Loss-of-function mutations in the transcription factor CREB3L3 (CREBH) associate with severe hypertriglyceridemia in humans. CREBH is believed to lower plasma triglycerides by augmenting the action of lipoprotein lipase (LPL). However, by using a mouse model of type 1 diabetes mellitus (T1DM), we found that greater liver expression of active CREBH normalized both elevated plasma triglycerides and cholesterol.Residual triglyceride-rich lipoprotein (TRL) remnants were enriched in apolipoprotein E (APOE) and impoverished in APOC3, an apolipoprotein composition indicative of increased hepatic clearance. The underlying mechanism was independent of LPL as CREBH reduced both triglycerides and cholesterol in LPL-deficient mice. Instead, APOE was critical for CREBH's ability to lower circulating remnant lipoproteins because it failed to reduce TRL cholesterol in Apoe -/mice. Importantly, humans with CREB3L3 loss-of-function mutations exhibited increased levels of remnant lipoproteins that were deprived of APOE. Recent evidence suggests that impaired clearance of TRL remnants promotes cardiovascular disease in patients with T1DM. Consistently, we found that hepatic expression of CREBH prevented the progression of diabetes-accelerated atherosclerosis. Our results support the proposal that CREBH acts through an APOEdependent pathway to increase hepatic clearance of remnant lipoproteins. They also implicate elevated levels of remnants in the pathogenesis of atherosclerosis in T1DM.

Background Use of lipoprotein(a) concentrations for identification of individuals at high risk of cardiovascular diseases is hampered by the size polymorphism of apolipoprotein(a), which strongly impacts immunochemical methods, resulting in discordant values. The availability of a reference method with accurate values expressed in SI units is essential for implementing a strategy for assay standardization. Method A targeted LC-MS/MS method for the quantification of apolipoprotein(a) was developed based on selected proteotypic peptides quantified by isotope dilution. To achieve accurate measurements, a reference material constituted of a human recombinant apolipoprotein(a) was used for calibration. Its concentration was assigned using an amino acid analysis reference method directly traceable to SI units through an unbroken traceability chain. Digestion time-course, repeatability, intermediate precision, parallelism, and comparability to the designated gold standard method for lipoprotein(a) quantification, a monoclonal antibody-based ELISA, were assessed. Results A digestion protocol providing comparable kinetics of digestion was established, robust quantification peptides were selected, and their stability was ascertained. Method intermediate imprecision was below 10% and linearity was validated in the 20–400 nmol/L range. Parallelism of responses and equivalency between the recombinant and endogenous apo(a) were established. Deming regression analysis comparing the results obtained by the LC-MS/MS method and those obtained by the gold standard ELISA yielded y = 0.98*ELISA +3.18 (n = 64). Conclusions Our method for the absolute quantification of lipoprotein(a) in plasma has the required attributes to be proposed as a candidate reference method with the potential to be used for the standardization of lipoprotein(a) assays.

Abstract:With the increasing prevalence of cardiovascular diseases (CVD) worldwide, finding reliable and clinically relevant biomarkers to predict acute cardiovascular events has been a major aim of the scientific and medical community. Improvements of the understanding of the pathophysiological pathways of the disease highlighted the major role of lipoprotein particles, and these past decades have seen the emergence of a number of new methodologies to separate, measure and quantitate lipoproteins. Those methods, also known as advanced lipoprotein testing methods (ALT), have gained acceptance in the field of CVD risk assessment and have proven their clinical relevance. In the context of worldwide standardization and harmonization of biological assays, efforts have been initiated toward standardization of ALT methods. However, the complexity of lipoprotein particles and the multiple approaches and methodologies reported to quantify them have rendered these initiatives a critical issue. In this context and to better understand these challenges, this review presents a summary of the major methods available for ALT with the aim to point out the major differences in terms of procedures and quantities actually measured and to discuss the resulting comparability issues.

Rationale: Hypertriglyceridemia (HyperTG) and low high-density lipoprotein cholesterol (HDL-C), both of which are regulated by lipoprotein lipase (LpL) activity, associate with increased cardiovascular disease (CVD). Genetic regulators of LpL actions track with CVD risk in humans. Whether this is due to changes in HDL-C or function, or circulating triglyceride (TG) levels is unresolved. Objective: We created HyperTG and HDL-C reduction in atherosclerotic mice to allow the assessment of how HyperTG and reduced HDL-C affect regression of atherosclerosis and the phenotype of plaque macrophages. Methods and Results: Atherosclerosis regression was studied in control LpL floxed ( Lpl fl/fl ) mice and tamoxifen-inducible whole-body LpL KO ( iLpl -/- ) mice with HyperTG (~500mg/dL) and reduced HDL-C (~50% reduction). Atherosclerosis regression was studied using two models in which advanced plaques resulting from hypercholesterolemia are exposed to normal LDL-C levels using aortic transplantation or treatments with oligonucleotides. In a subset of mice, we expressed human cholesterol ester transfer protein (hCETP) to humanize the relationship between apoB-lipoproteins and HDL. HDL particle number (HDL-P), cholesterol efflux capacity (CEC) and HDL proteome were measured in HyperTG mice and humans. Surprisingly, HyperTG and reduced HDL-C levels due to loss of LpL did not affect atherosclerosis lesion size or macrophage content (CD68+ cells) in either model. Expression of hCETP and further reduction of HDL-C did not alter lesions. Sera from iLpl -/- mice had a decrease in total CEC, but not ABCA1-mediated CEC. HyperTG humans, including those with LpL deficiency, had greater ABCA1-mediated CEC and total CEC per HDL-P. Conclusions: Atherosclerosis regression in mice is driven by LDL-C reduction and is not affected by HyperTG and plasma HDL-C levels.

This study demonstrates that ALT methods do not yet provide equivalent results for the measurement of non-HDL-P and apoB-100. The better agreement between methods harmonized to the WHO/IFCC reference material suggests that standardizing ALT methods by use of a common commutable calibrator will improve cross-platform comparability. This study provides further evidence that LC-MS/MS is the most suitable candidate reference measurement procedure to standardize apoB-100 measurement, as it would provide results with SI traceability. The absence of freezing and thawing effect suggests that frozen serum pools could be used as secondary reference materials.