Density Lipoprotein Particles Research Articles

Abstract 11198: Role of Different Lipid Parameters and Lipoproteins on Coronary Microvascular Dysfunction in the Young

Aim: Young with coronary microvascular dysfunction (CMD) have increased cardiovascular morbidity. Particularly, Asian patients with CMD are considerably younger than their Western peers. Blood lipids are traditionally considered risk factors for myocardial infarction (MI) of the elderly. However, little is known about CMD in the young. The purpose of our study was to test the associations between lipids and lipoprotein particles and CMD in the young. Methods: A cross-sectional study was conducted from 2014 to 2020, 1868 young participants were included. CMD was defined as angina but no obstructive coronary disease. All participants had baseline laboratory testing. Lipid parameters, including total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), small dense low density lipoprotein cholesterol (sd LDL-C), high-density lipoprotein (HDL), non-HDL, TC/HDL ratio, TG/HDL ratio and LDL/HDL ratio were measured. Both low- and high-density lipoprotein cholesterol (LDL-C and HDL-C) subfractions were measured using the Quantimetrix Lipoprint System. Logistic regression analysis was performed to determine associated factors for CMD. Results: Very low-, intermediate-, small dense-, and low- density lipoprotein particles were positively associated with CMD. HDL particles were inversely associated with CMD apart from small HDL. Cholesterol in large HDL was inversely associated with CMD (odds ratio [OR] = 0.75), whereas cholesterol in small HDL was not (OR = 1.08). TG within all lipoproteins, including most HDL particles, were positively associated with CMD. In multiple logistic regression analyses, non-HDL [OR = 1.56 (1.16-1.87)], sd LDL-C [OR = 1.80 (1.21-2.27)], TC [OR = 1.41 (1.25-1.93)], TC/HDL [OR = 1.26 (1.15-1.68)] were significantly associated with CMD. Conclusions: Lipids and lipoproteins showed similar associations with CMD. Within HDL particles, cholesterol concentrations were inversely associated, whereas triglyceride concentrations were positively associated with CMD. Non-HDL, sd LDL-C, TC, and TC/HDL were significantly associated with CMD than others. This result should be considered in future clinical lipid-lowering trials and a routine screening for CMD in the young.

893-P: Triglyceride-Rich Remnant Lipoproteins, Insulin Resistance, and Subclinical Inflammation in Youth with Type 2 Diabetes

Youth with type 2 diabetes (T2D-Y) have severe insulin resistance (IR) and increased risk for atherosclerotic cardiovascular disease (ASCVD). High levels of remnant triglyceride-rich lipoproteins (TRLp), the sum of small very low-density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) particles, may mediate this additional risk. Yet, the relationship of remnant TRLp with IR and inflammation in at-risk youth is unknown. We compared lipoprotein particles in 48 youth: 33 T2D-Y and 15 age/BMI matched peers without diabetes (age 15.5 ± 2.6 y, Tanner Stage IV-V, BMI 38.6 ± 7.4 kg/m2) and determined the relationship of remnant TRLp with IR and GlycA, a composite marker of inflammation. Fasting lipoprotein particle size and number, apolipoprotein B (apoB) and GlycA were derived from the NMR LipoProfile®. Insulin sensitivity was determined by the Matsuda index during a multi-sample 75g OGTT. T2D-Y had higher A1c (7.0 ± 1.2 vs. 5.6 ± 0.3%, P<0.01), lower Matsuda index (1.3 ± 1.0 vs. 2.5 ± 1.3, P<0.01) and higher GlycA (431 ± 73 vs. 390 ± 50 μmol/L, P=0.05). Triglyceride and low density (LDL) particles were higher in T2D-Y (ApoB: 74 ± 16 vs. 56 ± 12 mg/dL; total LDL particles: 1314 ± 298 vs. 983 ± 224 nmol/L; small LDL particles: 881 ± 348 vs. 492 ± 235 nmol/L; total TRLps: 111 ± 52 vs. 71 ± 30 nmol/L, and remnant TRLps: 97 ± 45 vs. 63 ± 29 nmol/L, all P<0.01). HDL particles were not different between groups. Matsuda index inversely correlated with GlycA (r=-0.4, P=0.01), total and remnant TRLps (both r=-0.5, P<0.01) and small LDL particles (r=-0.4, P=0.03), but not with total LDL or HDL particles. GlycA was associated with small LDLp (r=0.3, P=0.07 and remnant TRLp (all r=0.2, P=0.1) but this was not statistically significant. The high-risk profile marked by high remnant TRLps and small LDL particles in T2D-Y was associated with IR and an inflammatory marker, supporting the need for longitudinal studies to determine whether reducing TRLps and inflammation will reduce ASCVD risk. Disclosure A. Villalobos-perez: None. S. T. Chung: None. A. Zenno: None. C. K. Cravalho: None. S. Matta: None. A. B. Courville: None. L. Mabundo: None. V. R. Sharma: None. J. M. Dawson: None. M. W. Haymond: Advisory Panel; Self; Daiichi Sankyo, Zealand Pharma A/S, Other Relationship; Self; AstraZeneca, Stock/Shareholder; Self; Xeris Pharmaceuticals, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases

Physicochemical Properties of Lipoproteins Assessed by Nuclear Magnetic Resonance as a Predictor of Premature Cardiovascular Disease. PRESARV-SEA Study.

Some lipoprotein disorders related to the residual risk of premature cardiovascular disease (PCVD) are not detected by the conventional lipid profile. In this case-control study, the predictive power of PCVD of serum sdLDL-C, measured using a lipoprotein precipitation method, and of the physicochemical properties of serum lipoproteins, analyzed by nuclear magnetic resonance (NMR) techniques, were evaluated. We studied a group of patients with a first PCVD event (n = 125) and a group of control subjects (n = 190). Conventional lipid profile, the size and number of Very Low Density Lipoproteins (VLDL), Low Density Lipoproteins (LDL), High Density Lipoproteins (HDL) particles, and the number of particles of their subclasses (large, medium, and small) were measured. Compared to controls, PCVD patients had lower concentrations of all LDL particles, and smaller and larger diameter of LDL and HDL particles, respectively. PCVD patients also showed higher concentrations of small dense LDL-cholesterol (sdLDL), and triglycerides (Tg) in LDL and HDL particles (HDL-Tg), and higher concentrations of large VLDL particles. Multivariate logistic regression showed that sdLDL-C, HDL-Tg, and large concentrations of LDL particles were the most powerful predictors of PCVD. A strong relationship was observed between increased HDL-Tg concentrations and PCVD. This study demonstrates that beyond the conventional lipid profile, PCVD patients have other atherogenic lipoprotein alterations that are detected by magnetic resonance imaging (MRI) analysis.

Liraglutide in combination with metformin may improve the atherogenic lipid profile and decrease C-reactive protein level in statin treated obese patients with coronary artery disease and newly diagnosed type 2 diabetes: A randomized trial

Background and aimsAtherosclerosis in obesity and type 2 diabetes (T2DM) is associated with low-grade inflammation (LGI) and dyslipidemia, where especially small, dense lipoprotein particles are atherogenic. The glucagon-like peptide-1 receptor agonist, liraglutide, reduces cardiovascular events by poorly understood mechanisms. We investigated the effect of liraglutide combined with metformin on LGI and lipoprotein density profiles in patients with stable coronary artery disease (CAD) and newly diagnosed T2DM. MethodsWe conducted a randomized, double-blind, placebo-controlled, cross-over trial over a 12 + 12-week period, with ≥2-week wash-out. Intervention: liraglutide/metformin vs. placebo/metformin. Lipoproteins were separated by continuous density gradient ultracentrifugation, and LDL divided into five subfractions between 226 and 270 Å, considering particle size ≤255 Å as the atherogenic pattern. Plasma C-reactive protein and tumor necrosis factor-α were assessed by the enzyme-linked immunosorbent-assay. Results28 out of 41 randomized patients completed all visits. Intention-to-treat analysis was performed but one patient had statin dosage and was excluded from the analysis. 95% of the patients were on statin therapy. Overall, liraglutide did not affect lipid subfractions or markers of LGI compared to placebo. The combination of liraglutide and metformin reduced the total LDL subfractions, primarily by reducing the most atherogenic subfraction LDL5, and reduced CRP but not TNF-α. Explorative analyses suggested that the subfraction LDL5 during the wash-out period rebounded significantly at least in a per-protocol analysis of the sub-group of patients starting the liraglutide therapy. ConclusionsIn patients with CAD and newly diagnosed T2DM on stable statin therapy, liraglutide combined with metformin may improve the atherogenic LDL lipid profile and CRP.

Blueberries improve biomarkers of cardiometabolic function in participants with metabolic syndrome—results from a 6-month, double-blind, randomized controlled trial

ABSTRACTBackgroundAnthocyanin-rich blueberry intake is associated with reduced type 2 diabetes and cardiovascular disease (CVD) risk in prospective studies, although long-term randomized controlled trials (RCTs) have not been conducted in at-risk populations.ObjectiveIn the longest-duration RCT to date, we examined the effect of 6-mo blueberry intake on insulin resistance and cardiometabolic function in metabolic syndrome.MethodsA double-blind, parallel RCT (n = 115; age 63 ± 7 y; 68% male; body mass index 31.2 ± 3.0 kg/m2) was conducted, which fed 2 dietarily achievable blueberry intakes [equivalent to 1/2 and 1 cup/d (75/150 g)] compared with matched placebo. Insulin resistance was assessed via the homeostasis model assessment of insulin resistance (primary endpoint) and confirmed by [6-6-2H2]-glucose-labeled, 2-step hyperinsulinemic clamp (n = 20). Clinically relevant cardiometabolic endpoints [including flow-mediated dilatation, augmentation index, lipoprotein status (by nuclear magnetic resonance spectroscopy), and nitric oxide (NO)-related metabolite assay] and anthocyanin metabolism were assessed.ResultsA daily intake of 1 cup of blueberries improved endothelial function (flow-mediated dilatation: +1.45%; 95% CI: 0.83%, 2.1%; P = 0.003), systemic arterial stiffness (augmentation index: –2.24%; 95% CI: –3.97%, –0.61%; P = 0.04) and attenuated cyclic guanosine monophosphate concentrations. In statin nonusers (n = 71), elevated high-density lipoprotein cholesterol (+0.08 mmol/L; P = 0.03), high-density lipoprotein particle density (+0.48n, ×10–6; P = 0.002) and apolipoprotein A-I (+0.05 g/L; P = 0.01) concentrations were observed following the 1-cup/d intervention. Treatment compliance was 94.1% (wrapper returns) and total concentrations of anthocyanin-derived phenolic acid metabolites significantly increased, dose-dependently, in serum and 24-h urine (P < 0.01 and P < 0.001, respectively). Insulin resistance, pulse wave velocity, blood pressure, NO, and overall plasma thiol status were unaffected. Likewise, a half cup per day had no effect on any biomarkers.ConclusionsDespite insulin resistance remaining unchanged we show, to our knowledge, the first sustained improvements in vascular function, lipid status, and underlying NO bioactivity following 1 cup blueberries/d. With effect sizes predictive of 12–15% reductions in CVD risk, blueberries should be included in dietary strategies to reduce individual and population CVD risk. This study was registered at clinicaltrials.gov as NCT02035592.

Consumption of regular-fat vs reduced-fat cheese reveals gender-specific changes in LDL particle size - a randomized controlled trial

BackgroundRegular-fat cheese does not seem to increase low density lipoprotein cholesterol (LDL-C) concentrations compared to reduced-fat cheese. However, plasma LDL-C concentrations do not reflect levels and size of LDL particles, which might be a better predictor of cardiovascular risk.MethodsThe aim was to compare the effects of regular-fat cheese vs reduced-fat cheese and carbohydrate-rich foods on LDL particle size distribution in adults with ≥ 2 metabolic syndrome (MetS) risk factors. The study was part of a 12 weeks’ randomized controlled trial in which subjects had been randomly allocated to 1 of 3 intervention groups; regular-fat cheese (REG), reduced-fat cheese (RED) or a no-cheese/carbohydrate (CHO) group. Subjects in the REG and RED groups consumed 80 g cheese/d per 10 MJ, whereas subjects in the CHO consumed bread and jam corresponding to 90 g/d and 25 g/d per 10 MJ, respectively. Fasting blood samples at wk. 0 (baseline) and wk. 12 were analyzed for LDL particle size distribution and cholesterol content using nuclear magnetic resonance (NMR) spectroscopy.ResultsA total of 85 subjects [mean ± SD age: 54.0 ± 12.8 y; BMI: 28.7 ± 3.6 kg/m2] completed the study. Overall, regular-fat cheese did not impact lipoprotein particle number and size differently than reduced-fat cheese. In men (n = 23), the REG diet decreased total LDL particle number (LDL-P, − 223.2 ± 91.1 nmol/l, P = 0.01) compared with the RED diet. The reduction was primarily in the medium-sized LDL fraction (− 128.5 ± 51.8 nmol/l, P = 0.01). In women (n = 62), the REG diet increased the concentration of cholesterol in the small high density lipoprotein (HDL) particles compared with the CHO diet (2.9 ± 1.0 mg/dl, P = 0.006).ConclusionOverall, regular-fat cheese did not alter LDL particle size distribution compared to reduced-fat cheese after a 12 wk. intervention in subjects with ≥2 MetS risk factors. However, our results suggest that lipoprotein response to cheese intake is gender-specific. This warrants further investigation.Trial registrationThis trial was registered at Clinicaltrials.gov as NCT0261471. Registered 30 November 2015 - Retrospectively registered.

Abstract 440: Targeting LOX-1 Scavenger Receptor and oxLDL Uptake With Affimers

Introduction: Lectin-like oxidized low density lipoprotein receptor 1 (LOX-1, SREC-1, OLR1) is a class E scavenger receptor which binds oxidized low density lipoprotein (oxLDL) particles implicated in promoting atherosclerosis. Affimers are non-antibody synthetic protein-based scaffolds that enable recognition of a wide range of molecular targets. Aims: To investigate the ability of Affimers to bind LOX-1 and modulate oxLDL accumulation and effects on cellular responses. Methods: Recombinant soluble human LOX-1 was screened with an Affimer library and LOX-1-specific Affimers were isolated and characterized. Human embryonic kidney (HEK293T) cells inducibly expressing human LOX-1 were incubated with labeled oxLDL and Affimers to modulate LOX-1 and oxLDL uptake and trafficking. Furthermore, Affimer-mediated effects on oxLDL-regulated metabolism and apoptosis was evaluated. Results: Five Affimers specific for human LOX-1 were identified. Fluorescent labelled Affimers demonstrate specific binding to LOX-1 expressing HEK293T cells and modulate LOX-1 and oxLDL trafficking and accumulation in endosomes and lysosomes. LOX-1-specific Affimers also show effects in modulating oxLDL-mediated effects in specific cellular responses. Conclusion: LOX-1-specific Affimers are a new class of synthetic molecules that have therapeutic potential in targeting LOX-1, a key mediator of inflammation and arterial dysfunction in cardiovascular disease. Membrane specific binding is demonstrated only in induced LOX-1 expressing cells (LOX+). Increasing concentration of affimer prior to incubation with DiI labelled oxLDL.

Sialidases: Therapeutic and Antiatherogenic Potential.

This review focuses on the biological role and clinical relevance of relatively poor studied enzymes known as sialidases. We describe structure and function of sialic acid, in particular as a component of gangliosides and plasma lipoproteins. Several types of sialidases are known in mammals, of which trans-sialidase is of special interest, since it is capable of removing sialic acid from low density lipoprotein (LDL) particles and transferring it to different acceptors in blood plasma. Desialylation of LDL, in turn, endows it a capacity to accumulate in the smooth muscle cells of human aortic intima, and therefore is important for atherogenesis. Moreover, sialidases appear to be involved in a variety of pathological processes, including viral infections and cancer, which makes these enzymes an attractive therapeutic target.

The Relationship between LDL Particles and Their Surface Charge in Patients with Coronary Artery Disease

Introduction: According to recent researches, changes in the composition of LDL particles affect their surface charge and structural features (their diameters, for example), modifying their morphology and metabolism in the plasma. In the present study, the relationship of LDL particles size with their zeta potential were assessed in patients with coronary artery disease in comparison with control subjects. Materials & methods: In the case and control study, 30 patients with coronary artery disease and 30 control subjects were selected based on the clinical and angiographic parameters. The size and zeta potential of LDL particles were measured using zeta sizer instrument. Findings: Results of this study showed that, LDL mean particle size in patients (24.18 ± 1.6 nm) was significantly higher than that of control subjects (25.26 ± 0.63 nm)( P=./0001). Furthermore, the zeta potential level in patients (-25.08 ± 0.77 mV) was significantly lower than that of control subjects (-23.65 ± 1.56 mV) (P=./0001). This means that the negative charge of LDL particles was further in patients than in control subjects. On the other hand, there was a significant correlation between LDL mean particle size and zeta potential of the particles in patients. However, the correlation was not significant in control subjects. Discussion & conclusions: Low density lipoprotein (LDL) particles are heterogeneous entities based on their size, density and composition. It is conceived that among the particles the smaller and denser ones would be more atherogenic. The present study demonstrated that LDL particles with smaller size and more negative charges are associated with increased risk of cardiovascular disease. Also, changes in the diameter of LDL particles are associated with the changes in the zeta potentials of the particles.

Abstract 566: MicroRNA-146a Deficiency Prevents PCSK9 Gain-of-Function Mutation-induced Hypercholesterolemia in Mice

Background and Objective: Mimetic mediated activation of microRNA 146a (miR-146a) reduces atherosclerosis via suppression of nuclear factor-κB-driven inflammation in mice. The purpose of this study was to determine whether miR-146a influences plasma cholesterol in hypercholesterolemic mice. Methods and Results: To induce hypercholesterolemia, female C57BL/6 miR-146a WT (n=8) and miR-146a KO (n=8) mice were injected intraperitoneally with an adeno-associated viral vector (AAV) expressing the proprotein convertase subtilisin/kexin type 9 (PSCK9 D377Y) gain-of-function mutant at a dose of 3 x 10 10 genomic copies/mouse. After infection, mice were fed a Western diet (21% wt/wt milk fat; 0.15% wt/wt cholesterol) for sixteen weeks, and plasma PCSK9 and total cholesterol concentrations were monitored monthly using an enzymatic assay. Plasma PCSK9 concentrations were profoundly increased 4 weeks post injection (Baseline: WT - 179 ± 12 vs KO - 207 ± 12; Week 4: WT - 1700 ± 148 vs KO - 2689 ± 305 ng/ml) and remained significantly high during 16 weeks (WT - 882 ± 142 vs KO - 718 ± 109 ng/ml; p&lt;0.05 vs baseline) of Western diet feeding. Consistent with increased plasma PCSK9 concentrations, plasma cholesterol concentrations were increased in both groups of mice. Interestingly, miR-146a KO group mice showed less significant increase in plasma cholesterol compared to WT group (Baseline: WT - 88 ± 3 vs KO - 83 ± 3; Week 4: WT - 328 ± 25 vs KO - 195 ± 18 mg/dl) irrespective of the comparable plasma PCSK9 concentrations. Also, lipoprotein distribution analysis with size exclusion gel chromatography revealed that miR-146a KO mice showed a strong reduction in high density lipoprotein (HDL) particles while very low density lipoprotein (VLDL) and low density lipoprotein (LDL) particles were not affected. Conclusion: Our findings suggests that miR146a plays a critical role in the regulation of HDL particles in PCSK9 gain-of-function mutant-induced hypercholesterolemia in mice. Future studies will identify gene targets influenced by miR-146a in regulating HDL-cholesterol in hypercholesterolemic mice.

Abstract 421: Computational Modelling Suggests That Reversing Low HDL May Not Always Lead to Plaque Regression

Introduction: Clinical evidence suggests that high numbers of high density lipoprotein (HDL) particles in the bloodstream and high levels of HDL efficacy, reduce the risk of heart attack and stroke. Experimental evidence suggests that increasing HDL particle number too late in the life of the plaque does not lead to regression but only to reduced rates of plaque growth. Hypothesis: We postulate that nonlinear dynamics are intrinsic to how cells respond to HDL in reverse cholesterol transport (RCT) from macrophages and foam cells and that these dynamics have consequences for plaque growth, regression and the plaques’ responses to changes in the plaque environment, such as changes in low density lipoprotein (LDL) influx. Methods: We formulated a computational model for the endothelium and the inside of the artery wall. This model consisted of six partial differential equations and associated boundary conditions. We found what conditions led to model plaques remaining small and what led to model plaques growing unboundedly. We also determined the outcomes if the concentration or action of HDL changes at different times in the life of the model plaque. Results: Model plaques with high concentrations of HDL, highly efficacious HDL and low levels of LDL were small and did not grow. There was a tipping point (or bifurcation point) so that plaques with HDL concentrations below the tipping point grew unboundedly and plaques with HDL concentrations above the tipping point remained small. The position of this tipping point depended on the concentration of modified LDL experienced by the plaque, anti-inflammatory actions of HDL and the phenotypes of macrophages after RCT. Increasing HDL concentration after the model plaque had grown led to plaque regression only if the dynamics of the plaque were such that the model plaque was able to shrink to a small plaque that did not grow. If the reduction in HDL was too late, or not large enough, the model plaque could not regress and continued to grow. Conclusion: The nonlinear nature of the dynamics of plaque growth and regression may result in outcomes to treatment that seem unexpected or inexplicable. Computational modelling provides a means of exploring the consequences of these dynamics.

Can the effects of gender, menopause and ageing on lipid levels be differentiated?

The menopause, as well as ageing in both genders, can influence cardiovascular disease (CVD) risk1. An atherogenic lipoprotein profile, including small dense low density lipoprotein (sdLDL) particles, can be present even in normolipidaemic healthy young individuals (about 6%)2. If confirmed by larger studies, it will be necessary to consider different risk stratifications for those with atherogenic normolipidaemia and those with non-atherogenic hypercholesterolaemia. Furthermore, other changes associated with the menopause (increase in lipoprotein (a) [Lp(a)] levels, central obesity, endothelial dysfunction and systemic inflammation) may contribute to pro-atherogenic changes, while ethnicity and lifestyle habits (diet and exercise) cannot be ignored3. This article is protected by copyright. All rights reserved.

Lipidated APOE has effects on cognitive function that are independent of amyloid-β pathology.

This scientific commentary refers to ‘Opposing effects of Apoe/Apoa1 double deletion on amyloid-β pathology and cognitive performance in APP mice’, by Fitz et al. (doi:10.1093/brain/awv293). Since the discovery of the apolipoprotein E e4 allele ( APOE4 ) as the strongest genetic risk factor for Alzheimer’s disease (reviewed by Verghese et al. , 2011), there has been a dramatic increase in research on the intersection of lipid biology and Alzheimer pathology. APOE is the major cholesterol and lipid carrier in the brain, while apolipoprotein A1 (APOA1) is the major lipid carrier in the periphery. Efflux of cholesterol and phospholipids from cells onto lipid-poor APOE and APOA1 is regulated by the ATP binding cassette transporter A1 (ABCA1), which is essential for the generation of the vast majority of high density lipoprotein (HDL) particles. The importance of APOE in Alzheimer pathology is highlighted by the fact that crossing a transgenic Alzheimer’s disease mouse model with APOE-deficient mice produced a marked decrease in amyloid-β pathology in the offspring (Bales et al. , 1997). By contrast, crossing ABCA1-deficient mice with an Alzheimer’s disease model generated offspring with increased amyloid-β pathology, despite vastly reduced levels of APOE and APOA1 (reviewed by Koldamova et al. , 2014). This apparent contradiction is currently unresolved. However, in this issue of Brain , Fitz and co-workers provide substantial insight into the complex role of apolipoproteins and their lipidation state in the pathophysiology of Alzheimer’s disease (Fitz et al. , 2015 …

Abstract 527: Decay of Hepatic and Serum Apolipoprotein(a) Levels is Slower than ApolipoproteinB Following Inhibition of Lipid Synthesis

The association of Lipoprotein(a) (Lp(a)) levels and increased cardiovascular risk is substantiated by human epidemiology, genetics and interventional apheresis studies. Lp(a) is a plasma lipoprotein consisting of a Low Density Lipoprotein (LDL) particle with one molecule of apolipoprotein B100 (ApoB100) covalently linked to one molecule of apolipoprotein(a). Published studies show that apo(a) synthesis and secretion from hepatoma cells is coupled to triglyceride synthesis and secretion, illustrating a point of control for Lp(a) production. The objective of this study is to compare Lp(a), apo(a) and apoB production, secretion and half-life in vivo to understand mechanisms controlling circulating levels of Lp(a). Since rodent species do not express endogenous apo(a), human apo(a) transgenic mice were created and bred with human apoB100 transgenic mice to generate Lp(a)-producing mice. The circulating level of Lp(a) in the transgenic mice is ~3.7 mg/dL that is comparable to 5.4 mg/dL measured in a healthy human subject. To inhibit triglyceride and VLDL secretion in liver, mice were treated with a microsomal triglyceride transfer protein inhibitor (MTPi), and plasma was collected in a time-course. This treatment allowed measurement of plasma Lp(a), apo(a) and apoB half-lives since secretion of nascent VLDL and LDL was blocked. MTPi caused an 81% reduction in cholesterol and 68% reduction in triglycerides after 5 days of treatment. ApoB decreased significantly within 6 hours of treatment and remained low for 5 days. The calculated half-life was 4.6 hours. By contrast, apo(a) and Lp(a) decreased significantly after 3 days of treatment with half-lives of 2.4 and 2.5 days, respectively, illustrating delayed catabolism of apo(a)/Lp(a). Hepatic Lp(a), apo(a) and apoB were decreased significantly after 5 days dosing, indicating that the apolipoproteins were likely degraded and not accumulated in the liver. Treatment of stable HepG2 cells expressing apo(a) with MTPi caused similar difference in Lp(a) and apoB half-lives. These models illustrate rapid decay of apoB, but not apo(a), when lipoprotein particle assembly is blocked, and they provide methods for future mechanistic studies of apo(a) assembly into Lp(a) and its catabolism.

HPS2-THRIVE, AIM-HIGH and dal-OUTCOMES: HDL-cholesterol under attack.

A low level of high-density lipoprotein cholesterol (HDL-C) is an established predictor of the risk of coronary heart disease (CHD).1 HDL promotes cellular cholesterol efflux and reverse cholesterol transport from lipid-laden macrophages (Figure 1), and prevents lipoprotein oxidation. A linear inverse relation has been reported in many observational studies between plasma HDL-C level and incident CHD events, with a plateau effect at HDL-C values >90 mg/dL in men and 75 mg/dL in women.2 However, it remains uncertain whether raising HDL-C levels therapeutically (by either niacin or cholesterol ester transfer protein “CETP” inhibitors) would reduce subsequent cardiovascular (CV) events beyond that achieved with intensive statin therapy. Figure 1. Reverse Cholesterol Transport. ABCA1, ATP binding cassette A1 transporter; ABCG1, ATP binding cassette G1 transporter; CETP, cholesterol ester transfer protein; SR-B1: Scavenger receptor-B1; LDLR, low density lipoprotein receptor. Niacin has multiple beneficial effects on serum lipoproteins;3 (1) it raises HDL-C level primarily by promoting the production and retarding the catabolism of Apo A1 in the liver, and also by stimulating ATP-binding cassette transporter A1 which augments reverse cholesterol transport. (2) It decreases the secretion of very low density lipoprotein (VLDL) particles from the liver with subsequent reduction of both intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) particles; an effect mediated by inhibition of hormone-sensitive lipase in the adipose tissue, and hepatocyte diacylglycerol acyltransferase-2 (DGAT-2). (3) It is unique in lowering lipoprotein (a) levels by up to 30%. (4) It inhibits oxidative stress, key inflammatory genes, cytokines involved in atherosclerosis. Inhibition of CETP by torcetrapib, dalcetrapib, anacetrapib, and evacetrapib is another attractive strategy to raise plasma HDL-C level.4,5 CETP promotes the transfer of cholesterol esters from HDL to triglyceride-rich lipoprotein particles, in exchange for triglyceride, thereby reducing the circulating HDL-C concentration. Over the past years, numerous randomized clinical studies have been conducted to assess the clinical efficacy and safety of these drugs.

Abstract 420: Lysosomal Acid Lipase Deficiency Impairs Reverse Cholesterol Transport From Macrophages in LAL-Deficient Mice

The rate of flux of cholesterol out of lysosomes is a key regulator of oxysterol formation and subsequent activation of liver X-receptor to upregulate genes involved in reverse cholesterol transport (RCT). We previously demonstrated impaired regulation of the lipid transporter ATP-binding cassette transporter A1 (ABCA1), required to promote removal of cholesterol from cells and high density lipoprotein (HDL) particle formation, in the lysosomal cholesterol storage disorders Niemann Pick Disease Type C and Cholesteryl Ester Storage Disease, thus providing an explanation for the low plasma HDL-C seen in these diseases. To determine the importance of lysosomal acid lipase (LAL) in reverse cholesterol transport (RCT) from macrophages to feces in vivo, we performed intraperitoneal injection of immortalized peritoneal macrophages from LAL+/+ or LAL-/- mice that had been cholesterol loaded and labeled with acetylated LDL containing 3H-cholesteryl oleate into LAL+/+ and LAL-/- mice, respectively. LAL-/- macrophages exhibited reduced basal and cholesterol-stimulated ABCA1 expression in culture, and reduced ability to support RCT in LAL-/- mice (1.55 ± 0.35 % total injected 3H-cholesterol counts appearing in feces over 48 h, n=30) compared to LAL+/+ macrophages injected into LAL+/+ mice (5.38 ± 0.92 % 3H-cholesterol counts in feces at 48 hr, n=27), p &lt; 0.001. Injection of LAL-/- macrophages into LAL+/+ mice resulted in partial correction of RCT (2.60 ± 0.46 % 3H-cholesterol counts in feces at 48 hr, n=19), p &lt; 0.001 when compared to injection into LAL-/- mice. ABCA1 protein expression was reduced in lal-/- mouse liver and mRNA expression of several LXR-dependent genes involved in reverse cholesterol transport (ABCG1, ABCG5, ABCG8, CYP7A1, SR-B1) were differentially modulated compared to LAL+/+ controls. The results indicate a critical role of macrophage LAL in promoting ABCA1 expression and RCT in vivo, and the ability of secreted LAL to be taken up by macrophages and correct RCT in vivo. The premature atherosclerosis seen in LAL-deficient humans may therefore be reduced by correction of LAL deficiency.

Effects of Niacin Combination Therapy With Statin or Bile Acid Resin on Lipoproteins and Cardiovascular Disease

Two large studies in populations selected for cardiovascular disease (CVD) demonstrated that raising high-density lipoprotein (HDL) cholesterol with niacin added to statin therapy did not decrease CVD. We examine the association of lipoprotein subfractions with niacin and changes in coronary stenosis and CVD event risk. One hundred and seven patients from 2 previous studies using niacin in combination with either statin or bile acid-binding resin were selected to evaluate changes in lipoproteins separated by density-gradient ultracentrifugation to progression of coronary artery disease as assessed by quantitative coronary angiography. Improvement in coronary stenosis was significantly associated with the decrease of cholesterol in the dense low-density lipoprotein (LDL) particles and across most of the intermediate density lipoprotein (IDL) and very low density lipoprotein particle density range, but, not with any of the HDL fraction or of the more buoyant LDL fractions. Event-free survival was significantly associated with the decrease of cholesterol in the dense LDL and IDL; there was no association with changes in cholesterol in the HDL and buoyant LDL fractions. Niacin combination therapy raises HDL cholesterol and decreases dense LDL and IDL cholesterol levels. Changes in LDL and IDL are related to improvement in CVD. Lipoprotein subfraction analysis should be performed in larger studies utilizing niacin in combination with statins.

Nutraceuticals in dyslipidemia management

With the ever increasing epidemic of obesity, diabetes and hypertension among young adults, the risk of mortality and morbidity due to atherosclerotic heart disease is gradually increasing. Dyslipidemia is an additional risk factor for cardiovascular disease. Nutraceutical supplements can provide valid alternate to patients who are intolerant to statins or patients preferring alternative treatments. The combination of a lipid lowering diet and scientifically proven nutraceutical supplements can significantly reduce low density lipoprotein (LDL) cholesterol, increase LDL particle size, decreased LDL particle number decreased triglycerides and increased high density lipoprotein (HDL) particles. In addition, they address lipid induced vascular damage by suppressing inflammation, oxidative stress and immune response leading to additional antihypertension, antidiabetic properties. The current article reviews the evidence in support of different dietary supplements and their lipid lowering beneficial effects.

First Detection of Hypercholesterolemia Causing ApoB-100 R3527Q Mutation in a Family in Greece

Familial defective apolipoprotein B (FDB) is an autosomal dominant genetic disorder causing hypercholesterolemia in affected patients. It is occurring due to mutation of apoB gene leading to a decreased low density lipoprotein (LDL) particles clearance. The R3527Q mutation is one of the disease’s causative mutations. Data support mutation’s origin 6000-7000 years ago in Central Europe and its prevalence is decreasing in relation to the distance from where initially occurred. Until now, the presence of R3527Q mutation had not been confirmed in Greece. This is the first report of FDB due to R3527Q mutation in a family in Northwest Greece.

Abstract 263: Isotopic Labeling and Chemical Cross-Linking Reveals Intermolecular Contacts and Dynamics of Apolipoprotein A-I Structure in Reconstituted HDL Particles

Apolipoprotein (apo)A-I has been proposed to adopt a number of different, but related, structures when in contact with lipid. The technique of chemical cross-linking has recently been used to determine the spatial relationships between two molecules of apoA-I in reconstituted high density lipoprotein (rHDL) particles. However, this technique is limited in that it cannot unequivocally distinguish between intramolecular and intermolecular contacts. To address this issue, we have produced two forms of recombinant apoA-I that contain the naturally occurring isotope of nitrogen (N14) or a stable isotope (N15). These forms were mixed 1:1 and then used to produce reconstituted HDL particles with synthetic lipids. The resulting mass shifts (readily detectable in the mass spectrometer) were exploited to unambiguously distinguish between intramolecular (N14 to N14 or N15 to N15) and intermolecular (N14 to N15) cross-linked peptides. An additional benefit of this approach was the ability to identify cross-links with high certainty without the need for peptide fragmentation, allowing for dramatic increases in method sensitivity. We studied highly homogeneous rHDL particles made with the fully saturated phospholipid palmitoyl steroyl phosphatidylcholine (PSPC) to minimize apoA-I conformational dynamics. These particles were 98Å in diameter, contained two molecules of apoA-I, approximately 155 molecules of PC, and were discoidal in shape by cryo EM. We identified 30 cross-links (17 intramolecular, 13 intermolecular) that were overall consistent with the double belt model in which both apoA-I molecules wrap around a bilayer of lipids in an antiparallel orientation. Unambiguous intramolecular linkages between the N- and C-terminal regions of apoA-I were inconsistent with the ‘double super helix’ variant of the belt model. We identified several low abundance cross-links that suggest that the N-terminus may be conformationally dynamic and may spend some time folded back across the molecule as proposed for the ‘belt and buckle’ belt model.