Plasma Cell-Free DNA Predicts Survival and Maps Specific Sources of Injury in Pulmonary Arterial Hypertension.

BACKGROUND: Cell-free DNA (cfDNA) is a noninvasive marker of cellular injury. Its significance in pulmonary arterial hypertension (PAH) is unknown. METHODS: Plasma cfDNA was measured in 2 PAH cohorts (A, n=48; B, n=161) and controls (n=48). Data were collected for REVEAL 2.0 (Registry to Evaluate Early and Long-Term PAH Disease Management) scores and outcome determinations. Patients were divided into the following REVEAL risk groups: low (≤6), medium (7-8), and high (≥9). Total cfDNA concentrations were compared among controls and PAH risk groups by 1-way analysis of variance. Log-rank tests compared survival between cfDNA tertiles and REVEAL risk groups. Areas under the receiver operating characteristic curve were estimated from logistic regression models. A sample subset from cohort B (n=96) and controls (n=16) underwent bisulfite sequencing followed by a deconvolution algorithm to map cell-specific cfDNA methylation patterns, with concentrations compared using t tests. RESULTS: In cohort A, median (interquartile range) age was 62 years (47-71), with 75% female, and median (interquartile range) REVEAL 2.0 was 6 (4-9). In cohort B, median (interquartile range) age was 59 years (49-71), with 69% female, and median (interquartile range) REVEAL 2.0 was 7 (6-9). In both cohorts, cfDNA concentrations differed among patients with PAH of varying REVEAL risk and controls (analysis of variance P≤0.002) and were greater in the high-risk compared with the low-risk category (P≤0.002). In cohort B, death or lung transplant occurred in 14 of 54, 23 of 53, and 35 of 54 patients in the lowest, middle, and highest cfDNA tertiles, respectively. cfDNA levels stratified as tertiles (log-rank: P=0.0001) and REVEAL risk groups (log-rank: P<0.0001) each predicted transplant-free survival. The addition of cfDNA to REVEAL improved discrimination (area under the receiver operating characteristic curve, 0.72-0.78; P=0.02). Compared with controls, methylation analysis in patients with PAH revealed increased cfDNA originating from erythrocyte progenitors, neutrophils, monocytes, adipocytes, natural killer cells, vascular endothelium, and cardiac myocytes (Bonferroni adjusted P<0.05). cfDNA concentrations derived from erythrocyte progenitor cells, cardiac myocytes, and vascular endothelium were greater in patients with PAH with high-risk versus low-risk REVEAL scores (P≤0.02). CONCLUSIONS: Circulating cfDNA is elevated in patients with PAH, correlates with disease severity, and predicts worse survival. Results from cfDNA methylation analyses in patients with PAH are consistent with prevailing paradigms of disease pathogenesis.

A Translational Tool to Facilitate Use of Apolipoprotein B for Clinical Decision-Making.

BACKGROUND: Despite recent large-scale discordance studies showing definitively that atherosclerotic cardiovascular disease (ASCVD) risk correlates better with apolipoprotein B (apoB) than with low-density lipoprotein cholesterol (LDL-C), the latter remains the recommended metric for guiding lipid-lowering treatment decisions in the United States. A major barrier to change, in this regard, is the lack of guideline-recommended apoB treatment targets. We developed a simple method to "translate" apoB values into population-equivalent LDL-C units, allowing apoB-based treatment decisions to be made using LDL-C targets. METHODS: Sequentially collected, population-based samples underwent standard lipid panel analysis and apoB testing by immunoassay. Those with triglycerides greater than 1000 mg/dl were excluded, leaving a study cohort of 15 153 individuals. RESULTS: Linear regression of calculated LDL-C values against percentile-equivalent apoB values yielded an equation to convert apoB into percentile-equivalent LDL-C units: [LDL-C equivalents = 1.38(apoB) - 29] (R2 = 0.999). The extent of discordance between LDL-C and apoB was examined in subgroups with similar LDL-C, ranging from very low (55-70 mg/dL) to very high (175-190 mg/dL). Among individuals with very low LDL-C, 40% had discordantly higher apoB, indicating higher ASCVD risk. Of those with very high LDL-C, 49% had discordantly lower apoB. Across the range, a minority of patients (25%-40%) had concordant levels of apoB, confirming that discordance between these biomarkers is highly prevalent. Similar results were found in discordance analysis between apoB and non-high-density lipoprotein cholesterol (HDL-C). CONCLUSIONS: Providing visibility to discrepancies among LDL-C, non-HDL-C, and apoB should help to facilitate more rapid and widespread adoption of apoB for managing ASCVD risk.

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.

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.

Use of Apolipoprotein B in the Era of Precision Medicine: Time for a Paradigm Change?

Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of death worldwide and the risk of a major cardiovascular event is highest among those with established disease. Ongoing management of these patients relies on the accurate assessment of their response to any prescribed therapy, and their residual risk, in order to optimize treatment. Recent international guidelines and position statements concur that the plasma concentration of apolipoprotein B (apoB) is the most accurate measure of lipoprotein associated ASCVD risk. This is especially true for the growing number of individuals with diabetes, obesity, or the metabolic syndrome, and those on statin therapy. Most guidelines, however, continue to promote LDL-C as the primary risk marker due to uncertainty as to whether the greater accuracy of apoB is sufficient to warrant a paradigm shift. Recommendations regarding apoB measurement vary, and the information provided on how to interpret apoB results is sometimes insufficient, particularly for non-lipid specialists. Misinformation regarding the reliability of the assays is also frequently repeated despite its equivalent or better standardization than many other diagnostic assays. Thus, demand for apoB testing is relatively low, which means there is little incentive to increase its availability or reduce its cost. In this review, we examine the results of recent clinical outcomes studies and meta-analyses on the relative values of apoB, LDL-C, and non-HDL-C as markers of ASCVD risk. Although there is seemingly minimal difference among these markers when only population-based metrics are considered, it is evident from our analysis that, from a personalized or precision medicine standpoint, many individuals would benefit, at a negligible total cost, if apoB measurement were better integrated into the diagnosis and treatment of ASCVD.

Accuracy and Clinical Impact of Estimating Low-Density Lipoprotein-Cholesterol at High and Low Levels by Different Equations.

New more effective lipid-lowering therapies have made it important to accurately determine Low-density lipoprotein-cholesterol (LDL-C) at both high and low levels. LDL-C was measured by the ß-quantification reference method (BQ) (N = 40,346) and compared to Friedewald (F-LDL-C), Martin (M-LDL-C), extended Martin (eM-LDL-C) and Sampson (S-LDL-C) equations by regression analysis, error-grid analysis, and concordance with the BQ method for classification into different LDL-C treatment intervals. For triglycerides (TG) < 175 mg/dL, the four LDL-C equations yielded similarly accurate results, but for TG between 175 and 800 mg/dL, the S-LDL-C equation when compared to the BQ method had a lower mean absolute difference (mg/dL) (MAD = 10.66) than F-LDL-C (MAD = 13.09), M-LDL-C (MAD = 13.16) or eM-LDL-C (MAD = 12.70) equations. By error-grid analysis, the S-LDL-C equation for TG > 400 mg/dL not only had the least analytical errors but also the lowest frequency of clinically relevant errors at the low (<70 mg/dL) and high (>190 mg/dL) LDL-C cut-points (S-LDL-C: 13.5%, F-LDL-C: 23.0%, M-LDL-C: 20.5%) and eM-LDL-C: 20.0%) equations. The S-LDL-C equation also had the best overall concordance to the BQ reference method for classifying patients into different LDL-C treatment intervals. The S-LDL-C equation is both more analytically accurate than alternative equations and results in less clinically relevant errors at high and low LDL-C levels.

Atherosclerotic cardiovascular disease in hyperalphalipoproteinemia due to LIPG variants.

BACKGROUND: High density lipoprotein cholesterol (HDL-C) concentration correlates inversely with atherosclerotic cardiovascular disease (ASCVD) risk and is included in risk calculations. Endothelial lipase (EL) is a phospholipase that remodels HDL. Deficiency of EL due to mutations in its gene, LIPG, is associated with hyperalphalipoproteinemia. The effects of EL on HDL function and ASCVD risk remain poorly understood. OBJECTIVES: To determine whether hyperalphalipoproteinemia due to EL deficiency is protective against ASCVD. METHODS: We identified LIPG variants amongst patients with severe hyperalphalipoproteinemia (HDL-C >2.5 mmol/L) attending a referral lipid clinic in the Western Cape Province of South Africa. We analysed the clinical and biochemical phenotypes amongst primary hyperalphalipoproteinemia cases (males HDL-C >1.6 mmol/L; females HDL-C >1.8 mmol/L) due to LIPG variants, and the distribution of variants in normal and hyperalphalipoproteinemia ranges of HDL-C. RESULTS: 1007 patients with HDL-C concentration ranging from 1.2 to 4.5 mmol/L were included. Seventeen females had primary hyperalphalipoproteinemia. Vascular disease was prominent, but not associated with HDL-C concentration, LDL-C concentration or carotid artery intima media thickness. Two novel and three known LIPG variants were identified in severe hyperalphalipoproteinemia. Four additional variants were identified in the extended cohort. Two common variants appeared normally distributed across the HDL-C concentration range, while six less-common variants were found only at higher HDL-C concentrations. One rare variant had a moderate effect. CONCLUSION: Hyperalphalipoproteinemia due to LIPG variants is commoner in females and may not protect against ASCVD. Use of current risk calculations may be inappropriate in patients with hyperalphalipoproteinemia due to EL deficiency. Our study cautions targeting EL to reduce risk.

Zoo Animal Welfare: The Human Dimension.

Standards and policies intended to safeguard nonhuman animal welfare, whether in zoos, farms, or laboratories, have tended to emphasize features of the physical environment. However, research has now made it clear that very different welfare outcomes are commonly seen in facilities using similar environments or conforming to the same animal welfare requirements. This wide variation is almost certainly due, at least in part, to the important effects of the actions of animal care staff on animal welfare. Drawing mostly on the farm animal literature, we propose that this "human dimension" of animal welfare involves seven components: (1) positive human-animal interaction, (2) consistency and familiarity of keepers, (3) treating animals as individuals and taking account of their personalities, (4) the attitudes and personalities of keepers, (5) the keepers' knowledge and experience, (6) the keepers' own well-being, and (7) the influence of facility design on how keepers and others interact with the animals. We suggest that attention to these human factors provides major scope for improving the welfare of animals in zoos.