouse model of atherosclerosis was also rescued by disrupting CCL3. Taken together, our data indicate that the interaction between TTP and CCL3 mRNA plays an important role in modulating localized inflammatory processes in tissues that are dissociated from the systemic manifestations of chronic inflammation.

5A, an apolipoprotein A-I mimetic peptide, attenuates the induction of house dust mite-induced asthma.

New treatment approaches are needed for patients with asthma. Apolipoprotein A-I (apoA-I), the major structural protein of high-density lipoproteins, mediates reverse cholesterol transport and has atheroprotective and anti-inflammatory effects. In this study, we hypothesized that an apoA-I mimetic peptide might be effective at inhibiting asthmatic airway inflammation. A 5A peptide, which is a synthetic, bihelical apoA-I mimetic, was administered to wild-type A/J mice via osmotic mini-pump prior to the induction of house dust mite (HDM)-induced asthma. HDM-challenged mice that received the 5A apoA-I mimetic peptide had significant reductions in the number of bronchoalveolar lavage fluid eosinophils, lymphocytes, and neutrophils, as well as in histopathological evidence of airway inflammation. The reduction in airway inflammation was mediated by a reduction in the expression of Th2- and Th17-type cytokines, as well as in chemokines that promote T cell and eosinophil chemotaxis, including CCL7, CCL17, CCL11, and CCL24. Furthermore, the 5A apoA-I mimetic peptide inhibited the alternative activation of pulmonary macrophages in the lungs of HDM-challenged mice. It also abrogated the development of airway hyperresponsiveness and reduced several key features of airway remodeling, including goblet cell hyperplasia and the expression of collagen genes (Col1a1 and Col3a1). Our results demonstrate that the 5A apoA-I mimetic peptide attenuates the development of airway inflammation and airway hyperresponsiveness in an experimental murine model of HDM-induced asthma. These data support the conclusion that strategies using apoA-I mimetic peptides, such as 5A, might be developed further as a possible new treatment approach for asthma.

Sphingosine 1-phosphate lyase deficiency disrupts lipid homeostasis in liver.

The cleavage of sphingoid base phosphates by sphingosine-1-phosphate (S1P) lyase to produce phosphoethanolamine and a fatty aldehyde is the final degradative step in the sphingolipid metabolic pathway. We have studied mice with an inactive S1P lyase gene and have found that, in addition to the expected increase of sphingoid base phosphates, other sphingolipids (including sphingosine, ceramide, and sphingomyelin) were substantially elevated in the serum and/or liver of these mice. This latter increase is consistent with a reutilization of the sphingosine backbone for sphingolipid synthesis due to its inability to exit the sphingolipid metabolic pathway. Furthermore, the S1P lyase deficiency resulted in changes in the levels of serum and liver lipids not directly within the sphingolipid pathway, including phospholipids, triacyglycerol, diacylglycerol, and cholesterol. Even though lipids in serum and lipid storage were elevated in liver, adiposity was reduced in the S1P lyase-deficient mice. Microarray analysis of lipid metabolism genes in liver showed that the S1P lyase deficiency caused widespread changes in their expression pattern, with a significant increase in the expression of PPARgamma, a master transcriptional regulator of lipid metabolism. However, the mRNA expression of the genes encoding the sphingosine kinases and S1P phosphatases, which directly control the levels of S1P, were not significantly changed in liver of the S1P lyase-deficient mice. These results demonstrate that S1P lyase is a key regulator of the levels of multiple sphingolipid substrates and reveal functional links between the sphingolipid metabolic pathway and other lipid metabolic pathways that may be mediated by shared lipid substrates and changes in gene expression programs. The disturbance of lipid homeostasis by altered sphingolipid levels may be relevant to metabolic diseases.

Apolipoprotein E negatively regulates house dust mite-induced asthma via a low-density lipoprotein receptor-mediated pathway.

RATIONALE: Distinct sets of corticosteroid-unresponsive genes modulate disease severity in asthma. OBJECTIVES: To identify corticosteroid-unresponsive genes that provide new insights into disease pathogenesis and asthma therapeutics. METHODS: Experimental murine asthma was induced by nasal administration of house dust mite for 5 days per week. Dexamethasone and apolipoprotein E (apo E) mimetic peptides were administered via osmotic minipumps. MEASUREMENTS AND MAIN RESULTS: Genome-wide expression profiling of the lung transcriptome in a house dust mite-induced model of murine asthma identified increases in apo E mRNA levels that persisted despite corticosteroid treatment. House dust mite-challenged apo E⁻(/)⁻ mice displayed enhanced airway hyperreactivity and goblet cell hyperplasia, which could be rescued by administration of an apo E(130-149) mimetic peptide. Administration of the apo E(130-149) mimetic peptide to house dust mite-challenged apo E⁻(/)⁻ mice also inhibited eosinophilic airway inflammation, IgE production, and the expression of Th2 and Th17 cytokines. House dust mite-challenged low-density lipoprotein receptor (LDLR) knockout mice displayed a similar phenotype as apo E⁻(/)⁻ mice with enhanced airway hyperreactivity, goblet cell hyperplasia, and mucin gene expression, but could not be rescued by the apo E(130-149) mimetic peptide, consistent with a LDLR-dependent mechanism. CONCLUSIONS: These findings for the first time identify an apo E-LDLR pathway as an endogenous negative regulator of airway hyperreactivity and goblet cell hyperplasia in asthma. Furthermore, our results demonstrate that strategies that activate the apo E-LDLR pathway, such as apo E mimetic peptides, might be developed into a novel treatment approach for patients with asthma.

Apolipoprotein Mimetic Peptides: Potential New Therapies for Cardiovascular Diseases.

Since the seminal breakthrough of treating diabetic patients with insulin in the 1920s, there has been great interest in developing other proteins and their peptide mimetics as therapies for a wide variety of other medical disorders. Currently, there are at least 60 different peptides that have been approved for human use and over 150 peptides that are in various stages of clinical development. Peptides mimetic of the major proteins on lipoproteins, namely apolipoproteins, have also been developed first as tools for understanding apolipoprotein structure and more recently as potential therapeutics. In this review, we discuss the biochemistry, peptide mimetics design and clinical trials for peptides based on apoA-I, apoE and apoC-II. We primarily focus on applications of peptide mimetics related to cardiovascular diseases. We conclude with a discussion on the limitations of peptides as therapeutic agents and the challenges that need to be overcome before apolipoprotein mimetic peptides can be developed into new drugs.

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.

Trans-splicing into highly abundant albumin transcripts for production of therapeutic proteins in vivo.

Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo.

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.

Contribution of macromolecular structure to the retention of low-density lipoprotein at arterial branch points.

BACKGROUND: Extracellular deposition of low-density lipoprotein (LDL) in the arterial wall is an essential early step in atherosclerosis. This process preferentially occurs at arterial branch points, reflecting a regional variation in lipoprotein-arterial wall interactions. In this study, we characterized the submicron microstructure of arterial wall collagen and elastin to evaluate its potential role in regional LDL deposition. METHODS AND RESULTS: With 2-photon microscopy, we used the intrinsic optical properties of collagen and elastin to determine the arterial wall macromolecular microstructure in fresh porcine and murine arteries. This optical approach generated unique nondestructive en face 3-dimensional views of the wall. The collagen/elastin microstructure was found to vary with the topology of the arterial bed. A nearly confluent elastin surface layer was present throughout but was missing at atherosclerosis-susceptible branch points, exposing dense collagen-proteoglycan complexes. In LDL binding studies, this luminal elastin layer limited LDL penetration, whereas its absence at the branches resulted in extensive LDL binding. Furthermore, LDL colocalized with proteoglycans with a sigmoidal dose dependence (inflection point, approximately 130 mg LDL/dL). Ionic strength and competing anions studies were consistent with the initial interaction of LDL with proteoglycans to be electrostatic in nature. CONCLUSIONS: This optical sectioning approach provided a robust 3-dimensional collagen/elastin microstructure of the arterial wall in fresh samples. At atherosclerosis-susceptible vascular branch points, the absence of a luminal elastin barrier and the presence of a dense collagen/proteoglycan matrix contribute to increased retention of LDL.

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.

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.