apoB-containing Lipoproteins Research Articles

LDL Receptor Regulates the Reverse Transport of Macrophage-Derived Unesterified Cholesterol via Concerted Action of the HDL-LDL Axis: Insight From Mouse Models.

The HDL (high-density lipoprotein)-mediated stimulation of cellular cholesterol efflux initiates macrophage-specific reverse cholesterol transport (m-RCT), which ends in the fecal excretion of macrophage-derived unesterified cholesterol (UC). Early studies established that LDL (low-density lipoprotein) particles could act as efficient intermediate acceptors of cellular-derived UC, thereby preventing the saturation of HDL particles and facilitating their cholesterol efflux capacity. However, the capacity of LDL to act as a plasma cholesterol reservoir and its potential impact in supporting the m-RCT pathway in vivo both remain unknown. We investigated LDL contributions to the m-RCT pathway in hypercholesterolemic mice. Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL or ex vivo by plasma derived from subjects with familial hypercholesterolemia was assessed. In vivo, m-RCT was evaluated in mouse models of hypercholesterolemia that were naturally deficient in CETP (cholesteryl ester transfer protein) and fed a Western-type diet. LDL induced the efflux of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on HDL cholesterol efflux capacity in the familial hypercholesterolemia plasma. The m-RCT rates of the LDLr (LDL receptor)-KO (knockout), LDLr-KO/APOB100, and PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpressing mice were all significantly reduced relative to the wild-type mice. In contrast, m-RCT remained unchanged in HAPOB100 Tg (human APOB100 transgenic) mice with fully functional LDLr, despite increased levels of plasma APO (apolipoprotein)-B-containing lipoproteins. Hepatic LDLr plays a critical role in the flow of macrophage-derived UC to feces, while the plasma increase of APOB-containing lipoproteins is unable to stimulate m-RCT. The results indicate that, besides the major HDL-dependent m-RCT pathway via SR-BI (scavenger receptor class B type 1) to the liver, a CETP-independent m-RCT path exists, in which LDL mediates the transfer of cholesterol from macrophages to feces. Graphical Abstract: A graphical abstract is available for this article.

Remnant lipoproteins: are they equal to or more atherogenic than LDL?

To critically appraise new insights into the biology of remnant lipoproteins and their putative role in the pathophysiology of atherosclerotic cardiovascular disease, and to compare the atherogenicity of remnant particles with that of low-density lipoproteins (LDL). New in-vivo stable isotope tracer studies of the kinetics of apoB48 and apoB100-containing lipoproteins in postprandial conditions have revealed that apoB48-containing very low-density lipoproteins (VLDL) accumulated markedly in hypertriglyceridemic patients. These intestinally-derived particles were cleared slowly, and represented up to 25% of circulating VLDL; as part of the remnant particle population, they may increase cardiovascular risk. Importantly, the PCSK9 inhibitor, evolocumab, was shown to reduce remnant levels (-29%) during the postprandial period in diabetic patients on statin therapy - an effect which may be additive to that of LDL-cholesterol reduction in conferring cardiovascular benefit. In recent Mendelian randomization studies, the effect of lowering triglyceride-rich lipoproteins or LDL-cholesterol translated to similar clinical benefit per unit of apoB. Finally, in randomized trials involving statin-treated patients with atherosclerotic cardiovascular disease, remnant cholesterol levels were associated with coronary atheroma progression independently of LDL-cholesterol. Overall, data from observational studies in large cohorts, Mendelian randomization studies, meta-regression analyses, and post-hoc analyses of randomized trials are consistent with the contention that remnants are highly atherogenic particles and contribute to the atherosclerotic burden in an equivalent manner to that of LDL.

135-OR: The Effect of Fixed-Dose Combination of Insulin Degludec and Liraglutide on Serum Lipoprotein Metabolism in Patients with Type 2 Diabetes Mellitus

Aim: Administration of fixed-dose combination of Insulin Degludec and Liraglutide (IDegLira) has been associated with changes in the lipid-profile of diabetic patients. We assessed the effect of IDegLira on serum apolipoproteins, apoB-containing lipoprotein subclasses and HDL lipidome in patients with type 2 diabetes mellitus (T2DM). Methods: Sixty uncontrolled patients with T2DM on oral antidiabetic drugs were included. Of them, thirty received IDegLira and the remaining Insulin Degludec(IDeg). Lipid and carbohydrate metabolism parameters were assessed before and following 3-month-treatment. ApoB-containing lipoprotein subclasses levels were measured by LDL-Lipoprint method, whereas HDL lipidomic analysis via 1H-NMR spectroscopy. Results: Administration of IDegLira resulted in a significant reduction of total (TC) and LDL cholesterol (LDLC) by 14.68mg/dl and 10.45mg/dl, respectively. ApoA1 and apoB decreased in patients treated with IDegLira. A non-significant change in small-dense LDLC (sdLDL-C) levels was observed that was significantly correlated with changes in TG levels (r=0.468, p=0.007). Furthermore, IDegLira administration resulted in significant enrichment of HDL particles’ core in esterified cholesterol and depletion of TGs content. IDegLira induced an atheroprotective shift in fatty acid pattern of HDLs characterized by a statistically significant lower content in saturated and higher in unsaturated fatty acids (linoleic acid, eicosapentaenoic and arachidonic acids and docosahexaenoic acid). No significant changes were observed in the IDeg group. Conclusions: Administration of IDegLira is accompanied by changes in HDL lipdome, a decrease in TC, LDLC, apoA1 and B, while the levels of sdLDL were only reduced in patients experiencing a decrease in TGs. The contribution of these changes to cardiovascular risk reduction after treatment with liraglutide in clinical trials cannot be excluded. Disclosure E. Pappa: None. C. Kostara: None. C. Tellis: None. A.D. Tselepis: None. E. Bairaktari: None. V. Tsimihodimos: None.

The Beneficial Effects of Alpha Lipoic Acid Supplementation on Lp-PLA2 Mass and Its Distribution between HDL and apoB-Containing Lipoproteins in Type 2 Diabetic Patients: A Randomized, Double-Blind, Placebo-Controlled Trial.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a new specific vascular inflammation biomarker that is carried by the lipoproteins in the blood and plays a prominent role in the pathogenesis of atherosclerosis. Increased Lp-PLA2 levels and impaired Lp-PLA2 distribution across high-density lipoprotein (HDL) and non-HDL lipoproteins have been reported in diabetic patients, which is associated with the increase in cardiovascular disease (CVD) risk. This study is aimed at investigating the effect of alpha lipoic acid (ALA), as an antioxidant with potential cardioprotective properties, on the Lp-PLA2 mass and its distribution in diabetic patients. In a double-blind, randomized, placebo-controlled clinical trial, seventy diabetic patients were randomly allocated to ALA (1200 mg ALA as two 600 mg capsules/day) and placebo (two maltodextrin capsules/day) groups. The serum levels of total Lp-PLA2 mass, HDL-Lp-PLA2, oxidized low-density lipoproteins (ox-LDL), apolipoprotein A1 (apo A1), lipid profiles, fasting blood sugar (FBS), and insulin were measured, and apolipoprotein B- (apoB-) associated Lp-PLA2 and homeostasis model of assessment index (HOMA-IR) were calculated at the baseline and after 8 weeks of intervention. ALA significantly decreased the ox-LDL, total Lp-PLA2 mass, apoB-associated Lp-PLA2, and percent of apoB-associated Lp-PLA2 and triglyceride and increased the percent of HDL-Lp-PLA2 compared with the placebo group but had no significant effect on HDL-Lp-PLA2 mass, apo A1, lipid profiles, and glycemic indices. There was a positive correlation between the reduction in the ox-LDL level and total Lp-PLA2 mass in the ALA group. In conclusion, ALA may decrease the CVD risk by reducing the ox-LDL and Lp-PLA2 mass and improving the Lp-PLA2 distribution among lipoproteins in type 2 diabetic patients.

Model systems for studying the assembly, trafficking, and secretion of apoB lipoproteins using fluorescent fusion proteins

apoB exists as apoB100 and apoB48, which are mainly found in hepatic VLDLs and intestinal chylomicrons, respectively. Elevated plasma levels of apoB-containing lipoproteins (Blps) contribute to coronary artery disease, diabetes, and other cardiometabolic conditions. Studying the mechanisms that drive the assembly, intracellular trafficking, secretion, and function of Blps remains challenging. Our understanding of the intracellular and intraorganism trafficking of Blps can be greatly enhanced, however, with the availability of fusion proteins that can help visualize Blp transport within cells and between tissues. We designed three plasmids expressing human apoB fluorescent fusion proteins: apoB48-GFP, apoB100-GFP, and apoB48-mCherry. In Cos-7 cells, transiently expressed fluorescent apoB proteins colocalized with calnexin and were only secreted if cells were cotransfected with microsomal triglyceride transfer protein. The secreted apoB-fusion proteins retained the fluorescent protein and were secreted as lipoproteins with flotation densities similar to plasma HDL and LDL. In a rat hepatoma McA-RH7777 cell line, the human apoB100 fusion protein was secreted as VLDL- and LDL-sized particles, and the apoB48 fusion proteins were secreted as LDL- and HDL-sized particles. To monitor lipoprotein trafficking in vivo, the apoB48-mCherry construct was transiently expressed in zebrafish larvae and was detected throughout the liver. These experiments show that the addition of fluorescent proteins to the C terminus of apoB does not disrupt their assembly, localization, secretion, or endocytosis. The availability of fluorescently labeled apoB proteins will facilitate the exploration of the assembly, degradation, and transport of Blps and help to identify novel compounds that interfere with these processes via high-throughput screening.

Alirocumab, evinacumab, and atorvastatin triple therapy regresses plaque lesions and improves lesion composition in mice

Atherosclerosis-related CVD causes nearly 20 million deaths annually. Most patients are treated after plaques develop, so therapies must regress existing lesions. Current therapies reduce plaque volume, but targeting all apoB-containing lipoproteins with intensive combinations that include alirocumab or evinacumab, monoclonal antibodies against cholesterol-regulating proprotein convertase subtilisin/kexin type 9 and angiopoietin-like protein 3, may provide more benefit. We investigated the effect of such lipid-lowering interventions on atherosclerosis in APOE*3-Leiden.CETP mice, a well-established model for hyperlipidemia. Mice were fed a Western-type diet for 13 weeks and thereafter matched into a baseline group (euthanized at 13 weeks) and five groups that received diet alone (control) or with treatment [atorvastatin; atorvastatin and alirocumab; atorvastatin and evinacumab; or atorvastatin, alirocumab, and evinacumab (triple therapy)] for 25 weeks. We measured effects on cholesterol levels, plaque composition and morphology, monocyte adherence, and macrophage proliferation. All interventions reduced plasma total cholesterol (37% with atorvastatin to 80% with triple treatment; all P < 0.001). Triple treatment decreased non-HDL-C to 1.0 mmol/l (91% difference from control; P < 0.001). Atorvastatin reduced atherosclerosis progression by 28% versus control (P < 0.001); double treatment completely blocked progression and diminished lesion severity. Triple treatment regressed lesion size versus baseline in the thoracic aorta by 50% and the aortic root by 36% (both P < 0.05 vs. baseline), decreased macrophage accumulation through reduced proliferation, and abated lesion severity. Thus, high-intensive cholesterol-lowering triple treatment targeting all apoB-containing lipoproteins regresses atherosclerotic lesion area and improves lesion composition in mice, making it a promising potential approach for treating atherosclerosis.

Microsomal Triglyceride Transfer Protein: From Lipid Metabolism to Metabolic Diseases.

Microsomal triglyceride transfer protein (MTP) was first identified as an endoplasmic reticulum (ER) resident protein that helps in the transfer of neutral lipids to nascent apolipoprotein B (apoB). Its critical role in the assembly and secretion of apoB-containing lipoproteins was identified in abetalipoproteinemia patients who have mutations in MTP and completely lack apoB-containing lipoproteins in the circulation. It has been established now that MTP not only is involved in the transfer of neutral lipids but also plays a role in cholesterol ester and cluster of differentiation 1d (CD1d) biosynthesis. Besides neutral lipids, MTP may also help in the transfer of sphingolipids such as ceramides and sphingomyelin to the apoB-containing lipoproteins. MTP is a multifunctional protein, and its deregulation during pathophysiological conditions gives rise to different metabolic conditions. This book chapter discusses the physiological role and regulation of MTP to maintain the homeostasis of lipids and lipoproteins. It also reviews the regulation of MTP during certain pathophysiological conditions and provides a brief overview of therapeutic interventions that can be possibly used to target its activity or expression to alleviate some of these metabolic diseases.

Hyperalphalipoproteinemic scavenger receptor BI knockout mice exhibit a disrupted epidermal lipid barrier

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters (CE) from high-density lipoproteins (HDL). An impaired SR-BI function leads to hyperalphalipoproteinemia with elevated levels of cholesterol transported in the HDL fraction. Accumulation of cholesterol in apolipoprotein B (apoB)-containing lipoproteins has been shown to alter skin lipid composition and barrier function in mice. To investigate whether these hypercholesterolemic effects on the skin also occur in hyperalphalipoproteinemia, we compared skins of wild-type and SR-BI knockout (SR-BI−/−) mice. SR-BI deficiency did not affect the epidermal cholesterol content and induced only minor changes in the ceramide subclasses. The epidermal free fatty acid (FFA) pool was, however, enriched in short and unsaturated chains. Plasma CE levels strongly correlated with epidermal FFA C18:1 content. The increase in epidermal FFA coincided with downregulation of cholesterol and FFA synthesis genes, suggesting a compensatory response to increased flux of plasma cholesterol and FFAs into the skin. Importantly, the SR-BI−/− epidermal lipid barrier showed increased permeability to ethyl-paraminobenzoic acid, indicating an impairment of the barrier function. In conclusion, increased HDL-cholesterol levels in SR-BI−/− mice can alter the epidermal lipid composition and lipid barrier function similarly as observed in hypercholesterolemia due to elevated levels of apoB-containing lipoproteins.

Regulation of plasma glycero-lysophospholipid levels by lipoprotein metabolism.

Glycero-lysophospholipids, such as lysophosphatidic acids and lysophosphatidylserine, are gathering attention, since specific receptors have been identified. Most of these compounds have been proposed to be bound to albumin, while their associations with lipoproteins have not been fully elucidated. Therefore, in this study, we aimed to investigate the contents of glycero-lysophospholipids (lysophosphatidic acids, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine) on lipoproteins and the modulation of their metabolism by lipoprotein metabolism. We observed that moderate amounts of glycero-lysophospholipids, with the exception of lysophosphatidylserine, were distributed on the LDL and HDL fractions, and glycero-lysophospholipids that had bound to albumin were observed in lipoprotein fractions when they were co-incubated. The overexpression of cholesteryl ester transfer protein decreased the plasma levels of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, and lysophosphatidylinositol and it increased their contents in apoB-containing lipoproteins, while it decreased their contents in HDL and lipoprotein-depleted fractions in mice. The overexpression of the LDL receptor (LDLr) decreased the plasma levels of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, and lysophosphatidylinositol and decreased the contents of these compounds in the LDL, HDL, and lipoprotein-depleted fractions, while the knockdown of the LDLr increased them. These results suggest the potential importance of glycero-lysophospholipids in the pleiotropic effects of lipoproteins as well as the importance of lipoprotein metabolism in the regulation of glycero-lysophospholipids.

Complicated trafficking behaviors involved in paradoxical regulation of sortilin in lipid metabolism.

This review aims to summarize and discuss the most recent advances in our understanding of the underlying mechanisms of the paradoxical effects of sortilin on lipid metabolism. The vacuolar protein sorting 10 protein (Vps10p) domain in the sortilin protein is responsible for substrate binding. Its cytoplasmic tail interacts with adaptor molecules, and modifications can determine whether sortilin trafficking occurs via the anterograde or retrograde pathway. The complicated trafficking behaviors likely contribute to the paradoxical roles of sortilin in lipid metabolism. The anterograde pathway of sortilin trafficking in hepatocytes, enterocytes, and peripheral cells likely causes an increase in plasma lipid levels, while the retrograde pathway leads to the opposite effect. Hepatocyte sortilin functions via the anterograde or retrograde pathway in a complicated and paradoxical manner to regulate apoB-containing lipoprotein metabolism. Clarifying the regulatory mechanisms underlying the trafficking behaviors of sortilin is necessary and may lead to artificial sortilin intervention as a potential therapeutic strategy for lipid disorder diseases. Conclusively, the paradoxical regulation of sortilin in lipid metabolism is likely due to its complicated trafficking behaviors.

P4667Loss of function in PCSK9, atherogenic lipoprotein concentrations, and calcific aortic valve stenosis

Abstract Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition reduces plasma concentrations of most atherogenic lipoproteins such as low-density lipoprotein cholesterol (LDL-C), apolipoprotein B (apoB) and lipoprotein(a) [Lp(a)]. Atherogenic lipoprotein concentrations have also been linked with calcific aortic valve stenosis (CAVS). Purpose 1) To determine the association between genetic variants in PCSK9 and lipoprotein-lipid levels, 2) to determine whether loss of function (LOF) in PCSK9 is associated with CAVS and 3) to evaluate if PCSK9 could be implicated in aortic valve interstitial cells (VICs) calcification. Methods We built a weighted genetic risk score (wGRS) using 10 single nucleotide polymorphisms at the PCSK9 locus associated with LDL-C in the Global Lipids Genetics Consortium. We determined the association between the wGRS and LDL-C, apoB and Lp(a)] in 9692 participants of the EPIC-Norfolk study using linear regression. We investigated the association between the LOF PCSK9 R46L variant and CAVS risk in a meta-analysis of published (three Copenhagen studies, 1463 cases and 101,620 controls) and unpublished studies (UK Biobank, 1350 cases and 349,043 controls, Malmö Diet and Cancer study, 682 cases and 5963 controls and EPIC-Norfolk, 508 cases and 20,421 controls) prospective, population-based studies using logistic regression adjusted for age and sex. We evaluated PCSK9 expression and localization in explanted aortic valves by capillary Western blot and immunohistochemistry in patients with and without CAVS. Von Kossa staining was used to visualize aortic leaflet calcium deposits. We also assessed VICs calcification potential under oxidative stress condition. Results In EPIC-Norfolk, the wGRS was significantly associated with TC, LDL-C, and apoB (all p&lt;0.0001), but not with VLDL-C, HDL-C, triglycerides apoA-I, or Lp(a). Carriers of the R46L variant were at lower CAVS risk (odds ratio=0.71 (95% CI, 0.57–0.88, p&lt;0.001)). Aortic valves of patients with aortic sclerosis (n=12) and CAVS (n=8) presented elevated PCSK9 levels (log2 fold change [FC]=+28.6±5.1, p=0.008 and FC=+39.3±15.2, p=0.02, respectively) compared to controls (n=4).In calcified leaflets, PCSK9 expression co-localized with calcium deposits. PCSK9 expression in VICs was induced by oxidative stress (FC=+2.3±0.4, p=0.02), and subsequent increment in calcification potential was observed. Conclusion PCSK9LOF variants are associated with lifelong reductions in non-Lp(a) apoB-containing lipoprotein levels and a lower risk of coronary artery disease and CAVS. PCSK9 is abundant in fibrotic and calcified aortic leaflets. Oxidative stress increases PCSK9 expression in VICs. These results support randomized clinical trials of PCSK9 inhibition in the prevention of CAVS.

The crystal structure of human microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) plays an essential role in lipid metabolism, especially in the biogenesis of very low-density lipoproteins and chylomicrons via the transfer of neutral lipids and the assembly of apoB-containing lipoproteins. Our understanding of the molecular mechanisms of MTP has been hindered by a lack of structural information of this heterodimeric complex comprising an MTPα subunit and a protein disulfide isomerase (PDI) β-subunit. The structure of MTP presented here gives important insights into the potential mechanisms of action of this essential lipid transfer molecule, structure-based rationale for previously reported disease-causing mutations, and a means for rational drug design against cardiovascular disease and obesity. In contrast to the previously reported structure of lipovitellin, which has a funnel-like lipid-binding cavity, the lipid-binding site is encompassed in a β-sandwich formed by 2 β-sheets from the C-terminal domain of MTPα. The lipid-binding cavity of MTPα is large enough to accommodate a single lipid. PDI independently has a major role in oxidative protein folding in the endoplasmic reticulum. Comparison of the mechanism of MTPα binding by PDI with previously published structures gives insights into large protein substrate binding by PDI and suggests that the previous structures of human PDI represent the "substrate-bound" and "free" states rather than differences arising from redox state.

Identification of antisense transcripts of the microsomal triglyceride transfer protein genes in humans and mice

Microsomal triglyceride transfer protein (MTTP) is essential for the assembly and secretion of apoB-containing lipoproteins. Here, we report the presence of genes on the anti-sense strands of the human MTTP and mouse Mttp genes. The gene on the anti-sense strand of the human MTTP gene is called MTTP-AS1. It consists of 5 exons and 4 introns and codes for two different transcripts MTTP-AS1-Long and MTTP-AS1-Short. Exons 3 and 5 of the MTTP-AS1 gene are ancient and evolutionary conserved whereas exons 2 and 4 are primate specific. MTTP-AS1-Long is mainly in the liver and is in the cytoplasm of human hepatoma cells. MTTP-AS1-Short is in the testis. The MTTP-AS1-Long transcript shows complementarity with two different exons of the MTTP transcript. The gene on the opposite strand of the mouse Mttp gene is named as Mttpos. It consists of 2 exons and one intron and codes for one transcript. Partial sequence of the Mttpos exon 2 is homologous in several species from rodents to primates. Mttpos transcript is present in mouse liver, small intestine and testis. The Mttpos transcript shows significant complementarity with the corresponding mouse Mttp mRNA sequences. Further, we identified a conserved sequence in the human MTTP-AS1-Long and mouse Mttpos transcripts indicating for possible evolutionarily conserved regulatory function for these long noncoding RNAs. It is likely that these newly identified long noncoding RNAs interact with their complementary sequences in MTTP mRNAs and affect their stability or translation.

A combinated in vivo – in vitro approach to evaluate the influence of dietary plant oils on innate immune competence and eicosanoid metabolism process in common carp Cyprinus carpio

Omega-3 fatty acids are known to have several cardioprotective effects. Our aim was to investigate the effects of intakes of fish and Camelina sativa oil (CSO), rich in alpha-linolenic acid, on the atherogenic and anti-atherogenic functions of LDL and HDL particles.Altogether, 88 volunteers with impaired glucose metabolism were randomly assigned to CSO (10 g of alpha-linolenic acid/day), fatty fish (4 fish meals/week), lean fish (4 fish meals/week) or control group for 12 weeks. 79 subjects completed the study. The binding of lipoproteins to aortic proteoglycans, LDL aggregation and activation of endothelial cells by LDL and cholesterol efflux capacity of HDL were determined in vitro.Intake of CSO decreased the binding of lipoproteins to aortic proteoglycans in a non-normalized model (p = 0.006). After normalizing with serum concentrations of non-HDL cholesterol, apolipoprotein B (apoB) or LDL cholesterol, which decreased in the CSO group, the change was no longer statistically significant. In the fish groups, there were no changes in the binding of lipoproteins to proteoglycans. Regarding other lipoprotein functions, there were no changes in any of the groups.Intake of CSO decreases the binding of lipoproteins to aortic proteoglycans by decreasing serum LDL cholesterol concentration, which suggests that the level of apoB-containing lipoproteins in the circulation is the main driver of lipoprotein retention within the arterial wall. Intake of fish or CSO has no effects on other lipoprotein functions.

Pathways of smooth muscle foam cell formation in atherosclerosis.

Smooth muscle cells (SMCs) are the major cell type in human atherosclerosis-prone arteries and take up excess lipids, thereby contributing to luminal occlusion. Here we provide a focused review on pathways by which smooth muscle cells (SMCs) can become foam cells in atherosclerosis. A synthesis of recent and older investigations provides key mechanistic insights into SMC foam cell formation. LDL and other apoB-containing lipoproteins are modified by a diverse array of oxidative, enzymatic, and nonenzymatic processes present in the arterial intima. These modifications of LDL all promote the aggregation of LDL (agLDL), a key finding from analysis of arterial lesion particles. Scavenger receptor and phagocytic capacity of SMCs can vary greatly, perhaps related to differences in SMC phenotype or in-vitro cell culture environments, and can be increased with exposure to cytokines, growth factors, and cholesterol. Macrophages promote the formation of SMC foam cells in direct or indirect co-culture models. SMCs contribute significantly to the foam cell population in atherosclerosis. Further investigation and identification of key mechanisms of SMC foam cell formation will help drive new therapeutics to reduce cardiovascular disease.

Investigation of human apoB48 metabolism using a new, integrated non-steady-state model of apoB48 and apoB100 kinetics.

BackgroundTriglyceride‐rich lipoproteins and their remnants have emerged as major risk factors for cardiovascular disease. New experimental approaches are required that permit simultaneous investigation of the dynamics of chylomicrons (CM) and apoB48 metabolism and of apoB100 in very low‐density lipoproteins (VLDL).MethodsMass spectrometric techniques were used to determine the masses and tracer enrichments of apoB48 in the CM, VLDL 1 and VLDL 2 density intervals. An integrated non‐steady‐state multicompartmental model was constructed to describe the metabolism of apoB48‐ and apoB100‐containing lipoproteins following a fat‐rich meal, as well as during prolonged fasting.ResultsThe kinetic model described the metabolism of apoB48 in CM, VLDL 1 and VLDL 2. It predicted a low level of basal apoB48 secretion and, during fat absorption, an increment in apoB48 release into not only CM but also directly into VLDL 1 and VLDL 2. ApoB48 particles with a long residence time were present in VLDL, and in subjects with high plasma triglycerides, these lipoproteins contributed to apoB48 measured during fasting conditions. Basal apoB48 secretion was about 50 mg day−1, and the increment during absorption was about 230 mg day−1. The fractional catabolic rates for apoB48 in VLDL 1 and VLDL 2 were substantially lower than for apoB48 in CM.DiscussionThis novel non‐steady‐state model integrates the metabolic properties of both apoB100 and apoB48 and the kinetics of triglyceride. The model is physiologically relevant and provides insight not only into apoB48 release in the basal and postabsorptive states but also into the contribution of the intestine to VLDL pool size and kinetics.

Receptor-Independent Transfer of Low Density Lipoprotein Cargo to Biomembranes.

The fundamental task of lipoprotein particles is extracellular transport of cholesterol, lipids, and fatty acids. Besides, cholesterol-rich apoB-containing lipoprotein particles (i.e., low density lipoprotein LDL) are key players in progression of atherosclerotic cardiovascular disease and are associated with familial hypercholesterolemia (FH). So far, lipoprotein particle binding to the cell membrane and subsequent cargo transfer is directly linked to the lipoprotein receptors on the target cell surface. However, our observations showed that lipoprotein particle cargo transport takes place even in the absence of the receptor. This finding suggests that an alternative mechanism for lipoprotein-particle/membrane interaction, besides the receptor-mediated one, exists. Here, we combined several complementary biophysical techniques to obtain a comprehensive view on the nonreceptor mediated LDL-particle/membrane. We applied a combination of atomic force and single-molecule-sensitive fluorescence microscopy (AFM and SMFM) to investigate the LDL particle interaction with membranes of increasing complexity. We observed direct transfer of fluorescently labeled amphiphilic lipid molecules from LDL particles into the pure lipid bilayer. We further confirmed cargo transfer by fluorescence cross-correlation spectroscopy (FCCS) and spectral imaging of environment-sensitive probes. Moreover, the integration of the LDL particle into the membranes was directly visualized by high-speed atomic force microscopy (HS-AFM) and cryo-electron microscopy (cryo-EM). Overall, our data show that lipoprotein particles are able to incorporate into lipid membranes upon contact to transfer their cargo in the absence of specific receptors.

Normal serum ApoB48 and red cells vitamin E concentrations after supplementation in a novel compound heterozygous case of abetalipoproteinemia

Background and aimsAbetalipoproteinemia (ABL) is a rare recessive monogenic disease due to MTTP (microsomal triglyceride transfer protein) mutations leading to the absence of plasma apoB-containing lipoproteins. Here we characterize a new ABL case with usual clinical phenotype, hypocholesterolemia, hypotriglyceridemia but normal serum apolipoprotein B48 (apoB48) and red blood cell vitamin E concentrations. MethodsHistology and MTP activity measurements were performed on intestinal biopsies. Mutations in MTTP were identified by Sanger sequencing, quantitative digital droplet and long-range PCR. Functional consequences of the variants were studied in vitro using a minigene splicing assay, measurement of MTP activity and apoB48 secretion. ResultsIntestinal steatosis and the absence of measurable lipid transfer activity in intestinal protein extract supported the diagnosis of ABL. A novel MTTP c.1868G>T variant inherited from the patient's father was identified. This variant gives rise to three mRNA transcripts: one normally spliced, found at a low frequency in intestinal biopsy, carrying the p.(Arg623Leu) missense variant, producing in vitro 65% of normal MTP activity and apoB48 secretion, and two abnormally spliced transcripts resulting in a non-functional MTP protein. Digital droplet PCR and long-range sequencing revealed a previously described c.1067+1217_1141del allele inherited from the mother, removing exon 10. Thus, the patient is compound heterozygous for two dysfunctional MTTP alleles. The p.(Arg623Leu) variant may maintain residual secretion of apoB48. ConclusionsComplex cases of primary dyslipidemia require the use of a cascade of different methodologies to establish the diagnosis in patients with non-classical biological phenotypes and provide better knowledge on the regulation of lipid metabolism.

Association of Triglyceride-Lowering LPL Variants and LDL-C–Lowering LDLR Variants With Risk of Coronary Heart Disease

Triglycerides and cholesterol are both carried in plasma by apolipoprotein B (ApoB)-containing lipoprotein particles. It is unknown whether lowering plasma triglyceride levels reduces the risk of cardiovascular events to the same extent as lowering low-density lipoprotein cholesterol (LDL-C) levels. To compare the association of triglyceride-lowering variants in the lipoprotein lipase (LPL) gene and LDL-C-lowering variants in the LDL receptor gene (LDLR) with the risk of cardiovascular disease per unit change in ApoB. Mendelian randomization analyses evaluating the associations of genetic scores composed of triglyceride-lowering variants in the LPL gene and LDL-C-lowering variants in the LDLR gene, respectively, with the risk of cardiovascular events among participants enrolled in 63 cohort or case-control studies conducted in North America or Europe between 1948 and 2017. Differences in plasma triglyceride, LDL-C, and ApoB levels associated with the LPL and LDLR genetic scores. Odds ratio (OR) for coronary heart disease (CHD)-defined as coronary death, myocardial infarction, or coronary revascularization-per 10-mg/dL lower concentration of ApoB-containing lipoproteins. A total of 654 783 participants, including 91 129 cases of CHD, were included (mean age, 62.7 years; 51.4% women). For each 10-mg/dL lower level of ApoB-containing lipoproteins, the LPL score was associated with 69.9-mg/dL (95% CI, 68.1-71.6; P = 7.1 × 10-1363) lower triglyceride levels and 0.7-mg/dL (95% CI, 0.03-1.4; P = .04) higher LDL-C levels; while the LDLR score was associated with 14.2-mg/dL (95% CI, 13.6-14.8; P = 1.4 × 10-465) lower LDL-C and 1.9-mg/dL (95% CI, 0.1-3.9; P = .04) lower triglyceride levels. Despite these differences in associated lipid levels, the LPL and LDLR scores were associated with similar lower risk of CHD per 10-mg/dL lower level of ApoB-containing lipoproteins (OR, 0.771 [95% CI, 0.741-0.802], P = 3.9 × 10-38 and OR, 0.773 [95% CI, 0.747-0.801], P = 1.1 × 10-46, respectively). In multivariable mendelian randomization analyses, the associations between triglyceride and LDL-C levels with the risk of CHD became null after adjusting for differences in ApoB (triglycerides: OR, 1.014 [95% CI, 0.965-1.065], P = .19; LDL-C: OR, 1.010 [95% CI, 0.967-1.055], P = .19; ApoB: OR, 0.761 [95% CI, 0.723-0.798], P = 7.51 × 10-20). Triglyceride-lowering LPL variants and LDL-C-lowering LDLR variants were associated with similar lower risk of CHD per unit difference in ApoB. Therefore, the clinical benefit of lowering triglyceride and LDL-C levels may be proportional to the absolute change in ApoB.

The effect of apolipoprotein E polymorphism on serum metabolome \u2013 a population-based 10-year follow-up study

Apolipoprotein E (apoE) is the key regulator of plasma lipids, mediating altered functionalities in lipoprotein metabolism – affecting the risk of coronary artery (CAD) and Alzheimer’s diseases, as well as longevity. Searching pathways influenced by apoE prior to adverse manifestations, we utilized a metabolome dataset of 228 nuclear-magnetic-resonance-measured serum parameters with a 10-year follow-up from the population-based Young Finns Study cohort of 2,234 apoE-genotyped (rs7412, rs429358) adults, aged 24–39 at baseline. At the end of our follow-up, by limiting FDR-corrected p < 0.05, regression analyses revealed 180/228 apoE-polymorphism-related associations with the studied metabolites, in all subjects – without indications of apoE x sex interactions. Across all measured apoE- and apoB-containing lipoproteins, ε4 allele had consistently atherogenic and ε2 protective effect on particle concentrations of free/esterified cholesterol, triglycerides, phospholipids and total lipids. As novel findings, ε4 associated with glycoprotein acetyls, LDL-diameter and isoleucine – all reported biomarkers of CAD-risk, inflammation, diabetes and total mortality. ApoE-subgroup differences persisted through our 10-year follow-up, although some variation of individual metabolite levels was noticed. In conclusion, apoE polymorphism associate with a complex metabolic change, including aberrations in multiple novel biomarkers related to elevated cardiometabolic and all-cause mortality risk, extending our understanding about the role of apoE in health and disease.