Discordance Between Very Low-Density Lipoprotein Cholesterol and Low-Density Lipoprotein Cholesterol Increases Cardiovascular Disease Risk in a Geographically Defined Cohort.

BACKGROUND: Clinical risk scores are used to identify those at high risk of atherosclerotic cardiovascular disease (ASCVD). Despite preventative efforts, residual risk remains for many individuals. Very low-density lipoprotein cholesterol (VLDL-C) and lipid discordance could be contributors to the residual risk of ASCVD. METHODS AND RESULTS: Cardiovascular disease-free residents, aged ≥40 years, living in Olmsted County, Minnesota, were identified through the Rochester Epidemiology Project. Low-density lipoprotein cholesterol (LDL-C) and VLDL-C were estimated from clinically ordered lipid panels using the Sampson equation. Participants were categorized into concordant and discordant lipid pairings based on clinical cut points. Rates of incident ASCVD, including percutaneous coronary intervention, coronary artery bypass grafting, stroke, or myocardial infarction, were calculated during follow-up. The association of LDL-C and VLDL-C with ASCVD was assessed using Cox proportional hazards regression. Interaction between LDL-C and VLDL-C was assessed. The study population (n=39 098) was primarily White race (94%) and female sex (57%), with a mean age of 54 years. VLDL-C (per 10-mg/dL increase) was significantly associated with an increased risk of incident ASCVD (hazard ratio, 1.07 [95% CI, 1.05-1.09]; P<0.001]) after adjustment for traditional risk factors. The interaction between LDL-C and VLDL-C was not statistically significant (P=0.11). Discordant individuals with high VLDL-C and low LDL-C experienced the highest rate of incident ASCVD events, 16.9 per 1000 person-years, during follow-up. CONCLUSIONS: VLDL-C and lipid discordance are associated with a greater risk of ASCVD and can be estimated from clinically ordered lipid panels to improve ASCVD risk assessment.

Lipoprotein Insulin Resistance Score and Mortality Risk Stratification in Heart Failure.

BACKGROUND: Higher total serum cholesterol is associated with lower mortality in heart failure. Evaluating associations between lipoprotein subfractions and mortality among people with heart failure may provide insights into this observation. METHODS: We prospectively enrolled a community cohort of people with heart failure from 2003 to 2012 and assessed vital status through 2021. Plasma collected at enrollment was used to measure lipoprotein subfractions via nuclear magnetic resonance spectroscopy. A composite score of 6 lipoprotein subfractions was generated using the lipoprotein insulin resistance index (LP-IR) algorithm. Using covariate-adjusted proportional hazards regression models, we evaluated associations between LP-IR score and all-cause mortality. RESULTS: Among 1382 patients with heart failure (median follow-up 13.9 years), a one-standard-deviation (SD) increment in LP-IR score was associated with lower mortality (hazard ratio [HR] 0.93; 95% confidence interval [CI], 0.97-0.99). Among LP-IR parameters, mean high-density lipoprotein (HDL) particle size was significantly associated with lower mortality (HR per 1-SD decrement in mean HDL particle size = 0.83; 95% CI, 0.78-0.89), suggesting that the inverse association between LP-IR score and mortality may be driven by smaller mean HDL particle size. CONCLUSIONS: LP-IR score was inversely associated with mortality among patients with heart failure and may be driven by smaller HDL particle size.

Incremental Value of a Metabolic Risk Score for Heart Failure Mortality: A Population-Based Study.

BACKGROUND: Heart failure is heterogeneous syndrome with persistently high mortality. Nuclear magnetic resonance spectroscopy enables high-throughput metabolomics, suitable for precision phenotyping. We aimed to use targeted metabolomics to derive a metabolic risk score (MRS) that improved mortality risk stratification in heart failure. METHODS: Nuclear magnetic resonance was used to measure 21 metabolites (lipoprotein subspecies, branched-chain amino acids, alanine, GlycA (glycoprotein acetylation), ketone bodies, glucose, and citrate) in plasma collected from a heart failure community cohort. The MRS was derived using least absolute shrinkage and selection operator penalized Cox regression and temporal validation. The association between the MRS and mortality and whether risk stratification was improved over the Meta-Analysis Global Group in Chronic Heart Failure clinical risk score and NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels were assessed. RESULTS: The study included 1382 patients (median age, 78 years, 52% men, 43% reduced ejection fraction) with a 5-year survival rate of 48% (95% CI, 46%-51%). The MRS included 9 metabolites measured. In the validation data set, a 1 standard deviation increase in the MRS was associated with a large increased rate of death (hazard ratio, 2.2 [95% CI, 1.9-2.5]) that remained after adjustment for Meta-Analysis Global Group in Chronic Heart Failure score and NT-proBNP (hazard ratio, 1.6 [95% CI, 1.3-1.9]). These associations did not differ by ejection fraction. The integrated discrimination and net reclassification indices, and Uno's C statistic, indicated that the addition of the MRS improved discrimination over Meta-Analysis Global Group in Chronic Heart Failure and NT-proBNP. CONCLUSIONS: This MRS developed in a heart failure community cohort was associated with a large excess risk of death and improved risk stratification beyond an established risk score and clinical markers.

Frailty and Metabolic Vulnerability in Heart Failure: A Community Cohort Study.

BACKGROUND: Frailty is common in heart failure (HF) and is associated with death but not routinely captured clinically. Frailty is linked with inflammation and malnutrition, which can be assessed by a novel plasma multimarker score: the metabolic vulnerability index (MVX). We sought to evaluate the associations between frailty and MVX and their prognostic impact. METHODS AND RESULTS: In an HF community cohort (2003-2012), we measured frailty as a proportion of deficits present out of 32 physical limitations and comorbidities, MVX by nuclear magnetic resonance spectroscopy, and collected extensive longitudinal clinical data. Patients were categorized by frailty score (≤0.14, >0.14 and ≤0.27, >0.27) and MVX score (≤50, >50 and ≤60, >60 and ≤70, >70). Cox models estimated associations of frailty and MVX with death, adjusted for Meta-Analysis Global Group in Chronic Heart Failure (MAGGIC) score and NT-proBNP (N-terminal pro-B-type natriuretic peptide). Uno's C-statistic measured the incremental value of MVX beyond frailty and clinical factors. Weibull's accelerated failure time regression assessed whether MVX mediated the association between frailty and death. We studied 985 patients (median age, 77; 48% women). Frailty and MVX were weakly correlated (Spearman's ρ=0.21). The highest frailty group experienced an increased rate of death, independent of MVX, MAGGIC score, and NT-proBNP (hazard ratio, 3.3 [95% CI, 2.5-4.2]). Frailty improved Uno's c-statistic beyond MAGGIC score and NT-proBNP (0.69-0.73). MVX only mediated 3.3% and 4.5% of the association between high and medium frailty groups and death, respectively. CONCLUSIONS: In this HF cohort, frailty and MVX are weakly correlated. Both independently contribute to stratifying the risk of death, suggesting that they capture distinct domains of vulnerability in HF.

Circulating ketone bodies and mortality in heart failure: a community cohort study.

Background: The relationship between ketone bodies (KB) and mortality in patients with heart failure (HF) syndrome has not been well established. Objectives: The aim of this study is to assess the distribution of KB in HF, identify clinical correlates, and examine the associations between plasma KB and all-cause mortality in a population-based HF cohort. Methods: The plasma KB levels were measured by nuclear magnetic resonance spectroscopy. Multivariable linear regression was used to examine associations between clinical correlates and KB levels. Proportional hazard regression was employed to examine associations between KB (represented as both continuous and categorical variables) and mortality, with adjustment for several clinical covariates. Results: Among the 1,382 HF patients with KB measurements, the median (IQR) age was 78 (68, 84) and 52% were men. The median (IQR) KB was found to be 180 (134, 308) µM. Higher KB levels were associated with advanced HF (NYHA class III-IV) and higher NT-proBNP levels (both P < 0.001). The median follow-up was 13.9 years, and the 5-year mortality rate was 51.8% [95% confidence interval (CI): 49.1%-54.4%]. The risk of death increased when KB levels were higher (HRhigh vs. low group 1.23; 95% CI: 1.05-1.44), independently of a validated clinical risk score. The association between higher KB and mortality differed by ejection fraction (EF) and was noticeably stronger among patients with preserved EF. Conclusions: Most patients with HF exhibited KB levels that were consistent with those found in healthy adults. Elevated levels of KB were observed in patients with advanced HF. Higher KB levels were found to be associated with an increased risk of death, particularly in patients with preserved EF.

An improved method for estimating low LDL-C based on the enhanced Sampson-NIH equation.

BACKGROUND: The accurate measurement of Low-density lipoprotein cholesterol (LDL-C) is critical in the decision to utilize the new lipid-lowering therapies like PCSK9-inhibitors (PCSK9i) for high-risk cardiovascular disease patients that do not achieve sufficiently low LDL-C on statin therapy. OBJECTIVE: To improve the estimation of low LDL-C by developing a new equation that includes apolipoprotein B (apoB) as an independent variable, along with the standard lipid panel test results. METHODS: Using ß-quantification (BQ) as the reference method, which was performed on a large dyslipidemic population (N = 24,406), the following enhanced Sampson-NIH equation (eS LDL-C) was developed by least-square regression analysis: [Formula: see text] RESULTS: The eS LDL-C equation was the most accurate equation for a broad range of LDL-C values based on regression related parameters and the mean absolute difference (mg/dL) from the BQ reference method (eS LDL-C: 4.51, Sampson-NIH equation [S LDL-C]: 6.07; extended Martin equation [eM LDL-C]: 6.64; Friedewald equation [F LDL-C]: 8.3). It also had the best area-under-the-curve accuracy score by Regression Error Characteristic plots for LDL-C < 100 mg/dL (eS LDL-C: 0.953; S LDL-C: 0.920; eM LDL-C: 0.915; F LDL-C: 0.874) and was the best equation for categorizing patients as being below or above the 70 mg/dL LDL-C treatment threshold for adding new lipid-lowering drugs by kappa score analysis when compared to BQ LDL-C for TG < 800 mg/dL (eS LDL-C: 0.870 (0.853-0.887); S LDL-C:0.763 (0.749-0.776); eM LDL-C:0.706 (0.690-0.722); F LDL-C:0.687 (0.672-0.701). Approximately a third of patients with an F LDL-C < 70 mg/dL had falsely low test results, but about 80% were correctly reclassified as higher (≥ 70 mg/dL) by the eS LDL-C equation, making them potentially eligible for PCSK9i treatment. The M LDL-C and S LDL-C equations had less false low results below 70 mg/dL than the F LDL-C equation but reclassification by the eS LDL-C equation still also increased the net number of patients correctly classified. CONCLUSIONS: The use of the eS LDL-C equation as a confirmatory test improves the identification of high-risk cardiovascular disease patients, who could benefit from new lipid-lowering therapies but have falsely low LDL-C, as determined by the standard LDL-C equations used in current practice.

A High-Throughput NMR Method for Lipoprotein-X Quantification.

Lipoprotein X (LP-X) is an abnormal cholesterol-rich lipoprotein particle that accumulates in patients with cholestatic liver disease and familial lecithin-cholesterol acyltransferase deficiency (FLD). Because there are no high-throughput diagnostic tests for its detection, a proton nuclear magnetic resonance (NMR) spectroscopy-based method was developed for use on a clinical NMR analyzer commonly used for the quantification of lipoproteins and other cardiovascular biomarkers. The LP-X assay was linear from 89 to 1615 mg/dL (cholesterol units) and had a functional sensitivity of 44 mg/dL. The intra-assay coefficient of variation (CV) varied between 1.8 and 11.8%, depending on the value of LP-X, whereas the inter-assay CV varied between 1.5 and 15.4%. The assay showed no interference with bilirubin levels up to 317 mg/dL and was also unaffected by hemolysis for hemoglobin values up to 216 mg/dL. Samples were stable when stored for up to 6 days at 4 °C but were not stable when frozen. In a large general population cohort (n = 277,000), LP-X was detected in only 50 subjects. The majority of LP-X positive cases had liver disease (64%), and in seven cases, had genetic FLD (14%). In summary, we describe a new NMR-based assay for LP-X, which can be readily implemented for routine clinical laboratory testing.

Optimization of time interval for the measurement of plasma lipids for cardiovascular disease risk assessment.

BACKGROUND: Lipid testing for atherosclerotic cardiovascular disease (ASCVD) risk is often performed every 4-6 years, but we hypothesized that the optimum time interval may vary depending on baseline risk. RESEARCH DESIGN AND METHODS: Using lipid values and other risk factors from the National Health and Nutrition Examination Survey (NHANES) (n = 9,704), we calculated a 10-year risk score with the pooled-cohort equations. Future risk scores were predicted by increasing age and projecting systolic blood pressure (SBP) and lipid changes, using the mean-percentile age group change in NHANES for SBP (n = 17,329) and the Lifelines Cohort study for lipids (n = 133,540). The crossing of high and intermediate-risk thresholds were calculated by time to determine optimum intervals for lipid testing. RESULTS: Time to crossing risk thresholds depends on baseline risk, but the mean increase in the risk score plateaus at 1% per year for those with a baseline 10-year risk greater than 15%. Based on these findings, we recommend the following maximum time intervals for lipid testing: baseline risk < 15%: 5-years, 16%: 4-years, 17%: 3-years, 18%: 2-years, and 19%: ≤1-year. CONCLUSIONS: Testing patients for lipids who have a higher baseline risk more often could identify high-risk patients sooner, allowing for earlier and more effective therapeutic intervention.

The Metabolic Vulnerability Index: A Novel Marker for Mortality Prediction in Heart Failure.

BACKGROUND: Inflammation and protein energy malnutrition are associated with heart failure (HF) mortality. The metabolic vulnerability index (MVX) is derived from markers of inflammation and malnutrition and measured by nuclear magnetic resonance spectroscopy. MVX has not been examined in HF. OBJECTIVES: The authors sought to examine the prognostic value of MVX in patients with HF. METHODS: The authors prospectively assembled a population-based cohort of patients with HF from 2003 to 2012 and measured MVX scores with a nuclear magnetic resonance scan from plasma collected at enrollment. Patients were divided into 4 MVX score groups and followed until March 31, 2021. RESULTS: The authors studied 1,382 patients (median age: 78 years; 48% women). The median MVX score was 64.6. Patients with higher MVX were older, more likely to be male, have atrial fibrillation, have higher NYHA functional class, and have HF duration of >18 months. Higher MVX was associated with mortality independent of Meta-analysis Global Group in Chronic Heart Failure score, ejection fraction, and other prognostic biomarkers. Compared to those with the lowest MVX, the HRs for MVX groups 2, 3, and 4 were 1.2 (95% CI: 0.9-1.4), 1.6 (95% CI: 1.3-2.0), and 1.8 (95% CI: 1.4-2.2), respectively (Ptrend < 0.001). Measures of model improvement document the added value of MVX in HF for classifying the risk of death beyond the Meta-analysis Global Group in Chronic Heart Failure score and other biomarkers. CONCLUSIONS: In this HF community cohort, MVX was strongly associated with mortality independently of established clinical factors and improved mortality risk classification beyond clinically validated markers. These data underscore the potential of MVX to stratify risk in HF.

Performance of the enhanced Sampson-NIH equation for VLDL-C and LDL-C in a population with familial combined hyperlipidemia.

INTRODUCTION: Low-density cholesterol (LDL-C) has long been estimated by the Friedewald formula (F-LDL-C); however, this method underestimates LDL-C in patients with hypertriglyceridemia (HTG) or low LDL-C levels. The Martin (M-LDL-C) and Sampson (S-LDL-C) formulas partially resolve these limitations. Recently, Sampson et al. developed a new equation (eS-VLDL-C) that includes ApoB. This new equation could be particularly useful in FCHL, which is characterized by the predominance of triglyceride-rich VLDL and a discordance between LDL-C and ApoB. METHODS: Very low-density lipoproteins (VLDL-C) was measured in 336 patients with FCHL by sequential ultracentrifugation. LDL-C was estimated by subtracting VLDL-C, estimated by the different equations, from non-HDL cholesterol. Spearman correlations, R2, mean squared error (RMSE), and bias were used to compare the accuracy of the different equations. Concordance of the estimated LDL-C values with LDL-C thresholds and ApoB was also assessed by their kappa coefficients and ROC analysis. RESULTS: Overall population had a mean age of 47 years, and 61.5% were women. 19.5% had type 2 diabetes, hypertension was present in 20.8%, and only 12.2% were on statin treatment. Both S-LDL-C and eS-LDL-C performed similarly, and better than M-LDL-C and F-LDL-C. In Bland-Altman analysis, eS-LDL-C showed the lowest bias, better performance in HTG, and better concordance with LDL-C treatment goals compared to other formulas (e.g. ρ: 0.87, 95% CI 0.84-0.89). CONCLUSIONS: LDL-S and LDL-eS equations estimate the concentration of LDL-C with greater accuracy than other formulas. The LDL-eS has best performance in estimating LDL-C with lower RMSE than other formulas.

A virus-like particle-based bivalent PCSK9 vaccine lowers LDL-cholesterol levels in non-human primates.

Elevated low-density lipoprotein cholesterol (LDL-C) is an important risk factor in the development of atherosclerotic cardiovascular disease (ASCVD). Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of LDL-C metabolism, have emerged as promising approaches for reducing elevated LDL-C levels. Here, we evaluated the cholesterol-lowering efficacy of virus-like particle (VLP) based vaccines that target epitopes found within the LDL receptor (LDL-R) binding domain of PCSK9. In both mice and non-human primates, a bivalent VLP vaccine targeting two distinct epitopes on PCSK9 elicited strong and durable antibody responses and lowered cholesterol levels. In macaques, a VLP vaccine targeting a single PCSK9 epitope was only effective at lowering LDL-C levels in combination with statins, whereas immunization with the bivalent vaccine lowered LDL-C without requiring statin co-administration. These data highlight the efficacy of an alternative, vaccine-based approach for lowering LDL-C.

Reducing Lipid Panel Error Allowances to Improve the Accuracy of Cardiovascular Risk Stratification.

BACKGROUND: The standard lipid panel forms the backbone of atherosclerotic cardiovascular disease risk assessment. Suboptimal analytical performance, along with biological variability, could lead to erroneous risk assessment and management decisions. The current National Cholesterol Education Program (NCEP) performance recommendations have remained unchanged for almost 3 decades despite improvements in assay technology. We investigated the potential extent of risk misclassification when the current recommendations are met and explored the impact of improving analytical performance goals. METHODS: We extracted lipid panel data for 8506 individuals from the NHANES database and used these to classify subjects into 4 risk groups as recommended by the 2018 US Multisociety guidelines. Analytical bias and imprecision, at the allowable limits, as well as biological variability, were introduced to the measured values to determine the impact on misclassification. Bias and imprecision were systematically reduced to determine the degree of improvement that may be achieved. RESULTS: Using the current performance recommendations, up to 10% of individuals were misclassified into a different risk group. Improving proportional bias by 1%, and fixing imprecision to 3% across all assays reduced misclassifications by up to 10%. The effect of biological variability can be reduced by taking the average of serial sample measurements. CONCLUSIONS: The current NCEP recommendations for analytical performance of lipid panel assays allow for an unacceptable degree of misclassification, leading to possible mismanagement of cardiovascular disease risk. Iteratively reducing allowable error can improve this.

Associations between neighborhood socioeconomic deprivation, IFNγ, and high-density lipoprotein particle size: Data from the Washington, D.C. cardiovascular health and needs assessment.

INTRODUCTION: Neighborhood socioeconomic deprivation is associated with increased cardiovascular risk factors, including inflammation. Inflammation plays an important role in modifying the cardioprotective function of high-density lipoprotein (HDL). Moreover, recent studies suggest that very high HDL is associated with adverse cardiovascular disease (CVD) outcomes. Thus, we sought to explore the relationships between neighborhood socioeconomic deprivation as a marker of chronic stress, inflammation, proprotein convertase subtilisin/kexin type 9 (PCSK9) (a core component of the HDL proteome), HDL characterisitcs, and biological aging as a predictor of CVD and all-cause mortality. METHODS: Sixty African American subjects were recruited to the NIH Clinical Center as part of a community-based participatory research-designed observational study. Neighborhood deprivation index (NDI), a marker of neighborhood socioeconomic deprivation, was measured using US Census data. HDL characteristics (cholesterol, particle number, size, subspecies) were determined from NMR lipoprotein profiling, and plasma cytokines (IL-1ß, IL-6, IL-8, TNFα, IFNγ) were measured using an ELISA-based multiplex technique. Epigenetic clock biomarkers of aging were measured using DNA methylation data obtained from participants' buffy coat samples. We used linear regression modeling adjusted for atherosclerotic cardiovascular disease (ASCVD) risk score, body mass index (BMI), and lipid-lowering medication use to investigate relationships of interest. RESULTS: NDI directly associated with large HDL particle count (H7P) and IFNγ and trended toward significance with HDL-C and PCSK9. IFNγ and PCSK9 then directly associated with H7P. H7P also directly associated with higher DNA methylation phenotypic age (PhenoAge). CONCLUSION: We highlight associations between neighborhood socioeconomic deprivation, IFNγ, PCSK9, HDL subspecies, and epigenetic biomarkers of aging. Taken together, our findings suggest indirect pathways linking neighborhood deprivation-related stress and inflammation to HDL and immune epigenetic changes. Moreover, these results add to recent work showing the pathogenicity of high HDL levels and underscore the need to understand how chronic stress-related inflammation and lipoprotein subspecies relate to CVD risk across diverse populations.

Association of Lipoprotein Subfractions With Presence and Severity of Coronary Artery Disease in Patients Referred for Coronary Angiography.

Lipoprotein subfractions (LS) can be used for better risk stratification in subjects deemed not at high risk for coronary artery disease (CAD). In this study, we evaluated the correlation between LS with CAD presence and severity. This is a prospective case-control study of 157 patients referred for coronary angiography who were not on lipid-lowering therapy and had LS measured by nuclear magnetic resonance spectroscopy. Synergy between PCI with Taxus and Cardiac Surgery (SYNTAX) scores were calculated to estimate CAD severity. Univariate and multivariable regression analysis was performed to determine correlation of LS with CAD presence and severity and acute coronary syndrome (ACS). There was significant association of certain LS (positive for total low-density lipoprotein particle [LDL-P], small LDL-P and apolipoprotein B, negative for large high-density lipoprotein particle [HDL-P] and apolipoprotein A1 [ApoA1]) with the presence of obstructive CAD and CAD severity. Small LDL-P and HDL-P were still predictive for obstructive CAD after adjusting for traditional risk factors, 10-year atherosclerotic cardiovascular disease risk score and in those with low-density lipoprotein cholesterol <100 mg/100 ml. Total LDL-P and ApoA1 were predictive of CAD severity on multivariable analysis. Higher small LDL-P and lower large HDL-P were associated with ACS presence, although only large HDL-P had a significant inverse correlation with ACS on adjusted analysis (odds ratio 0.74 95% confidence interval 0.58, 0.95) In conclusion, in our cohort of patients referred for coronary angiography, total LDL-P, small LDL-P, and apolipoprotein B had significant direct correlation, and large HDL-P and ApoA1 had significant inverse correlation with obstructive CAD and CAD severity.

Identification of ligand-receptor pairs that drive human astrocyte development.

Extrinsic signaling between diverse cell types is crucial for nervous system development. Ligand binding is a key driver of developmental processes. Nevertheless, it remains a significant challenge to disentangle which and how extrinsic signals act cooperatively to affect changes in recipient cells. In the developing human brain, cortical progenitors transition from neurogenesis to gliogenesis in a stereotyped sequence that is in part influenced by extrinsic ligands. Here we used published transcriptomic data to identify and functionally test five ligand-receptor pairs that synergistically drive human astrogenesis. We validate the synergistic contributions of TGFß2, NLGN1, TSLP, DKK1 and BMP4 ligands on astrocyte development in both hCOs and primary fetal tissue. We confirm that the cooperative capabilities of these five ligands are greater than their individual capacities. Additionally, we discovered that their combinatorial effects converge in part on the mTORC1 signaling pathway, resulting in transcriptomic and morphological features of astrocyte development. Our data-driven framework can leverage single-cell and bulk genomic data to generate and test functional hypotheses surrounding cell-cell communication regulating neurodevelopmental processes.

Cell-free, high-density lipoprotein-specific phospholipid efflux assay predicts incident cardiovascular disease.

BACKGROUNDCellular cholesterol efflux capacity (CEC) is a better predictor of cardiovascular disease (CVD) events than HDL-cholesterol (HDL-C) but is not suitable as a routine clinical assay.METHODSWe developed an HDL-specific phospholipid efflux (HDL-SPE) assay to assess HDL functionality based on whole plasma HDL apolipoprotein-mediated solubilization of fluorescent phosphatidylethanolamine from artificial lipid donor particles. We first assessed the association of HDL-SPE with prevalent coronary artery disease (CAD): study I included NIH severe-CAD (n = 50) and non-CAD (n = 50) participants, who were frequency matched for sex, BMI, type 2 diabetes mellitus, and smoking; study II included Japanese CAD (n = 70) and non-CAD (n = 154) participants. We also examined the association of HDL-SPE with incident CVD events in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study comparing 340 patients with 340 controls individually matched for age, sex, smoking, and HDL-C levels.RESULTSReceiver operating characteristic curves revealed stronger associations of HDL-SPE with prevalent CAD. The AUCs in study I were as follows: HDL-SPE, 0.68; apolipoprotein A-I (apoA-I), 0.62; HDL-C, 0.63; and CEC, 0.52. The AUCs in study II were as follows: HDL-SPE, 0.83; apoA-I, 0.64; and HDL-C, 0.53. Also longitudinally, HDL-SPE was significantly associated with incident CVD events independent of traditional risk factors with ORs below 0.2 per SD increment in the PREVEND study (P < 0.001).CONCLUSIONHDL-SPE could serve as a routine clinical assay for improving CVD risk assessment and drug discovery.TRIAL REGISTRATIONClinicalTrials.gov NCT01621594.FUNDINGNHLBI Intramural Research Program, NIH (HL006095-06).

A Virus-like particle-based bivalent PCSK9 vaccine lowers LDL-cholesterol levels in Non-Human Primates.

Elevated low-density lipoprotein cholesterol (LDL-C) is an important risk factor in the development of atherosclerotic cardiovascular disease (ASCVD). Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of LDL-C metabolism, have emerged as promising approaches for reducing elevated LDL-C levels. Here, we evaluated the cholesterol lowering efficacy of virus-like particle (VLP) based vaccines that target epitopes found within the LDL receptor (LDL-R) binding domain of PCSK9. In both mice and non-human primates, a bivalent VLP vaccine targeting two distinct epitopes on PCSK9 elicited strong and durable antibody responses and lowered cholesterol levels. In macaques, a VLP vaccine targeting a single PCSK9 epitope was only effective at lowering LDL-C levels in combination with statins, whereas immunization with the bivalent vaccine lowered LDL-C without requiring statin co-administration. These data highlight the efficacy of an alternative, vaccine-based approach for lowering LDL-C.

Estimated sdLDL-C for predicting high-risk coronary plaque features in psoriasis: a prospective observational study.

BACKGROUND: Psoriasis (PSO) is a skin disorder with systemic inflammation and high coronary artery disease risk. A distinct lipid phenotype occurs in psoriasis, which is characterized by high plasma triglycerides (TGs) with typically normal or even low LDL-C. The extent to which cholesterol on LDL subfractions, such as small dense LDL-C (sdLDL-C), are associated with vulnerable coronary plaque characteristics in PSO remains elusive. METHODS: A recently developed equation for estimating sdLDL-C from the standard lipid panel was utilized in a PSO cohort (n = 200) with 4-year follow-up of 75 subjects. Coronary plaque burden was assessed by quantitative coronary computed tomography angiography (CCTA). Multivariate regression analyses were used for establishing associations and prognostic value of estimated sdLDL-C. RESULTS: Estimated sdLDL-C was positively associated with non-calcified burden (NCB) and fibro-fatty burden (FFB), which remained significant after multivariate adjustment for NCB (ß = 0.37; P = 0.050) and LDL-C adjustment for FFB (ß = 0.29; P < 0.0001). Of note, total LDL-C calculated by the Friedewald equation was not able to capture these associations in the study cohort. Moreover, in the regression modelling estimated sdLDL-C was significantly predicting necrotic burden progression over 4 years follow-up (P = 0.015), whereas LDL-C did not. Finally, small LDL particles (S-LDLP) and small HDL particles (S-HDLP), along with large and medium TG-rich lipoproteins (TRLPs) had the most significant positive correlation with estimated sdLDL-C. CONCLUSIONS: Estimated sdLDL-C has a stronger association than LDL-C with high-risk features of coronary atherosclerotic plaques in psoriasis patients. CLINICAL TRIAL REGISTRATION: URL: https://www. CLINICALTRIALS: gov . Unique identifiers: NCT01778569.

Constitutive and conditional epitope-tagging of endogenous G protein coupled receptors in Drosophila.

To visualize the cellular and subcellular localization of neuromodulatory G-protein coupled receptors (GPCRs) in Drosophila , we implement a molecular strategy recently used to add epitope tags to ionotropic receptors at their endogenous loci. Leveraging evolutionary conservation to identify sites more likely to permit insertion of a tag, we generated constitutive and conditional tagged alleles for Drosophila 5-HT1A, 5-HT2A, 5-HT2B, Octß1R, Octß2R, two isoforms of OAMB, and mGluR. The conditional alleles allow for the restricted expression of tagged receptor in specific cell types, an option not available for any previous reagents to label these proteins. We show that 5-HT1A and 5-HT2B localize to the mushroom bodies and central complex respectively, as predicted by their roles in sleep. By contrast, the unexpected enrichment of Octß1R in the central complex and of 5-HT1A and 5-HT2A to nerve terminals in lobular columnar cells in the visual system suggest new hypotheses about their function at these sites. Using an additional tagged allele of the serotonin transporter, a marker of serotonergic tracts, we demonstrate diverse spatial relationships between postsynaptic 5-HT receptors and presynaptic 5-HT neurons, consistent with the importance of both synaptic and volume transmission. Finally, we use the conditional allele of 5-HT1A to show that it localizes to distinct sites within the mushroom bodies as both a postsynaptic receptor in Kenyon cells and a presynaptic autoreceptor. Significance Statement: In Drosophila , despite remarkable advances in both connectomic and genomic studies, antibodies to many aminergic GPCRs are not available. We have overcome this obstacle using evolutionary conservation to identify loci in GPCRs amenable to epitope-tagging, and CRISPR/Cas9 genome editing to generated eight novel lines. This method also may be applied to other GPCRs and allows cell-specific expression of the tagged locus. We have used the tagged alleles we generated to address several questions that remain poorly understood. These include the relationship between pre- and post-synaptic sites that express the same receptor, and the use of relatively distant targets by pre-synaptic release sites that may employ volume transmission as well as standard synaptic signaling.