Sally P. A. McCormick

High-Density Lipoprotein Reduces the Human Monocyte Inflammatory Response

Objective—Whereas the anti–inflammatory effects of high-density lipoprotein (HDL) on endothelial cells are well described, such effects on monocytes are less studied. Methods and Results—Human monocytes were isolated from whole blood followed by assessment of CD11b activation/expression and cell adhesion under shear-flow. HDL caused a dose-dependent reduction in the activation of CD11b induced by PMA or receptor-dependent agonists. The constituent of HDL responsible for the antiinflammatory effects on CD11b activation was found to be apolipoprotein A-I (apoA-I). Cyclodextrin, but not cyclodextrin/cholesterol complex, also inhibited PMA-induced CD11b activation implicating cholesterol efflux as the main mechanism. This was further confirmed with the demonstration that cholesterol content of lipid rafts diminished after treatment with the cholesterol acceptors. Blocking ABCA1 with an anti-ABCA1 antibody abolished the effect of apoA-I. Furthermore, monocytes derived from a Tangier disease patient definitively confirmed the requirement of ABCA1 in apoA-I–mediated CD11b inhibition. The antiinflammatory effects of apoA-I were also observed in functional models including cell adhesion to an endothelial cell monolayer, monocytic spreading under shear flow, and transmigration. Conclusions—HDL and apoA-I exhibit an antiinflammatory effect on human monocytes by inhibiting activation of CD11b. ApoA-I acts through ABCA1, whereas HDL may act through several receptors.

Molecular Basis of the Interaction between Plasma Platelet-activating Factor Acetylhydrolase and Low Density Lipoprotein

The platelet-activating factor acetylhydrolases are enzymes that were initially characterized by their ability to hydrolyze platelet-activating factor (PAF). In human plasma, PAF acetylhydrolase (EC 3.1.1.47) circulates in a complex with low density lipoproteins (LDL) and high density lipoproteins (HDL). This association defines the physical state of PAF acetylhydrolase, confers a long half-life, and is a major determinant of its catalytic efficiency in vivo. The lipoprotein–associated enzyme accounts for all of the PAF hydrolysis in plasma but only two-thirds of the protein mass. To characterize the enzyme–lipoprotein interaction, we employed site-directed mutagenesis techniques. Two domains within the primary sequence of human PAF acetylhydrolase, tyrosine 205 and residues 115 and 116, were important for its binding to LDL. Mutation or deletion of those sequences prevented the association of the enzyme with lipoproteins. When residues 115 and 116 from human PAF acetylhydrolase were introduced into mouse PAF acetylhydrolase (which normally does not associate with LDL), the mutant mouse PAF acetylhydrolase associated with lipoproteins. To analyze the role of apolipoprotein (apo) B100 in the formation of the PAF acetylhydrolase–LDL complex, we tested the ability of PAF acetylhydrolase to bind to lipoproteins containing truncated forms of apoB. These studies indicated that the carboxyl terminus of apoB plays a key role in the association of PAF acetylhydrolase with LDL. These data on the molecular basis of the PAF acetylhydrolase–LDL association provide a new level of understanding regarding the pathway for the catabolism of PAF in human blood.

Overexpression of Hepatic Lipase in Transgenic Mice Decreases Apolipoprotein B-containing and High Density Lipoproteins EVIDENCE THAT HEPATIC LIPASE ACTS AS A LIGAND FOR LIPOPROTEIN UPTAKE

To determine the mechanisms by which human hepatic lipase (HL) contributes to the metabolism of apolipoprotein (apo) B-containing lipoproteins and high density lipoproteins (HDL)in vivo, we developed and characterized HL transgenic mice. HL was localized by immunohistochemistry to the liver and to the adrenal cortex. In hemizygous (hHLTg +/0) and homozygous (hHLTg +/+) mice, postheparin plasma HL activity increased by 25- and 50-fold and plasma cholesterol levels decreased by 80% and 85%, respectively. In mice fed a high fat, high cholesterol diet to increase endogenous apoB-containing lipoproteins, plasma cholesterol decreased 33% (hHLTg +/0) and 75% (hHLTg +/+). Both apoB-containing remnant lipoproteins and HDL were reduced. To extend this observation, the HL transgene was expressed in human apoB transgenic (huBTg) and apoE-deficient (apoE −/−) mice, both of which have high plasma levels of apoB-containing lipoproteins. (Note that thehuBTg mice that were used in these studies were all hemizygous for the human apoB gene.) In both thehuBTg,hHLTg +/0 mice and theapoE −/−,hHLTg +/0mice, plasma cholesterol decreased by 50%. This decrease was reflected in both the apoB-containing and the HDL fractions. To determine if HL catalytic activity is required for these decreases, we expressed catalytically inactive HL (HL-CAT) in apoE −/−mice. The postheparin plasma HL activities were similar in theapoE −/− and theapoE −/−,HL-CAT +/0mice, reflecting the activity of the endogenous mouse HL and confirming that the HL-CAT was catalytically inactive. However, the postheparin plasma HL activity was 20-fold higher in theapoE −/−,hHLTg +/0mice, indicating expression of the active human HL. Immunoblotting demonstrated high levels of human HL in postheparin plasma of bothapoE −/−,hHLTg +/0and apoE −/−,HL-CAT +/0mice. Plasma cholesterol and apoB-containing lipoprotein levels were ∼60% lower inapoE −/−,HL-CAT +/0mice than in apoE −/− mice. However, the HDL were only minimally reduced. Thus, the catalytic activity of HL is critical for its effects on HDL but not for its effects on apoB-containing lipoproteins. These results provide evidence that HL can act as a ligand to remove apoB-containing lipoproteins from plasma.

Transgenic Mice That Overexpress Mouse Apolipoprotein B EVIDENCE THAT THE DNA SEQUENCES CONTROLLING INTESTINAL EXPRESSION OF THE APOLIPOPROTEIN B GENE ARE DISTANT FROM THE STRUCTURAL GENE

An 87-kilobase (kb) P1 bacteriophage clone (p649) spanning the mouse apolipoprotein (apo) B gene was used to generate transgenic mice that express high levels of mouse apoB. Plasma levels of apoB, low density lipoprotein cholesterol, and low density lipoprotein triglycerides were increased, and high density lipoprotein cholesterol levels were decreased in the transgenic mice, compared with nontransgenic littermate controls. Although p649 contained 33 kb of 5′-flanking sequences and 11 kb of 3′-flanking sequences, the tissue pattern of transgene expression was different from that of the endogenous apoB gene. RNA slot blots and RNase protection analysis indicated that the transgene was expressed in the liver but not in the intestine, whereas the endogenous apoB gene was expressed in both tissues. To confirm the absence of transgene expression in the intestine, the mouse apoB transgenic mice were mated with the apoB knockout mice, and transgenic mice that were homozygous for the apoB knockout mutation were obtained. Because of the absence of transgene expression in the intestine, those mice lacked all intestinal apoB synthesis, resulting in a marked accumulation of fats within the intestinal villus enterocytes. The current studies, along with prior studies of human apoB transgenic animals, strongly suggest that the DNA sequence element(s) controlling intestinal expression of the apoB gene is located many kilobases from the structural gene.

Overexpression of Human Apolipoprotein B-100 in Transgenic Rabbits Results in Increased Levels of LDL and Decreased Levels of HDL

In this study, and 80-kb human genomic DNA fragment spanning the human apoB gene was used to generate transgenic New Zealand White rabbits that expressed human apoB-100. The concentration of human apoB in the plasma of the transgenic rabbits ranged between 5 and 100 mg/dL. The transgenic rabbits had nearly threefold elevations in the plasma levels of triglycerides and cholesterol compared with nontransgenic controls. Nearly all the cholesterol and human apoB in the plasma was in the LDL fraction. Pronounced triglyceride enrichment of the LDL fraction was a striking feature of human apoB overexpression in the transgenic rabbits, in which the LDL fraction contained more than 75% of the plasma triglycerides. The triglyceride-enriched LDL particles were smaller and more dense than the native rabbit LDL and contained markedly increased amounts of apoE and apoC-III. In the nontransgenic control animals most of the triglycerides were in the VLDL, and most of the apoE and apoC-III were in the VLDL and HDL fractions. In addition to increased LDL levels, overexpression of human apoB in rabbits resulted in lower plasma levels of HDL cholesterol and apoA-I. In our prior studies on transgenic mice expressing human apoB, we documented triglyceride-rich LDL and reduced levels of HDL cholesterol. These prior findings in mice, together with the present findings in transgenic rabbits, suggest that triglyceride-rich LDL and lowered levels of HDL cholesterol may be hallmark features of apoB overexpression.

Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain

The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein‐folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggregation assay. All analogs inhibited PDI, but the activity (IC50) ranged from 20 μm for bacitracin F to 1050 μm for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI‐TOF/TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate‐binding domain of PDI.

Interaction of the inflammasome genes CARD8 and NLRP3 in abdominal aortic aneurysms

OBJECTIVE Cholesterol crystals have been shown to cause inflammation, and ultimately atherosclerotic lesions through the activation of the NLRP3 inflammasome. As cholesterol crystals have also been found in the walls of patients with abdominal aortic aneurysms (AAA), it is possible that the NLRP3 inflammasome is involved in AAA and genetic variability within this protein complex could alter disease risk. The primary objective of this study was to assess whether there is genetic evidence for a role of the NLRP3 inflammasome in AAA by testing for association of AAA with functional single nucleotide polymorphisms (SNPs) in the CARD8 and NLRP3 genes. METHODS AAA patients (n=1151) and controls (n=727) were genotyped for CARD8 SNP rs2043211 and NLRP3 SNP rs35829419 using TaqMan SNP assays. IL1-β, C-reactive protein (CRP), and lipoprotein (a) [Lp(a)] were measured in the plasma of a subset of study participants. The Kruskal-Wallis Rank test was conducted to test for differences in mean concentration of IL1-β, CRP and Lp(a). Logistic regression was used to test for interaction between CARD8 and NLRP3. RESULTS Significantly higher mean concentration of plasma IL1-β was observed in study participants who were homozygous for the common C allele of NLRP3 rs35829419 (p=0.010). Interaction between rs2043211 and rs35829419 was observed in this dataset (χ(2)=6.22; p=0.044), which strengthened when adjusted for age, gender, smoking, diabetes, hypertension, and dyslipidemia (χ(2)=14.75; p=0.012); and separately for NOD2 genotype (χ(2)=14.06; p=0.015). CONCLUSION Our finding suggests genetic variability within the NLRP3 inflammasome may be important in the pathophysiology of AAA.

Novel rare mutations and promoter haplotypes in ABCA1 contribute to low-HDL-C levels

The ATP‐binding cassette A1 (ABCA1) protein regulates plasma high‐density lipoprotein (HDL) levels. Mutations in ABCA1 can cause HDL deficiency and increase the risk of premature coronary artery disease. Single nucleotide polymorphisms (SNPs) in ABCA1 are associated with variation in plasma HDL levels. We investigated the prevalence of mutations and common SNPs in ABCA1 in 154 low‐HDL individuals and 102 high‐HDL individuals. Mutations were identified in five of the low‐HDL subjects, three having novel variants (I659V, R2004K, and A2028V) and two with a previously identified variant (R1068H). Analysis of four SNPs in the ABCA1 gene promoter (C‐564T, G‐407C, G‐278C, and C‐14T) identified the C‐14T SNP and the TCCT haplotype to be over‐represented in low‐HDL individuals. The R1587K SNP was over‐represented in low‐HDL individuals, and the V825I and I883M SNPs over‐represented in high‐HDL individuals. We conclude that sequence variation in ABCA1 contributes significantly to variation in HDL levels.

Proteomics of Lipoprotein(a) identifies a protein complement associated with response to wounding

Lipoprotein(a) [Lp(a)] is a major independent risk factor for cardiovascular disease. Twenty percent of the general population exhibit levels above the risk threshold highlighting the importance for clinical and basic research. Comprehensive proteomics of human Lp(a) will provide significant insights into Lp(a) physiology and pathogenicity. Using liquid chromatography-coupled mass spectrometry, we established a high confidence Lp(a) proteome of 35 proteins from highly purified particles. Protein interaction network analysis and functional clustering revealed proteins assigned to the two major biological processes of lipid metabolism and response to wounding. The latter includes the processes of coagulation, complement activation and inflammatory response. Furthermore, absolute protein quantification of apoB-100, apo(a), apoA1, complement C3 and PON1 gave insights into the compositional stoichiometry of associated proteins per particle. Our proteomics study has identified Lp(a)-associated proteins that support a suggested role of Lp(a) in response to wounding which points to mechanisms of Lp(a) pathogenicity at sites of vascular injury and atherosclerotic lesions. This study has identified a high confidence Lp(a) proteome and provides an important basis for further comparative and quantitative analyses of Lp(a) isolated from greater numbers of plasma samples to investigate the significance of associated proteins and their dynamics for Lp(a) pathogenicity.

Effects of Extended-Release Niacin on the Postprandial Metabolism of Lp(a) and ApoB-100–Containing Lipoproteins in Statin-Treated Men With Type 2 Diabetes Mellitus

Objective—The effects of extended-release niacin (ERN; 1–2 g/d) on the metabolism of lipoprotein(a) (Lp(a)) and apolipoprotein (apo) B-100–containing lipoproteins were investigated in 11 statin-treated white men with type 2 diabetes mellitus in a randomized, crossover trial of 12-weeks duration. Approach and Results—The kinetics of Lp(a) and very low–density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) apoB-100 were determined following a standardized oral fat load (87% fat) using intravenous administration of D3-leucine, gas chromatography–mass spectrometry, and compartmental modeling. ERN significantly decreased fasting plasma total cholesterol, LDL cholesterol, and triglyceride concentrations. These effects were achieved without significant changes in body weight or insulin resistance. ERN significantly decreased plasma Lp(a) concentration (−26.5%) and the production rates of apo(a) (−41.5%) and Lp(a)-apoB-100 (−32.1%); the effect was greater in individuals with elevated Lp(a) concentration. ERN significantly decreased VLDL (−58.7%), intermediate-density lipoprotein (−33.6%), and LDL (−18.3%) apoB-100 concentrations and the corresponding production rates (VLDL, −49.8%; intermediate-density lipoprotein, −44.7%; LDL, −46.1%). The number of VLDL apoB-100 particles secreted increased in response to the oral fat load. Despite this, total VLDL apoB-100 production over the 10-hour postprandial period was significantly decreased with ERN (−21.9%). Conclusions—In statin-treated men with type 2 diabetes mellitus, ERN decreased plasma Lp(a) concentrations by decreasing the production of apo(a) and Lp(a)-apoB-100. ERN also decreased the concentrations of apoB-100–containing lipoproteins by decreasing VLDL production and the transport of these particles down the VLDL to LDL cascade. Our study provides further mechanistic insights into the lipid-regulating effects of ERN.