Inverse Correlation of Cholesterol Efflux Capacity with Peripheral Plaque Volume Measured by 3D Ultrasound.

INTRODUCTION: Cardiovascular disease (CVD) is a systemic multifocal illness called atherosclerosis that causes artery constriction and blockage. By causing cholesterol to build up in the artery wall, hypercholesterolemia is a major factor in the pathophysiology of atherosclerotic plaque development. Reverse cholesterol transport is the process of transporting cholesterol from the periphery back to the liver through cholesterol efflux mediated by high-density lipoprotein (HDL). It was suggested that the cholesterol efflux capacity (CEC), which is inversely linked with cardiovascular risk, can serve as a stand-in measure for reverse cholesterol transport. In this work, we sought to investigate a potential link between the peripheral plaque volume (PV) and CEC. METHODS: Since lipid-lowering therapy interferes with CEC, we performed a cross-sectional study of 176 patients (48.9% females) with one cardiovascular risk factor or known CVD that did not currently take lipid-lowering medication. CEC was determined using cAMP-treated 3H-cholesterol-labeled J774 cells. Cholesterol ester transfer protein (CETP)-mediated cholesterol ester transfer was measured by quantifying the transfer of cholesterol ester from radiolabeled exogenous HDL cholesterol to Apolipoprotein B-containing lipoproteins. PV in the carotid and the femoral artery, defined as the total PV, was measured using a 3D ultrasound system equipped with semi-automatic software. RESULTS: In our patients, we discovered an inverse relationship between high total PV and CEC (p = 0.027). However, there was no connection between total PV and low-density lipoprotein cholesterol, lipoprotein (a), or CETP-mediated cholesterol ester transfer. CONCLUSION: In patients not receiving lipid-lowering treatment, CEC inversely correlates with peripheral atherosclerosis, supporting its role in the pathophysiology of atherosclerosis.

Independent Effects of Kidney Function and Cholesterol Efflux on Cardiovascular Mortality.

BACKGROUND: Impaired renal function is associated with cardiovascular and all-cause mortality. In the general population, HDL-cholesterol is associated with cardiovascular events, which is not true in patients with chronic kidney disease (CKD). This has been attributed to abnormal HDL function in CKD. METHODS: In this study, we analyzed the association of genetic markers for kidney function with cholesterol efflux capacity as one of the major HDL functions, as well as with cardiovascular mortality, in 2469 patients of the Ludwigshafen Risk and Cardiovascular Health Study who all underwent coronary angiography. RESULTS: A genetic score of 53 SNPs associated with GRF and the uromodulin SNP rs12917707 were inversely correlated with cholesterol efflux capacity. This was in line with the observed association between cholesterol efflux capacity and kidney function in these patients. Adjustment for eGFR and uromodulin as markers of kidney function did not affect the relationship between cholesterol efflux and cardiovascular mortality. CONCLUSIONS: Our data propose the view that cholesterol efflux and kidney function are exerting their effects on cardiovascular mortality via different and independent pathways. Decreased cholesterol efflux may therefore not mediate the effects of impaired kidney function on cardiovascular mortality.

Adipocyte GPX4 protects against inflammation, hepatic insulin resistance and metabolic dysregulation.

OBJECTIVES: Metabolic inflammation is a hallmark of obesity and related disorders, afflicting substantial morbidity and mortality to individuals worldwide. White visceral and subcutaneous adipose tissue not only serves as energy storage but also controls metabolism. Adipose tissue inflammation, commonly observed in human obesity, is considered a critical driver of metabolic perturbation while molecular hubs are poorly explored. Metabolic stress evoked by e.g. long-chain fatty acids leads to oxidative perturbation of adipocytes and production of inflammatory cytokines, fuelling macrophage infiltration and systemic low-grade inflammation. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation, accumulation of oxygen-specific epitopes and cell death, collectively referred to as ferroptosis. Here, we explore the function of adipocyte GPX4 in mammalian metabolism. METHODS: We studied the regulation and function of GPX4 in differentiated mouse adipocytes derived from 3T3-L1 fibroblasts. We generated two conditional adipocyte-specific Gpx4 knockout mice by crossing Gpx4fl/fl mice with Adipoq-Cre+ (Gpx4-/-AT) or Fabp4-Cre+ (Gpx4+/-Fabp4) mice. Both models were metabolically characterized by a glucose tolerance test and insulin resistance test, and adipose tissue lipid peroxidation, inflammation and cell death were assessed by quantifying oxygen-specific epitopes, transcriptional analysis of chemokines, quantification of F4/80+ macrophages and TUNEL labelling. RESULTS: GPX4 expression was induced during and required for adipocyte differentiation. In mature adipocytes, impaired GPX4 activity spontaneously evoked lipid peroxidation and expression of inflammatory cytokines such as TNF-α, interleukin 1ß (IL-1ß), IL-6 and the IL-8 homologue CXCL1. Gpx4-/-AT mice spontaneously displayed adipocyte hypertrophy on a chow diet, which was paralleled by the accumulation of oxygen-specific epitopes and macrophage infiltration in adipose tissue. Furthermore, Gpx4-/-AT mice spontaneously developed glucose intolerance, hepatic insulin resistance and systemic low-grade inflammation, when compared to wildtype littermates, which was similarly recapitulated in Gpx4+/-Fabp4 mice. Gpx4-/-AT mice exhibited no signs of adipocyte death. CONCLUSION: Adipocyte GPX4 protects against spontaneous metabolic dysregulation and systemic low-grade inflammation independent from ferroptosis, which could be therapeutically exploited in the future.

Cholesterol Efflux Capacity Associates with the Ankle-Brachial Index but Not All-Cause Mortality in Patients with Peripheral Artery Disease.

BACKGROUND: Cholesterol efflux is an important mechanism by which high-density lipoproteins (HDLs) protect against cardiovascular disease. As peripheral artery disease (PAD) is associated with high mortality rates, mainly due to cardiovascular causes, we investigated whether cholesterol efflux capacity (CEC) of apolipoprotein B (apoB)-depleted plasma, a widely used surrogate of HDL function, may serve as a predictive marker for mortality in this patient population. METHODS: In this prospective single-center study (median follow-up time: 9.3 years), apoB-containing lipoproteins were precipitated from plasma of 95 patients with PAD and incubated with J744-macrophages, which were loaded with radiolabeled cholesterol. CEC was defined as the fractional radiolabel released during 4 h of incubation. RESULTS: Baseline CEC was lower in PAD patients that currently smoked (p = 0.015) and had a history of myocardial infarction (p = 0.011). Moreover, CEC showed a significant correlation with HDL-cholesterol (p = 0.003) and apolipoprotein A-I levels (p = 0.001) as well as the ankle-brachial index (ABI, p = 0.018). However, CEC did not differ between survivors and non-survivors. Neither revealed Kaplan-Meier and Cox regression analyses any significant association of CEC with all-cause mortality rates. CONCLUSION: Taken together, CEC is associated with ABI but does not predict all-cause mortality in patients with PAD.

Matcha Green Tea Powder does not Prevent Diet-Induced Arteriosclerosis in New Zealand White Rabbits Due to Impaired Reverse Cholesterol Transport.

INTRODUCTION: Green tea is associated with decreased risk for cardiovascular disease and stroke. Matcha is a special kind of powdered green tea known for its use in the Japanese tea ceremony. Due to its influence on lipoprotein parameters, it has been postulated to exert antiatherogenic effects. This study investigates whether it modulates the high-density lipoprotein (HDL) function and thereby influences the atherogenic process in an animal model with a strong influence on humans' situation. METHODS AND RESULTS: After a pretreatment phase based on a standard diet, 10 female New Zealand White (NZW) rabbits are fed a high-fat diet for 20 weeks. The treatment group is additionally administered 1% matcha during the whole experiment. Long-term matcha treatment leads to lowered HDL cholesterol, impaired cholesterol transport manifested by reduced in vitro cholesterol efflux capacity, reduced cholesteryl ester transfer protein (CETP)-mediated cholesterol ester (CE) transfer between HDL and triglyceride-rich particles, and reduced macrophage-specific in vivo transfer, where ian increased absorption of cholesterol in the liver but a decreased secretion into bile is observed. Pulse wave velocity, assessed by nuclear magnetic resonance, is increased in matcha-treated animals, and a similar trend is observed for atherosclerotic lesion formation. CONCLUSION: Long-term matcha green tea treatment of hypercholesterolemic rabbits cause impaired reverse cholesterol transport and increased vascular stiffness, and susceptibility for atherosclerotic lesion development.

Cholesterol Efflux Capacity and Cardiovascular Disease: The Ludwigshafen Risk and Cardiovascular Health (LURIC) Study.

(1) Background and Aims: Efforts to reduce coronary artery disease (CAD) by raising high-density lipoprotein (HDL) cholesterol (HDL-C) have not been uniformly successful. A more important factor than HDL-C may be cellular cholesterol efflux mediated by HDL, which has been shown to be associated with CAD. In this report, we analyzed the influence of cardiovascular biomarkers and risk factors on cholesterol efflux in a prospective observational study of patients referred to coronary angiography. (2) Methods: HDL-mediated efflux capacity was determined for 2468 participants of the Ludwigshafen Risk and Cardiovascular Health (LURIC) study who were referred to coronary angiography at baseline between 1997 and 2000. Median follow-up time was 9.9 years. Primary and secondary endpoints were cardiovascular and all-cause mortality, respectively. (3) Results: Cholesterol efflux strongly correlated with HDL-related markers including HDL cholesterol, HDL phospholipids, and apolipoproteins AI and AII, as well as HDL particle concentration, which was not seen for low density lipoprotein (LDL) markers including LDL cholesterol and apoB. Cholesterol efflux was associated negatively with C-reactive protein (CRP), fibrinogen, interleukin-6 (IL-6), and serum amyloid A. Cardiovascular mortality was higher in patients in the lowest cholesterol efflux quartile. This association was weakened, but not fully abolished, after adjustment for HDL cholesterol. (4) Conclusions: We demonstrate that cholesterol efflux was associated with HDL-composition as well as inflammatory burden in patients referred for coronary angiography, and that this inversely predicts cardiovascular mortality independently of HDL cholesterol.

HDL cholesterol efflux capacity is inversely associated with subclinical cardiovascular risk markers in young adults: The cardiovascular risk in Young Finns study.

The atherogenic process begins already in childhood and progresses to symptomatic condition with age. We investigated the association of cholesterol efflux capacity (CEC) and vascular markers of subclinical atherosclerosis in healthy, young adults. CEC was determined in 2282 participants of the Young Finns study using cAMP treated 3H-cholesterol-labeled J774 cells. The CEC was correlated to baseline and 6-year follow-up data of cardiovascular risk factors and ultrasound measurements of arterial structure and function. CEC was higher in women, correlated with total cholesterol, HDL-C, and apolipoprotein A-I, but not with LDL-C or apolipoprotein B. Compared to the lowest CEC quartile, the highest CEC quartile was significantly associated with high CRP levels and inversely associated with adiponectin. At baseline, high CEC was associated with decreased flow-mediated dilation (FMD) and carotid artery distensibility, as well as an increased Young's modulus of elasticity, indicating adverse changes in arterial structure, and function. The association reversed with follow-up FMD data, indicating the interaction of preclinical parameters over time. A higher CEC was directly associated with a lower risk of subclinical atherosclerosis at follow-up. In young and healthy subjects, CEC was associated with important lipid risk parameters at baseline, as in older patients and CAD patients, but inversely with early risk markers for subclinical atherosclerosis.

Common APOC3 variants are associated with circulating ApoC-III and VLDL cholesterol but not with total apolipoprotein B and coronary artery disease.

BACKGROUND AND AIMS: Very rare loss-of-function mutations in the apolipoprotein C3 (APOC3) gene have been associated with low circulating apoC-III, low triglycerides, and reduced cardiovascular risk. We aimed to analyze the impact of common APOC3 variants on key parameters of lipid metabolism and coronary artery disease in the largest sample so far. METHODS: Common variants in APOC3 were tested for associations with circulating apoC-III, lipids, and apolipoprotein B (apoB) in 3041 participants of the LUdwigshafen RIsk and Cardiovascular health study (LURIC). These variants were then tested for associations with coronary artery disease in a meta-analysis comprising up to 332,389 participants of the CARDIOGRAMplusC4D consortium and the UK Biobank. RESULTS: The mean (standard deviation) apoC-III concentration was 14.6 (5.1) mg/dl. Seven common variants in APOC3 (rs734104, rs4520, rs5142, rs5141, rs5130, rs5128, and rs4225) were associated with circulating apoC-III (all p < 0.05). The alleles that modestly raised apoC-III were also associated with markedly higher total triglycerides and very low density lipoprotein (VLDL) triglycerides and cholesterol (all p < 0.05), but not with low density lipoprotein (LDL) cholesterol and total apoB (all p > 0.05). These variants were not associated with coronary artery disease in the CARDIOGRAMplusC4D consortium and the UK Biobank (all p > 0.1). CONCLUSIONS: Modest, genetically caused elevations of apoC-III are associated with a marked increase of triglyceride-rich lipoproteins but not with an increase of LDL cholesterol, total apoB, and coronary artery disease. Whether effective inhibition of apoC-III production with antisense oligomers will be instrumental to reduce cardiovascular risk remains to be demonstrated.

N-Phenethyl Substitution in 14-Methoxy-N-methylmorphinan-6-ones Turns Selective µ Opioid Receptor Ligands into Dual µ/δ Opioid Receptor Agonists.

Morphine and structurally-derived compounds are µ opioid receptor (µOR) agonists, and the most effective analgesic drugs. However, their usefulness is limited by serious side effects, including dependence and abuse potential. The N-substituent in morphinans plays an important role in opioid activities in vitro and in vivo. This study presents the synthesis and pharmacological evaluation of new N-phenethyl substituted 14-O-methylmorphinan-6-ones. Whereas substitution of the N-methyl substituent in morphine (1) and oxymorphone (2) by an N-phenethyl group enhances binding affinity, selectivity and agonist potency at the µOR of 1a and 2a, the N-phenethyl substitution in 14-methoxy-N-methylmorphinan-6-ones (3 and 4) converts selective µOR ligands into dual µ/δOR agonists (3a and 4a). Contrary to N-methylmorphinans 1-4, the N-phenethyl substituted morphinans 1a-4a produce effective and potent antinociception without motor impairment in mice. Using docking and molecular dynamics simulations with the µOR, we establish that N-methylmorphinans 1-4 and their N-phenethyl counterparts 1a-4a share several essential receptor-ligand interactions, but also interaction pattern differences related to specific structural features, thus providing a structural basis for their pharmacological profiles. The emerged structure-activity relationships in this class of morphinans provide important information for tuning in vitro and in vivo opioid activities towards discovery of effective and safer analgesics.

Structure-function relationships of HDL in diabetes and coronary heart disease.

High-density lipoproteins (HDL) contain hundreds of lipid species and proteins and exert many potentially vasoprotective and antidiabetogenic activities on cells. To resolve structure-function-disease relationships of HDL, we characterized HDL of 51 healthy subjects and 98 patients with diabetes (T2DM), coronary heart disease (CHD), or both for protein and lipid composition, as well as functionality in 5 cell types. The integration of 40 clinical characteristics, 34 nuclear magnetic resonance (NMR) features, 182 proteins, 227 lipid species, and 12 functional read-outs by high-dimensional statistical modeling revealed, first, that CHD and T2DM are associated with different changes of HDL in size distribution, protein and lipid composition, and function. Second, different cellular functions of HDL are weakly correlated with each other and determined by different structural components. Cholesterol efflux capacity (CEC) was no proxy of other functions. Third, 3 potentially novel determinants of HDL function were identified and validated by the use of artificially reconstituted HDL, namely the sphingadienine-based sphingomyelin SM 42:3 and glycosylphosphatidylinositol-phospholipase D1 for the ability of HDL to inhibit starvation-induced apoptosis of human aortic endothelial cells and apolipoprotein F for the ability of HDL to promote maximal respiration of brown adipocytes.

APOE-knockout in rabbits causes loss of cells in nucleus pulposus and enhances the levels of inflammatory catabolic cytokines damaging the intervertebral disc matrix.

Rabbits with naturally high levels of cholesterol ester transfer protein (CETP), unlike rodents, have become an interesting animal model for the study of lipid metabolism and atherosclerosis, as they have similarities to humans in lipid metabolism, cardiovascular physiology and susceptibility to develop atherosclerosis. Rodents, such as mice, are not prone to atherosclerosis as they lack the mass and activity of CETP, as a key player in lipoprotein metabolism. Recently, APOE-knockout in rabbits has been shown to promote atherosclerosis and associated premature IVD degeneration that mimic the symptoms of atherosclerosis and structural changes of IVDs in humans. Here we examined whether APOE-knockout promoted IVD degeneration in rabbits is associated with imbalanced inflammatory catabolic activities, as the underlying problem of biological deterioration that mimic the symptoms of advanced IVD degeneration in humans. We analysed in lumbar nucleus pulposus (NP) of APOE-knockout rabbits the cell viabilities and the intracellular levels of inflammatory, catabolic, anti-catabolic and anabolic proteins derogating IVD matrix. Grades of IVD degeneration were evaluated by magnetic resonance imaging. NP cells were isolated from homozygous APOE-knockout and wild-type New Zealand White rabbits of similar age. Three-dimensional cell culture with low-glucose was completed in alginate hydrogel. Cell proliferation and intracellular levels of target proteins were examined by MTT and ELISA assays. Alike human NP cells of different disc degeneration grades, NP cells of APOE-knockout and wild-type rabbits showed significantly different in vivo cell population densities (p<0.0001) and similar in vitro proliferation rates. Furthermore, they showed differences in overexpression of selective inflammatory and catabolic proteins (p<0.0001) similar to those found in human NP cells of different disc degeneration grades, such as IL-1ß, TNF-α, ADAMTS-4, ADAMTS-5 and MMP-3. This study showed that premature IVD degeneration in APOE-knockout rabbits was promoted by the accumulation of selective inflammatory catabolic factors that enhanced imbalances between catabolic and anabolic factors mimicking the symptoms of advanced IVD degeneration in humans. Thus, APOE-knockout rabbits could be used as a promising model for therapeutic approaches of degenerative disc disorders.

A Novel Candidate for Prevention and Treatment of Atherosclerosis: Urolithin B Decreases Lipid Plaque Deposition in apoE-/- Mice and Increases Early Stages of Reverse Cholesterol Transport in ox-LDL Treated Macrophages Cells.

SCOPE: HDL cholesterol is inversely related to the incidence of atherosclerosis. Polyphenols including ellagitannins have been shown to exert antiatherogenic properties. Urolithin B is formed from ellagitannins by components of the gut microbiota, and urolithins might be involved in beneficial effects against cardiovascular diseases in vitro. In this study, the influence of urolithin B on several parameters involved in the lipid plaque deposition and the reverse cholesterol transport is investigated. METHODS AND RESULTS: In apoE-/- mice and two different macrophage cell lines, the influence of urolithin B and its phase II conjugated metabolite on lipid plaque deposition, cholesterol uptake, and expression of ABCA1 and SR-BI is tested. It is shown that urolithin B decreases lipid plaque deposition, both urolithin B and urolithin B sulfate modulate expression of SR-BI and ABCA1, and cholesterol efflux increases from cholesterol laden macrophages to HDL particles as well as to reverse lipid uptake by stimulated THP-1 macrophages. CONCLUSIONS: Urolithin B can decrease lipid plaque deposition, and urolithin B and urolithin B sulfate are able to induce reverse cholesterol transport by influencing expression of key proteins of this pathway. Urolithin B may represent the basis for development of new drugs for prevention and treatment of atherosclerosis in humans.

HDL cholesterol efflux capacity and cholesteryl ester transfer are associated with body mass, but are not changed by diet-induced weight loss: A randomized trial in abdominally obese men.

BACKGROUND AND AIMS: Obesity is associated with a lower HDL-mediated cholesterol efflux from macrophages and a higher CETP (cholesteryl ester transfer protein) activity, but effects of weight loss are not clear. In addition, associations with visceral and subcutaneous adipose tissue are not known. We therefore investigated effects of diet-induced weight loss on HDL-mediated cholesterol efflux and cholesterol ester (CE) transfer in abdominally obese men. Differences between normal-weight and abdominally obese men were also examined. METHODS: Twenty-five apparently healthy, normal-weight men (waist circumference: <94 cm) and 52 abdominally obese men (waist circumference: 102-110 cm) were included. Abdominally obese subjects were randomly allocated to a dietary weight-loss intervention group or a no-weight loss control group. Individuals from the intervention group followed a very-low-calorie diet for 6 weeks to obtain a waist circumference below 102 cm, followed by a 2-week weight-stable period. Cholesterol efflux was measured in BODIPY-labeled murine J774 macrophages. CE transfer was measured by quantifying the transfer of CE from radiolabeled exogenous HDL to apoB-containing lipoproteins. RESULTS: Cholesterol efflux capacity was 9 percentage point (pp) lower in abdominally obese than in normal-weight men (p≤0.001), while CE transfer was 5 pp higher (p≤0.01). Diet-induced weight-loss of 10.3 kg did not change cholesterol efflux and CE transfer. In addition, stepwise regression analysis did not suggest that the different fat depots are differently related to efflux capacity and CE transfer. CONCLUSIONS: After a 2-week weight-stable period, dietary weight loss of 10 kg did not improve ABCA1-mediated cholesterol efflux and CE transfer in abdominally obese men.

Subclases de Lipoproteínas de Alta Densidad, Enfermedad Coronaria y Mortalidad Cardiovascular / High Density Lipoprotein Subclasses, Coronary Artery Disease, and Cardiovascular Mortality

Antecedentes: La relación inversa entre el colesterol HDL y la mortalidad cardiovascular se debilita en presencia de enfermedad coronaria (EC). El objetivo de este trabajo fue investigar las asociaciones de las concentraciones de partículas de HDL con la mortalidad cardiovascular y el impacto de la EC en estas asociaciones. También se buscó evaluar comparativamente las concentraciones de colesterol HDL y partículas de HDL en la predicción de la mortalidad cardiovascular. Métodos: Las concentraciones totales de HDL y sus sub-clases se midieron mediante espectroscopía de resonancia magnética nuclear en 2.290 participantes del estudio LUdwigshafen RIsk and Cardiovascular Health remitidos para angiografía coronaria. Los participantes fueron seguidos prospectivamente durante una mediana (rango intercuartílico) con una duración de 10,0 (6,1-10,6) años. Resultados: La media de la edad (DE) de los participantes (1.575 hombres, 715 mujeres) fue de 62,9 (10,4) años, índice de masa corporal 27,6 (4,1) kg/m², colesterol-HDL 39 (11) mg/dL [1 (0,29) mmol/L], y la concentración total de partículas de HDL 24,1 (5,8) μmol/L. Cuatroscientos treinta y cuatro de los participantes murieron de enfermedad cardiovascular. En análisis multivariados, los tercilos de las concentraciones totales de partículas de HDL se relacionaron inversamente con la mortalidad cardiovascular (Hazard Ratio para 3° frente a 1° tercilo = 0,55; p<0,001). Esta asociación fue mediada principalmente por las partículas de HDL pequeñas (p<0,001). La adición a los modelos de predicción multivariada de las concentraciones de partículas HDL totales o pequeñas, en lugar de colesterol HDL, mejoró las métricas de rendimiento para predicción de mortalidad cardiovascular. La presencia de EC no tuvo impacto en las asociaciones entre las concentraciones de partículas de HDL y la mortalidad cardiovascular. Conclusiones: La alta concentración de partículas de HDL se encuentra asociada con una disminución de la mortalidad cardiovascular de manera consistente e independiente de la EC. Sin embargo, si esta relación inversa entre la concentración de partículas de HDL y la mortalidad cardiovascular se puede traducir en nuevas estrategias terapéuticas está aún bajo investigación.

Background: The inverse relationship between HDL cholesterol and cardiovascular mortality is weakened in coronary artery disease (CAD). We aimed to investigate the associations of HDL particle concentrations with cardiovascular mortality and the impact of CAD on these associations. We also sought to comparatively evaluate HDL cholesterol and HDL particle concentrations in predicting cardiovascular mortality. Methods: Total and subclass HDL particle concentrations were measured by nuclear magnetic resonance spectroscopy in 2,290 participants of the LUdwigshafen RIsk and Cardiovascular Health study referred for coronary angiography. The participants were prospectively followed over a median (interquartile range) duration of 10.0 (6.1-10.6) years. Results: The mean (SD) age of the participants (1,575 males, 715 females) was 62.9 (10.4) years, body mass index 27.6 (4.1) kg/m², HDL cholesterol 39 (11) mg/dL [1 (0.29) mmol/L], and total HDL particle concentration 24.1 (5.8) μmol/L. 434 persons died from cardiovascular diseases. In multivariate analyses, tertiles of total HDL particle concentrations were inversely related to cardiovascular mortality (HR for 3rd vs. 1st tertile = 0.55, P<0.001). This association was primarily mediated by small HDL particles (P<0.001). Adding total or small HDL particle concentrations rather than HDL cholesterol to multivariate prediction models improved performance metrics for cardiovascular mortality. The presence of CAD had no impact on the associations between HDL particle concentrations and cardiovascular mortality. Conclusions: High HDL particle concentration is consistently and independently of CAD associated with decreased cardiovascular mortality. Whether the inverse relationship between HDL particle concentration and cardiovascular mortality may be translated into novel therapies is under investigation.

Expression, Purification, and Biochemical Characterization of Human Afamin.

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.

Determinants of cholesterol efflux capacity in humans.

High-density lipoproteins (HDL) play an important role in the reverse cholesterol transport (RCT) pathway, contributing to the efflux of excess cellular cholesterol. However, the classic hypothesis that raising HDL-cholesterol (HDL-C) concentrations reduces cardiovascular disease (CVD) risk has been challenged by recent intervention studies. Nowadays, improving cholesterol efflux capacity of HDL particles is considered to be a better target for the prevention of CVD. We will first briefly summarize assays that have been developed in order to quantify HDL-mediated cholesterol efflux from macrophages. However, the main purpose of this review is to discuss factors and ABC-transporters that are associated with HDL-mediated cholesterol efflux, such as HDL particle characteristics (i.e. HDL size and composition), subjects' characteristics (i.e. gender, BMI and age), HDL-C raising drugs, lifestyle, genetic background, as well as acute and low-grade systemic inflammation. Results suggested that factors associated with small HDL particles efficiently promote cholesterol efflux via the ABCA1 transporter. This appears to contradict findings from epidemiological studies suggesting that in particular large HDL2 particles are related to a reduced CVD risk. It is therefore essential not only to understand targets to increase cholesterol efflux capacity, but also to prove causality between cholesterol efflux capacity and the prevention of CVD risk.

Knockout of Apolipoprotein E in rabbit promotes premature intervertebral disc degeneration: A new in vivo model for therapeutic approaches of spinal disc disorders.

Intervertebral disc (IVD) degeneration that accelerates the loss of disc structural and functional integrities is recognized as one of the major factors of chronic back pain. Cardiovascular risk factors, such as deficits of apolipoproteins that elevate the levels of cholesterol and triglycerides, are considered critical for the progress of atherosclerosis; notably in the abdominal aorta and its lumbar branching arteries that supply lumbar vertebrae and IVDs. Obstruction of the lumbar arteries by atherosclerosis is presumed to promote lumbar disc degeneration and low back pain. APOE-knockout rabbits have recently been shown to generate hyperlipidemia with increased levels of cholesterol and triglycerides that mimic the symptoms of atherosclerosis in humans. Here, we analysed IVD degeneration in the lumbar spines of ten homozygous APOE-knockout and four wild-type New Zealand White rabbits of matching age to prove accelerated IVD degeneration in APOE-knockout rabbits, since APOE-knockout rabbits could be a beneficial model for therapeutic approaches of degenerative IVD disorders. Experiments were performed using T1/T2-weighted magnetic resonance imaging, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, glucose-oxidase assay, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot. APOE-knockout lumbar spines showed more advanced IVD degeneration, obstructed lumbar arteries and lower enhancement of contrast agent in IVDs. Moreover, lower concentration of glucose, lower number of viable cells and lower concentrations of aggrecan, collagen II and higher concentration of collagen I were detected in APOE-knockout IVDs (p < 0.0001). APOE-knockout in rabbits could induce structurally deteriorating premature IVD degeneration that mimics the symptoms of accelerated IVD degeneration in humans. APOE-knockout rabbits could be used as beneficial model, as they can bypass the standard surgical interventions that are commonly applied in research animals for the induction of enhanced IVD degeneration. Their parallel use in therapeutic approaches of IVD disorders and atherosclerosis could reduce the number of research animals to be used and contribute to the principles of 3Rs (Replacement, Reduction and Refinement).

High-Density Lipoprotein Subclasses, Coronary Artery Disease, and Cardiovascular Mortality.

BACKGROUND: The inverse relationship between HDL cholesterol and cardiovascular mortality is weakened in coronary artery disease (CAD). We aimed to investigate the associations of HDL particle concentrations with cardiovascular mortality and the impact of CAD on these associations. We also sought to comparatively evaluate HDL cholesterol and HDL particle concentrations in predicting cardiovascular mortality. METHODS: Total and subclass HDL particle concentrations were measured by nuclear magnetic resonance spectroscopy in 2290 participants of the LUdwigshafen RIsk and Cardiovascular Health study referred for coronary angiography. The participants were prospectively followed over a median (interquartile range) duration of 10.0 (6.1-10.6) years. RESULTS: The mean (SD) age of the participants (1575 males, 715 females) was 62.9 (10.4) years; body mass index, 27.6 (4.1) kg/m2; HDL cholesterol, 39 (11) mg/dL [1 (0.29) mmol/L]; and total HDL particle concentration, 24.1 (5.8) µmol/L. Of the participants, 434 died from cardiovascular diseases. In multivariate analyses, tertiles of total HDL particle concentrations were inversely related to cardiovascular mortality (hazard ratio for third vs first tertile = 0.55, P < 0.001). This association was primarily mediated by small HDL particles (P < 0.001). Adding total or small HDL particle concentrations rather than HDL cholesterol to multivariate prediction models improved performance metrics for cardiovascular mortality. The presence of CAD had no impact on the associations between HDL particle concentrations and cardiovascular mortality. CONCLUSIONS: High HDL particle concentration is consistently and independently of CAD associated with decreased cardiovascular mortality. Whether the inverse relationship between HDL particle concentration and cardiovascular mortality may be translated into novel therapies is under investigation.

Symmetric dimethylarginine, high-density lipoproteins and cardiovascular disease.

AIMS: The vascular effects of high-density lipoproteins (HDL) differ under certain clinical conditions. The composition of HDL is modified in patients with chronic kidney disease (CKD). As a consequence, uremic HDL induces endothelial dysfunction. We have previously shown that accumulation of symmetric dimethylarginine (SDMA) in HDL causes these adverse effects of HDL in CKD. The aim of the study is to determine the impact of the accumulation of SDMA on the association between HDL and mortality. METHODS AND RESULTS: Mortality, renal function, serum SDMA and HDL-cholesterol (HDL-C) were assessed in the LURIC study including 3310 subjects undergoing coronary angiography. All-cause mortality was 30.0% during median follow-up of 9.9 years. Serum SDMA levels significantly predicted all-cause and cardiovascular mortality, and were significantly correlated with SDMA accumulation in HDL. Notably, higher serum SDMA was independently associated with lower cholesterol efflux (P = 0.004) as a measure of HDL functionality. In subjects with low SDMA levels, higher HDL-C was associated with significantly lower mortality. In contrast, in subjects with high SDMA, HDL-C was associated with higher mortality. These findings were confirmed in 1424 participants of the MONICA/KORA S3 cohort. Of note, we derived an algorithm allowing for calculation of biologically effective HDL-C' based on measured HDL-C and SDMA. We corroborated these clinical findings with invitro evidence showing that SDMA accumulation abolishes the anti-inflammatory and regenerative properties of HDL. CONCLUSION: The data identify SDMA as a marker of HDL dysfunction. These findings highlight on the pivotal role of SDMA accumulation in HDL as a mediator of pre-mature cardiovascular disease in patients with CKD.