HDL Particles Research Articles

AQP1 as a novel biomarker to predict prognosis and tumor immunity in glioma patients

Abstract Background Glioma is a kind of nervous system cancer with a low overall survival rate. Aquaporin 1 (AQP1) is linked to a number of cancers. Its prognostic relevance and immunological consequences in gliomas, however, are unclear. Objectives Our objective was to thoroughly examine the modified expression of AQP1, its prognostic significance, and its correlation with immune cells and markers to discover innovative molecular immunotherapy strategies for glioma patients. Methods RNA sequencing data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used. In addition, we used real-time reverse transcription polymerase chain reaction (RT-PCR) and Western Blot methods to monitor AQP1 expression in glioma tissues. Results AQP1 expression was greater in gliomas than in traumatized brain tissues. The increased AQP1 expression in gliomas was additionally confirmed through immunohistochemical analysis in the Human Protein Atlas (HPA) repository. An elevated level of AQP1 expression was identified as a separate determinant of the overall survival (OS) and prognosis of individuals with glioma. AQP1 expression was shown to be tightly linked to the tumor immune milieu, immune checkpoint blockade (ICB) and temozolomide drug reaction. In conclusion, the 50 genes that show coexpression with AQP1 indicate that the predominant functions and pathways are related to anterior pattern specification, pattern specification, regionalization, high-density lipoprotein particles, protein–lipid complexes, glycosaminoglycan binding, DNA-binding transcription repressor specific activation to RNA polymerase II, DNA-binding transcription repressor activity, nitrogen metabolism, alpha-linolenic acid metabolism, and fat digestion and absorption. Conclusions The results indicate that AQP1 could serve as both a predictive marker and a potential treatment target in glioma.

Functional characterization of missense variants affecting the extracellular domains of ABCA1 using a fluorescence-based assay

Excess cholesterol originating from nonhepatic tissues is transported within HDL particles to the liver for metabolism and excretion. Cholesterol efflux is initiated by lipid-free or lipid-poor apolipoprotein A1 interacting with the transmembrane protein ABCA1, a key player in cholesterol homeostasis. Defective ABCA1 results in reduced serum levels of HDL cholesterol, deposition of cholesterol in arteries, and an increased risk of early onset CVD. Over 300 genetic variants in ABCA1 have been reported, many of which are associated with reduced HDL cholesterol levels. Only a few of these have been functionally characterized. In this study, we have analyzed 51 previously unclassified missense variants affecting the extracellular domains of ABCA1 using a sensitive, easy, and low-cost fluorescence-based assay. Among these, only 12 variants showed a distinct loss-of-function phenotype, asserting their direct association with severe HDL disorders. These findings emphasize the crucial role of functional characterization of genetic variants in pathogenicity assessment and precision medicine. The functional rescue of ABCA1 loss-of-function variants through proteasomal inhibition or by the use of the chemical chaperone 4-phenylbutyric acid was genotype specific. Genotype-specific responses were also observed for the ability of apolipoprotein A1 to stabilize the different ABCA1 variants. In view of personalized medicine, this could potentially form the basis for novel therapeutic strategies.

High‐density lipoprotein particle size distribution among patients with Alzheimer’s disease, mild cognitive impairment, and age‐matched controls

AbstractBackgroundHigh‐density lipoproteins (HDL) are protective against several chronic diseases, including Alzheimer’s disease (AD). The size distribution of HDL particles is one of the determining factors of HDL function since HDL of various sizes subgroups play unique roles. Yet not much is known about HDL particle size distribution in the context of AD.MethodIn this study negative‐stained transmission electron microscopy (NS‐TEM) was used to explore the particle size distribution of isolated HDL samples from 194 subjects with AD (n = 71), mild cognitive impairment (MCI, n = 40), or no cognitive impairment (n = 83). HDL particles were isolated using density‐adjusted ultracentrifugation followed by size exclusion chromatography and analyzed by NS‐TEM followed by image analysis using the software ImageJ. Individual particle diameter data were obtained, grouped into different size‐based categories and the relative abundances of each subgroup were compared among diagnostic groups and across clinical parameters.ResultPatients with AD and MCI had a significantly higher percentage of 7‐8 nm HDL particles and a lower percentage of 9‐10 nm, 12‐13 nm, and 13‐14 nm particles. Body mass index, clinical dementia rating score, and white matter hyperintensity volume were positively correlated with the percentage of 7‐8 nm particles and negatively correlated with the percentage of 9‐10 nm particles, whereas executive score, semantic memory score, and verbal memory score were negatively correlated with the percentage of 7‐8 nm particles, and positively correlated with the percentage of 9‐10 nm particles.ConclusionThese findings show that specific alterations in HDL particle size distribution in patients with AD and MCI are linked with measures of cognitive function and may be a promising direction for the discovery of new biomarkers and therapeutic targets in AD.

Mechanisms of reduced Apolipoprotein E4 lipidation in iPSC‐derived astrocytes

AbstractBackgroundCarrying the apolipoprotein E ε4 (APOE4) allele is the highest known genetic risk factor for late‐onset Alzheimer’s disease (LOAD). Although how APOE4 leads to LOAD is not known, dysregulated endo‐lysosomal trafficking and lipid metabolism are key defects associated with APOE4. Cholesterol accumulation in glia instigates intracellular trafficking defects, increases cellular stress, and disrupts secretion. Additionally, enlarged endosomes were observed in the frontal lobes of APOE4‐carrying individuals, decades before the appearance of Aβ fibrilization. Therefore, we asked if cholesterol and endo‐lysosomal trafficking dysregulation in APOE4 carriers were due to differential lipidation of APOE3 and APOE4.MethodLocalization of ATP Binding Cassette Subfamily A Member 1 (ABCA1) in early, late, and recycling endosomal compartments were analyzed in human isogenic induced pluripotent stem cell (iPSC)‐derived neural cells and in cell lines treated with recombinant APOE in microscopic images. Membrane and cytoplasmic ABCA1 were compared using Western blots. Secreted high‐density lipoprotein (HDL) particles containing fluorescently tagged APOE3 or APOE4 were analyzed using ion‐mobility spectrometry. CS‐6253, an ABCA1 agonist peptide, was used to increase APOE lipidation.ResultThe protein levels of ABCA1, which mediates cholesterol and phospholipid efflux to the nascent apolipoprotein particles, were lower in APOE4/4 iPSC‐derived astrocytes compared to astrocytes derived from isogenic APOE3/3 iPSCs. Advanced image analysis revealed lower ABCA1 protein levels in Rab11+ recycling endosomes and less colocalization between ABCA1 and Rab7, a late endosome marker, in APOE4/4 astrocytes than APOE3/3 astrocytes. Consequently, less ABCA1 was detected at the cell membrane. Combined with previous data, these findings suggest APOE4 lipidation was reduced due to reduced ABCA1 activity. Additionally, BHK cells treated with recombinant APOE3 or APOE4 showed differences in ABCA1 localization that were altered by increasing APOE lipidation by activating ABCA1 via CS‐6253. Finally, fluorescently tagged APOE3 and APOE4 were incorporated in HDL particles and were secreted whose sizes were altered after CS‐6253 treatment, suggesting differences in lipidation levels.ConclusionWe demonstrated that diminished APOE4 lipidation is, at least in part, due to dysregulation of endo‐lysosomal trafficking of ABCA1 and that increasing APOE4 lipidation could alleviate cellular defects.

Transfer Proteins, Lipoprotein Remodeling, and Antioxidant Action on Pregnant Women: Comparison from the First to the Third Trimester of Gestation

Introduction: During pregnancy, maternal lipid metabolism undergoes several transformations originated by hormonal changes. There are two distinct phases: initial and late, finally driven to hypertriglyceridemia. Maternal hyperlipidemia, especially hypertriglyceridemia, is associated with an increased hepatic very-low-density lipoprotein (VLDL) synthesis stimulated by high estrogen levels during gestation. Objectives: This study aimed to evaluate the action of possible lipoproteins remodeling modulators, such as phospholipid and cholesteryl-ester transfer proteins (PLTP and CETP), paraoxonase-1 (PON-1), and apolipoproteins (apo), during gestation. Methods: An observational prospective cohort study composed of 40 pregnant women was conducted. Blood samples were collected in two moments: at the first and third trimesters of gestation, followed by the biochemical determination of apo A-I, apo B, lipid profile, PON-1, PLTP and CETP activities, and HDL particle size. Results: The majority of pregnant women in the third trimester showed dyslipidemia, mainly hypertriglyceridemia. In the third trimester of gestation, we observed an increase in CETP activity (70.5 ± 9.6 pmol/µL/h; p <0.001) and TG/HDL-C ratio (2.7 [2.0-3.8]), but on the other hand, PON-1 activity (65.4 [46.5-105] nMol of p-nitrophenol/min/mL) decreased. High-density level lipoprotein (HDL) particle size and PLTP activity did not differ in the two studied moments. The serum triglyceride concentration and CETP activity showed a positive linear correlation in the first trimester of gestation (r2=0.65, p<0.001). Conclusion: Data suggest that the CETP activity, antioxidant profile, and low-density lipoprotein (LDL) remodeling are modified by physiological metabolic changes during an uncomplicated gestational period.

Flipped C-Terminal Ends of APOA1 Promote ABCA1-Dependent Cholesterol Efflux by Small HDLs.

Cholesterol efflux capacity (CEC) predicts cardiovascular disease independently of high-density lipoprotein (HDL) cholesterol levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 (ATP-binding cassette transporter A1) pathway, but the underlying mechanisms are unclear. We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 (apolipoprotein A1) in the different particles, and the CECs of plasma and isolated HDLs. We quantified macrophage and ABCA1 CEC of 4 distinct sizes of reconstituted HDL. CEC increased as particle size decreased. Tandem mass spectrometric analysis of chemically cross-linked peptides and molecular dynamics simulations of APOA1, the major protein of HDL, indicated that the mobility of C-terminus of that protein was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs (like reconstituted HDLs) are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3- to 5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC. We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the 2 antiparallel molecules of APOA1 are "flipped" off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased cardiovascular disease risk. Thus, extra-small and small HDLs may be key mediators and indicators of the cardioprotective effects of HDL.

Effect of Exercise Training on Lipoprotein Subclass Particle Concentrations and Sizes in Older Women: Results from a Randomized Controlled Trial.

Evidence suggests that lipoprotein subclass particles are critical markers of cardiovascular disease (CVD) risk. Older women have increased CVD risk related to age. The purpose of this study was to determine whether low and moderate doses of exercise influence lipoprotein subclasses. Women (60-75 years) were randomized into groups for 16 weeks of moderate-intensity exercise training at a low or moderate dose (33.6 and 58.8 kJ/kg body weight weekly, respectively). Lipoprotein subclasses were determined by nuclear magnetic resonance spectroscopy before and after the training. The average weekly exercise duration was 109 and 164 min, for low- and moderate-dose groups, respectively. In the low-dose group, high-density lipoprotein particle (HDL-P) concentration decreased (Δ = -1.9 ± 3.1 µmol/L, mean ± SD, p = 0.002) and mean HDL-P size increased (Δ = 0.1 ± 0.3 nm, p = 0.028). In the moderate-dose group, mean HDL-P size (Δ = 0.1 ± 0.2 nm; p = 0.024) and low-density lipoprotein particle size increased (Δ = 0.4 ± 3.9 nm; p = 0.007). Baseline body mass index, peak oxygen consumption and age were associated with changes in a few lipoprotein subclasses. In this sample of inactive older women, moderate-intensity exercise training at a dose equivalent to or even lower than the minimally recommended level by public health agencies induced changes in lipoprotein subclasses in line with reduced CVD risk. However, higher doses are encouraged for greater health benefits.

Human HDL subclasses modulate energy metabolism in skeletal muscle cells

In addition to its antiatherogenic role, HDL reportedly modulates energy metabolism at the whole-body level. HDL functionality is associated with its structure and composition, and functional activities can differ between HDL subclasses. Therefore, we studied if HDL2 and HDL3, the two major HDL subclasses, are able to modulate energy metabolism of skeletal muscle cells. Differentiated mouse and primary human skeletal muscle myotubes were used to investigate the influences of human HDL2 and HDL3 on glucose and fatty uptake and oxidation. HDL-induced changes in lipid distribution and mRNA expression of genes related to energy substrate metabolism, mitochondrial function, and HDL receptors were studied with human myotubes. Additionally, we examined the effects of apoA-I and discoidal, reconstituted HDL particles on substrate metabolism. In mouse myotubes, HDL subclasses strongly enhanced glycolysis upon high and low glucose concentrations. HDL3 caused a minor increase in ATP-linked respiration upon glucose conditioning but HDL2 improved complex I–mediated mitochondrial respiration upon fatty acid treatment. In human myotubes, glucose metabolism was attenuated but fatty acid uptake and oxidation were markedly increased by both HDL subclasses, which also increased mRNA expression of genes related to fatty acid metabolism and HDL receptors. Finally, both HDL subclasses induced incorporation of oleic acid into different lipid classes. These results, demonstrating that HDL subclasses enhance fatty acid oxidation in human myotubes but improve anaerobic metabolism in mouse myotubes, support the role of HDL as a circulating modulator of energy metabolism. Exact mechanisms and components of HDL causing the change, require further investigation.

High-density lipoprotein remodeling affects the osmotic properties of plasma in goldfish under critical salinity.

To investigate the stress response and physiological adaptations of goldfish (Carassius auratus) to critical salinity (CS) waters, we analyzed high-density lipoprotein (HDL) stoichiometry, stress markers (cortisol, glucose), and plasma osmotic properties (Na+ , osmolality, water content) using ichthyology, biochemistry, and proteomics approaches. After 21 days of exposure to CS, plasma concentrations of cortisol, glucose, and Na+ increased, indicating stress. Total plasma osmolality (Osmtotal ) and osmolality generated by inorganic (Osminorg ) and organic osmolytes (Osmorg ) also increased, the latter by ~2%. We associated the increase of Osmorg with (1) increased metabolite concentration (glucose), (2) dissociation of HDL particles resulting in increased HDL number per unit plasma volume (~1.5-2-fold) and (3) increased HDL osmotic activity. HDL remodeling may be the reason for the redistribution of bound and free water in plasma, which may contribute to water retention in plasma and, at the same time, to hemodynamic disturbances under CS conditions. The study's findings suggest that HDL remodeling is an important mechanism for maintaining osmotic homeostasis in fish, which is consistent with current capillary exchange models in vertebrates.

Causal role of lipid metabolome on the risk of ischemic stroke, its etiological subtypes, and long-term outcome: A Mendelian randomization study

Background and aimsThe lipid profile is consistently associated with coronary artery disease (CAD) and ischemic stroke (IS). However, the lipoprotein subfractions have not been deeply explored in stroke subtypes, especially in IS outcome. MethodsWe performed two-sample Mendelian randomization (MR) analysis using 92 lipid traits measured by nuclear magnetic resonance in 115,000 subjects from the UK Biobank. Data for genetic associations with IS, its subtypes, and long-term outcome (LTO) were obtained from three cohorts of European ancestry: GIGASTROKE (73,652 cases, 1,234,808 controls), GODS (n = 1791) and GISCOME (n = 6165). Results obtained using CARDIoGRAMPlusC4D were used to identify differences with CAD. ResultsGenetically determined low concentration of medium high-density lipoprotein (HDL) particles (odds ratio (OR) = 0.92, 95% CI 0.88–0.96; p = 3.6 × 10−4) and its cholesterol content (OR = 0.92, 95% CI 0.88–0.96; p = 1.9 × 10−4) showed causal associations with an increased risk of stroke. Genetic predisposition to high apolipoprotein (apo)B to apoA-I ratio was causally associated with an increased risk of IS (OR = 1.12, 95% CI 1.06–1.18, p = 1.1 × 10−4), and a highly suggestive association was found between non-esterified cholesterol in low-density lipoprotein (LDL) and increased risk of atherothrombotic stroke (LAS) (OR = 1.35, 95% CI 1.10–1.66; p = 4.0 × 10−3). Low cholesterol in small and medium LDL was suggestively associated with poor LTO. ConclusionsOur results support that low medium HDL concentration was causally associated with an increased stroke risk, while high levels of non-esterified cholesterol in LDL were suggestively associated with an increased risk of LAS and with a better LTO.

Adherence to the Mediterranean diet and nuclear magnetic resonance spectroscopy biomarkers in older individuals at high cardiovascular disease risk: cross-sectional and longitudinal analyses

BackgroundThe Mediterranean diet (MedDiet) has been related to a decreased risk of cardiovascular disease (CVD) and diabetes. ObjectivesWe aimed to prospectively assess the relationship between adherence to the MedDiet and advanced lipoprotein subclass profiles and glucose metabolism and inflammation markers, as determined by nuclear magnetic resonance (NMR) spectroscopy. DesignWe conducted cross-sectional and longitudinal analyses within the framework of the PREvención con DIeta MEDiterránea study in 196 participants from the Reus-Tarragona center. Adherence to the MedDiet was assessed using a 14-item validated questionnaire [Mediterranean Diet Adherence Score (MEDAS)]. Plasma lipoprotein subclasses and molecular metabolite profiles were determined using NMR spectra collected on a Vantera Clinical Analyzer at baseline and after 1 y of follow-up. Baseline and 1-y categories of MEDAS were related to measures of lipoprotein atherogenicity and diabetes risk using multivariable-adjusted analysis of covariance models. ResultsCompared with participants in the lowest category of baseline MEDAS, those in the highest category showed higher concentrations of total high-density lipoprotein (HDL) particles and H1P HDL, lower concentrations of very low-density lipoprotein (VLDL)-triglyceride, smaller size of VLDL, and lower concentrations of very large VLDL, as well as lower concentrations of branched-chain amino acids, leucine, and GlycA and reduced Diabetes Risk Index (DRI) scores. In addition, participants who increased by 3 or more points in their 1-y MEDAS showed an increase in concentrations of H7P-HDL, H5P-HDL, and citrate, and reduced acetone and DRI scores compared with those with lesser adherence increases. ConclusionsIn older adults at high cardiometabolic risk, higher MEDAS was associated with modest beneficial changes in lipoprotein and glucose metabolism. The results suggest that lipoprotein subclass distribution and glycemic control are potential mechanisms behind the well-known salutary effects of MedDiet on CVD and diabetes risk. Future clinical trials exploring the effects of the MedDiet on advanced lipoprotein subclass profiles and glucose metabolism markers are needed to confirm the results of our study. Trial registration numberThis trial was registered at controlled-trials.com as ISRCTN35739639.

Synergistic effect of Interleukin-1beta and cholesterol efflux capacity on outcomes in acute myocardial infarction patients

Abstract Background Low cholesterol efflux capacity and elevated levels of Interleukin-1ß (IL-1ß) are both associated with residual cardiovascular risk in patients with acute myocardial infarction (MI) and may be used as new biomarkers to identify patients at higher cardiovascular risk. Both pathways are currently evaluated as new therapeutic targets in ongoing clinical trials. Objectives We evaluated potential synergetic effect of cholesterol efflux capacity and IL-1ß on recurrent major cardiovascular events (MACE) at one-year in patients with acute ST-segment elevation MI who underwent primary percutaneous coronary intervention. Methods Cholesterol efflux capacity and IL-1ß were measured among 2012 ST-segment elevation MI patients enrolled in a prospective cohort. The association of these biomarkers with the primary outcome of MACE (MI, Stroke, or cardiovascular death) was evaluated at one-year follow-up using a multivariate-cox proportional regression analysis. In addition, in-vitro analysis of anti-atherogenic properties of HDL particles was also performed. Results Patients with both, a reduced cholesterol efflux capacity and an elevated level of IL-1b, exhibited the highest risk of recurrent MACE at one year compared to patients with both high cholesterol efflux capacity and low IL-1b (adjusted HR: 3.28; 95% CI: 1.83 to 5.89, p<0.0001) (Figure). In this high-risk subgroup, patients exhibited HDL particles defective for ABCA1-dependent efflux capacity with enhanced pro-inflammatory activity as a potential explanation for our clinical findings. Conclusion Defective cholesterol efflux capacity and elevated IL-1β increase synergistically the residual cardiovascular risk in MI patients.

The concentration of small-diameter HDL particles (HDLp1) predicts the incidence of coronary calcium score in diabetic subjects without established cardiovascular disease

Abstract Introduction Type 2 diabetes (DM2) decisively influences the evolution of coronary atherosclerosis. However, this is a heterogeneous and poorly understood process. While in 50% of diabetics the chance of incident Coronary Calcium Score (CAC) in 5 years exceeds 40%, in 10% of diabetics the chance of incident CAC is less than 10%. To better understand the progression of coronary atherosclerosis measured by CAC in diabetics without cardiovascular disease, we studied the impact of the concentration of the small-diameter subfraction of HDL (HDLp1, 7 to 8nm). HDLp1 has been implicated as a central mediator of the anti-inflammatory effect of the HDL pool and could play a specific role in CAC incidence among diabetics. Methods From 2370 individual with two CAC evaluations and hydrogen spectroscopy/magnetic resonance (H1-NMR) plasma profile available, we found 461 diabetic individuals, non-users of statins, without previous cardiovascular disease, with CAC=0 in the first measurement and a second CAC measurement after an average period of 5 years. [HDLp1] concentration was measured by H1-NMR and divided into tertiles. The incidence of CAC was defined as an initial CAC of 0 followed by CAC>0 in the second measurement. Stepwise forward logistic regressions were used to identify risk factors for CAC incidence. Discrimination analysis was performed by calculating the area under the ROC curve (AUROC). Results A total of 143 (31.0%) patients had incident CAC. In stepwise logistic regression, alongside factors such as age, gender, systolic blood pressure, smoking, LDL-C, C-reactive protein and HbA1c, the 3rd tertile of [HDLp1] was associated with 55.2% (95% CI 15-110%, p =0.0039) and 140% (95% CI 32-341%, p<0.001) higher risk of CAC incidence compared to the 2nd tertile and 1st tertile, respectively. When compared to a model composed of 10 confounding variables (age, gender, systolic blood pressure, BMI, smoking, Lp(a), LDL-C,triglycerides, C-reactive protein and HbA1c) for predicting the incidence of CAC, the diameter of HDLp1 substantially increased the AUROC from 0.651 (95% CI 0.597-0.705) to 0.684 (95% CI 0.631–0.736), p=0.027 for the DeLong test comparing AUROCs. A restricted cubic spline demonstrated a strong association between [HDLp1] and CAC incidence. Conclusion The concentration of HDLp1 improves the predictive capacity for the incidence of CAC in diabetic individuals, non-users of statins, without cardiovascular disease.Spline HDLp1 concentration vs CAC incideROC for incident CAC

Abstract 17049: Specific Subspecies of High Density Lipoprotein are Associated With Arterial Stiffness in Youth With and Without Diabetes

Introduction: Early vascular aging (EVA), a risk factor for cardiovascular disease (CVD), is evident in youth with diabetes. The mechanisms responsible for EVA are not well understood. Causal relationship of HDL-C with CVD is doubtful, but HDL particle concentration (HDL-P) and HDL function (cholesterol efflux capacity, CEC) are associated with incident and prevalent CVD independent of HDL-C. We investigated whether these metrics associate with EVA in youth. Methods: We studied three cohorts of youth with T1D (n=173), T2D (n=214) and healthy controls (n=116). We quantified total HDL-P and 6 HDL subspecies by differential ion mobility, serum HDL CEC (total and ABCA1-specific), and investigated their association with arterial stiffness (tonometric carotid-femoral pulse wave velocity, PWV) with multivariate regression. Results: PWV was not associated with total HDL-P in any of the three groups, but was associated positively with concentration of extra-small (xsHDL, 7.6nm, r=0.22, P=0.004) in T1D, small (sHDL, 8.2nm) particles in T1D (r=0.31, P<0.0001) and T2D (r=0.17, P=0.02), and negatively with large HDL particles (lHDL, 11.5 nm) in T1D (r=-0.19, P=0.01). None of the HDL subspecies associated with PWV in healthy subjects. The association of sHDL with PVW remained significant after adjustment for age, sex, BMIz, mean arterial pressure (MAP), and diabetes status when analyzing T1D and controls or T2D and control subjects (Table 1). Both HDL-CEC metrics were significantly associated with higher PWV in T1D cohort but not T2D cohort, however, in multivariate models neither remained significant after adjusting the model for MAP (Table 1). Conclusions: A specific population of small HDL particles, but not HDL CEC, is associated with EVA, a predictor of CVD, independent of traditional risk factors and distending pressure (MAP). How the small HDL particles relate to EVA and whether they may be a novel target for intervention to prevent EVA in youth remains to be investigated.

Identification of genetic drivers of plasma lipoprotein size in the Diversity Outbred mouse population

Despite great progress in understanding lipoprotein physiology, there is still much to be learned about the genetic drivers of lipoprotein abundance, composition, and function. We used ion mobility spectrometry to survey 16 plasma lipoprotein subfractions in 500 Diversity Outbred mice maintained on a Western-style diet. We identified 21 quantitative trait loci (QTL) affecting lipoprotein abundance. To refine the QTL and link them to disease risk in humans, we asked if the human homologs of genes located at each QTL were associated with lipid traits in human genome-wide association studies. Integration of mouse QTL with human genome-wide association studies yielded candidate gene drivers for 18 of the 21 QTL. This approach enabled us to nominate the gene encoding the neutral ceramidase, Asah2, as a novel candidate driver at a QTL on chromosome 19 for large HDL particles (HDL-2b). To experimentally validate Asah2, we surveyed lipoproteins in Asah2−/− mice. Compared to wild-type mice, female Asah2−/− mice showed an increase in several lipoproteins, including HDL. Our results provide insights into the genetic regulation of circulating lipoproteins, as well as mechanisms by which lipoprotein subfractions may affect cardiovascular disease risk in humans.

Novel Size-Based High-Density Lipoprotein Subspecies and Incident Vascular Events.

Background High-density lipoprotein (HDL) particle concentration likely outperforms HDL cholesterol in predicting atherosclerotic cardiovascular events. Whether size-based HDL subspecies explain the atheroprotective associations of HDL particle concentration remains unknown. Our objective was to assess whether levels of specific size-based HDL subspecies associate with atherosclerotic cardiovascular disease in a multiethnic pooled cohort and improve risk prediction beyond traditional atherosclerotic cardiovascular disease risk factors. Methods and Results Seven HDL size-based subspecies were quantified by nuclear magnetic resonance (LP4 algorithm; H1=smallest; H7=largest) among participants without prior atherosclerotic cardiovascular disease in ARIC (Atherosclerosis Risk in Communities), MESA (Multi-Ethnic Study of Atherosclerosis), PREVEND (Prevention of Renal and Vascular Endstage Disease), and DHS (Dallas Heart Study) cohorts (n=15 371 people). Multivariable Cox proportional hazards models were used to evaluate the association between HDL subspecies and incident myocardial infarction (MI) or ischemic stroke at follow-up (average 8-10 years) adjusting for HDL cholesterol and risk factors. Improvement in risk prediction was assessed via discrimination and reclassification analysis. Within the pooled cohort (median age 57 years; female 54%; Black 22%) higher H1 (small) and H4 (medium) concentrations were inversely associated with incident MI (hazard ratio [HR]/SD, H1 0.88 [95% CI, 0.81-0.94]; H4 0.89 [95% CI, 0.82-0.97]). H4 but not H1 improved risk prediction indices for incident MI. Increasing H2 and H4 were inversely associated with improved risk prediction indices for composite end point of stroke, MI, and cardiovascular death (HR/SD, H2 0.94 [95% CI, 0.88-0.99]; H4 0.91 [95% CI, 0.85-0.98]). Levels of the large subspecies (H6 and H7) were not associated with any vascular end point. Conclusions Two of 7 HDL size-based subspecies modestly improved risk prediction for MI and composite vascular end points in a large multiethnic pooled cohort. These findings support assessment of precise HDL subspecies for future studies regarding clinical utility.

QiShenYiQi pill inhibits atherosclerosis by promoting TTC39B-LXR mediated reverse cholesterol transport in liver

BackgroundTetranucleotide repeat domain protein 39B (TTC39B) was found to combine with ubiquitin ligase E3, and promote the ubiquitination modification of liver X receptor (LXR), which led to the inhibition of reverse cholesterol transport and development of atherosclerosis. QiShenYiQi pill (QSYQ) is a modern Chinese patent drug for treating ischemic cardiovascular diseases, the underlying mechanism is found to promote the expression of LXR-α/ ATP-binding cassette transporter G5 (ABCG5) in the liver of atherosclerotic mice. PurposeThe aim of this study is to investigate the effect of QSYQ on TTC39B-LXR mediated reverse cholesterol transport in atherosclerotic mice. Study design and methodsMale apolipoprotein E gene knockout mice (7 weeks old) were fed with high-fat diet and treated with low dose of QSYQ (QSYQ-l, 0.3 g/kg·d), high dose of QSYQ (QSYQ-H, 1.2 g/kg·d) and LXR-α agonist (LXR-A, GW3965 10 mg/kg·d) for 8 weeks. C57BL/6 J mice were fed with normal diet and used as negative control. Oil red O staining, HE staining, ELISA, RNA sequencing, western blot, immunohistochemistry, RT-PCR, cell culture and RNA interference were performed to analyze the effect of QSYQ on atherosclerosis. ResultsHE staining showed that QSYQ reduced the atherosclerotic lesion significantly when compared to the control group. ELISA measurement showed that QSYQ decreased serum VLDL and increased serum ApoA1. Oil Red O staining showed that QSYQ reduced the lipid content of liver and protect liver function. Comparative transcriptome RNA-sequence of liver showed that DEGs after QSYQ treatment enriched in high-density lipoprotein particle, ubiquitin ligase complex, bile secretion, etc. Immunohistochemical staining and western blot proved that QSYQ increased the protein expression of hepatic SR-B1, LXR-α, LXR-β, CYP7A1 and ABCG5. Targeted inhibiting Ttc39b gene in vitro further established that QSYQ inhibited the gene expression of Ttc39b, increased the protein expression of SR-B1, LXR-α/β, CYP7A1 and ABCG5 in rat hepatocyte. ConclusionOur results demonstrated the new anti-atherosclerotic mechanism of QSYQ by targeting TTC39B-LXR mediated reverse cholesterol transport in liver. QSYQ not only promoted reverse cholesterol transport, but also improved fatty liver and protected liver function.

Lipid Handling Induces Ferroptosis and Conveys Sensitivity to Anti-BCMA Immunotherapy in Multiple Myeloma

Lipid metabolic changes in cancer are attracting increased attention. BAFF/APRIL axis is a multifaceted immune regulator of adipose tissue function and an emerging target for anti-BCMA therapy in multiple myeloma (MM). However, lipid analyses are scarce in MM onset and response to treatment. First, we measured metabolites and proteins in sera obtained from matched bone marrow and peripheral blood of 17 newly diagnosed and 9 relapsed/refractory MM patients, by LC-MS/MS. String analysis identified crucial pathways including fibrinogen regulation, inflammatory response, structural integrity of lipoproteins and lipid transport, with a significant increase of apoliproteins APO-A1 (key component of HDL particle and cofactor for lecithin-cholesterol acyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters), A2,C2 (which hydrolyzes triglycerides to provide free fatty acids for cells), C3, components of HDL and LDL, confirming a deranged lipid handling in RR-MM patients. Second, we studied the lipid metabolic changes upon anti-BCMA exposure with Elranatamab (EL), an investigational, off-the-shelf, humanized BCMA-CD3-directed bispecific antibody engineered to elicit potent T-cell-mediated anti-MM activity. Combining in vitro assays, FACS, qRT-PCR, immunofluorescence, Western Blot and RNA-seq we evaluated the response to EL treatment in 3 human myeloma cell lines U266, NCI-H929 and OPM2 (3 cell lines chosen for their different basal bioenergetics profile). MM cells were co-incubated with various concentrations of EL in presence of CD3+T cells isolated from healthy donors in 96- well U-bottom plates for 4 h and 24 h at an E:T ratio of 1:5 and 1:10, then stained with anti-CD138-PE antibody . CD138 + cells were detected by flow cytometry and counted as surviving MM cells. BODIPY™ 581/591 C11 (Lipid Peroxidation Sensor) and BODIPY 493/503 for Neutral Lipid Droplets Staining for FACS and immunofluorescence were used to quantify lipid peroxidation and cytoplasmatic LDs on surviving CD138 + cells. Despite the BCMA target was equally expressed on U266, NCI-H929 and OPM2, the sensitivity to EL was different, inducing cell cycle arrest in the G2/M phase in U266 and NCI-H929 cell lines but not in OPM2, in E:T ratio independent-manner. The expression of the activation markers CD69 and CD25 on T cells and the release of cytokines by T cells were target-dependent and occurred only in the presence of BCMA-positive cells. Oil-Red-O staining revealed an increased lipid content after EL exposure in all tested MM cell lines, with OPM2 the richest in basal lipids content. To dissect the contribution of lipophagy versus lipolysis we performed an autophagy flux analysis by western blot and RNA-seq essay, showing that lipolysis was the emerging response to EL treatment. In EL-sensitive U266 we found an increase of lipid peroxides, hallmark of ferroptosis, a form of cell death due to missed control of membrane lipid peroxidation, a phenomenon negligible in EL-resistant OPM2. Lipidomics demonstrated higher levels of monounsaturated fatty acids (MUFA) than polyunsaturated fatty acids (PUFA) in OPM2, associated to an increased expression of Stearoyl CoA desaturase 1 (SCD1), the enzyme that catalyzes the rate-limiting step in the production of MUFA. While the PUFA oxidation is pro-ferroptotic, MUFA activation promotes a ferroptosis-resistant cell state. Indeed, a ferroptosis-primed resistant phenotype in EL-sensitive U266 was associated to downregulation of lipid peroxides scavengers GPX4 and SLC7A11 and up-regulation of pro-ferroptotic acyl-CoA synthetase long-chain family member 4 (ACSL4). Exogenous MUFA (via supplementation with palmitic acid 125 µM conjugated to BSA 1:6) alleviated ferroptosis induction in U266 making them resistant to EL, a phenotype associated to overexpression of CD36, the main free fatty acids (FFA) transporter. Conversely, in OPM2, inducing iron-dependent lipid peroxidation through RSL3 (3 µM) treatment rescued EL resistance, resulting in a time-dependent increase in cytosolic lipid peroxidation beginning at 6 hours, and overt cell death via ferroptosis at 18 hours. Taken together, our findings suggest that MUFA/PUFA ratio drives ferroptosis and mediates EL sensitivity, addressing lipid handling as a novel means to tailor immunotherapy in RRMM.

Postprandial glycemic and lipidemic effects of black rice anthocyanin extract fortification in foods of varying macronutrient compositions and matrices

Anthocyanin (ACN) fortification of commonly consumed foods is significant as a dietary strategy against the development of metabolic complications by delivering ACNs at high doses. However, its bioactivity and translated metabolic effects in the presence of varying food matrices and macro-constituents is particularly unclear. This end-to-end study investigates the metabolic effects of black rice ACN extract (BRAE) fortification—from in-vitro enzyme inhibitory activities and digestibility, to downstream in vivo impacts on GI, postprandial glycemia and lipidemia. The in vivo effects were investigated in two separate crossover randomised controlled trials (RCT) of 24 healthy participants each—the first RCT determined the postprandial blood glucose, insulin, and ACN bioavailability to a starch-rich single food over 2 h, while the second RCT determined the postprandial blood glucose, insulin, lipid panel, and lipoprotein particles and subfractions to a starch- and fat-rich composite meal over 4 h. In-vitro findings confirmed the inhibitory activities of major black rice ACNs on carbohydrases (p = 0.0004), lipases (p = 0.0002), and starch digestibility (p < 0.0001). in vivo, a 27-point mean GI reduction of wheat bread was observed with BRAE fortification, despite a non-significant attenuation in postprandial glycemia. Conversely, there were no differences in postprandial glycemia when fortified bread was consumed as a composite meal, but acute lipid profiles were altered: (1) improved plasma HDL-c, ([0.0140 mmol/L, 95% CI: (0.00639, 0.0216)], p = 0.0028), Apo-A1 ([0.0296 mmol/L, 95% CI: (0.00757, 0.0515)], p = 0.0203), and Apo-B ([0.00880 mmol/L, 95% CI: (0.00243, 0.0152)], p = 0.0185), (2) modified LDL and HDL subfractions (p < 0.05), and (3) remodelled lipid distributions in HDL and LDL particles. This end-to-end study indicates the potential of ACN fortification in GI reduction and modulating postprandial lipoprotein profiles to starch- and fat-rich composite meals.

ANGPTL3 (Angiopoietin-Like 3) Preferentially Resides on High-Density Lipoprotein in the Human Circulation, Affecting Its Activity.

Background ANGPTL3 (angiopoietin-like protein 3) is an acknowledged crucial regulator of lipid metabolism by virtue of its inhibitory effect on lipoprotein lipase and endothelial lipase. It is currently unknown whether and to which lipoproteins ANGPTL3 is bound and whether the ability of ANGPTL3 to inhibit lipase activity is affected by binding to lipoproteins. Methods and Results Incubation of ultracentrifugation-isolated low-density lipoprotein (LDL) and high-density lipoprotein (HDL) fractions from healthy volunteers with recombinant ANGPTL3 revealed that ANGPTL3 associates with both HDL and LDL particles ex vivo. Plasma from healthy volunteers and a patient deficient in HDL was fractionated by fast protein liquid chromatography, and ANGPTL3 distribution among lipoprotein fractions was measured. In healthy volunteers, ≈75% of lipoprotein-associated ANGPTL3 resides in HDL fractions, whereas ANGPTL3 was largely bound to LDL in the patient deficient in HDL. ANGPTL3 activity was studied by measuring lipolysis and uptake of 3H-trioleate by brown adipocyte T37i cells. Unbound ANGPTL3 did not suppress lipase activity, but when given with HDL or LDL, ANGPTL3 suppressed lipase activity by 21.4±16.4% (P=0.03) and 25.4±8.2% (P=0.006), respectively. Finally, in a subset of the EPIC (European Prospective Investigation into Cancer) Norfolk study, plasma HDL cholesterol and amount of large HDL particles were both positively associated with plasma ANGPTL3 concentrations. Moreover, plasma ANGPTL3 concentrations showed a positive association with incident coronary artery disease (odds ratio, 1.25 [95% CI, 1.01-1.55], P=0.04). Conclusions Although ANGPTL3 preferentially resides on HDL, its activity was highest once bound to LDL particles.